Role of FGFR2b expression and signaling in keratinocyte differentiation: sequential involvement of PKCδ and PKCα

Role of FGFR2b expression and signaling in keratinocyte differentiation: sequential involvement... The tumor suppressor epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) induces human keratinocyte early differentiation. Moreover, protein kinases C (PKCs) are known to regulate the differentiation program in several cellular contexts, including keratinocytes. Therefore, in this paper we propose to clarify if FGFR2b could play a role also in the late steps of keratinocyte differentiation and to assess if this receptor-induced process would sequentially involve PKCδ and PKCα isoforms. Immunofluorescence, biochemical, and molecular approaches, performed on 2D cultures or 3D organotypic rafts of human keratinocytes overexpressing FGFR2b by stable transduction, showed that receptor signaling induced the precocious onset and an accelerated progression of keratinocyte differentiation, indicating that FGFR2b is a crucial regulator of the entire program of keratinocyte differentiation. In addition, the use of specific inhibitors and gene silencing approaches through specific siRNA demonstrated that PKCδ controls the onset of FGFR2b-triggered differentiation, while PKCα plays a role restricted to the terminal stages of the process. Molecular analysis revealed that the two PKC isoforms sequentially act via induction of KLF4 and DLX3, two transcription factors linked by negative loops to p63, suggesting that p63 would represent the hub molecule at the crossroad of an intricate signaling network downstream FGFR2b, involving multiple PKC-induced transcription factors. 3,4 Introduction roles . In the case of FGFR2, altered isoform switching The family of fibroblast growth factor receptors and aberrant expression of the mesenchymal FGFR2c (FGFRs) includes four transmembrane receptor tyrosine isoform in epithelial cells induce epithelial-mesenchymal 5,6 7,8 kinases (FGFR1–4) involved in the regulation of crucial transition (EMT) and are involved in cancerogenesis , biological processes, such as proliferation, migration, while the epithelial FGFR2b variant appears to exert 1,2 9,10 survival, and differentiation . Alternative splicing of the tumor suppressive functions . extracellular IgIII loop in FGFR1-3 generates epithelial In agreement with the knowledge of the key role played 11–13 FGFRb and mesenchymal FGFRc isoforms, determining by FGFR2b in the regulation of skin homeostasis , ligand specificity . Deregulation of the expression and studies from our group have demonstrated that FGFR2b signaling of FGFRs is well known to play oncogenic is up-regulated in epidermal suprabasal layers and is able to induce early differentiation in normal human keratinocytes . We have also suggested that FGFR2b Correspondence: Francesca Belleudi (francesca.belleudi@uniroma1.it) exerts its differentiative function via the repression of Department of Clinical and Molecular Medicine,Laboratory affiliated to Istituto p63 , a transcription factor whose down-modulation Pasteur Italia—Fondazione Cenci Bolognetti, Sapienza University of Rome, Roma, Italy represents the main molecular mechanism known to drive S. Andrea University Hospital, Rome, Italy These authors contributed equally: Benedetta Rosato, Danilo Ranieri. These authors jointly supervised this work: Maria Rosaria Torrisi, Francesca Belleudi. Edited by E Candi. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Rosato et al. Cell Death and Disease (2018) 9:565 Page 2 of 16 early differentiation of stratified epithelia . Although the Results role of FGFR2b in early stages of keratinocyte differ- FGFR2b controls not only the early, but also the late steps entiation has been deeply investigated, its involvement in of keratinocyte differentiation 18–20 the later steps of the process still remains debated Studies from our group have demonstrated that and to be clarified. FGFR2b expression and activation promote keratinocyte 15,16 With the aim to analyze the possible signaling candi- early differentiation . Here, in order to investigate if dates for the regulation of FGFR2b-mediated early dif- FGFR2b might also control the later stages of differ- ferentiation,wehavepreviously demonstratedthe entiation, we first analyzed the impact of the receptor involvement of the PI3K/AKT pathway . However, the signaling on the expression and distribution throughout keratinocyte differentiation is a complex program the epidermal layers of two distinct markers of the most implying several steps of coordinated molecular events early or the most late differentiation steps: the early driven by an intricate signaling network; therefore, to cytokeratin K1 and the terminal differentiation FIL. For verify if additional pathways downstream FGFR2b could this purpose we reproduced in vitro the keratinocyte participate to such network, we focused our attention on differentiation program occurring in vivo, using 3D protein kinases C (PKCs) and in particular to the PKCδ organotypic skin equivalents prepared with human and PKCα isoforms. In fact, these signaling substrates HaCaT keratinocytes stably transduced with pBp- are involved in FGFR2-mediated differentiation in the FGFR2b retroviral constructs or with empty pBp vector 21,22 6 bone context and it has been proposed that PKCδ as negative control . In fact, through this model of and PKCα could regulate keratinocyte differentiation , receptor-overexpressing cells we are able to force the during which they appear to exert temporally and spa- system toward the differentiated phenotype. The orga- tially distinct roles. notypic cultures were left untreated or stimulated with A function in the onset of keratinocyte differentiation FGF7 as reported in Materials and methods section. 23–29 has been proposed for PKCδ , which is expressed Quantitative immunofluorescence analysis showed that, starting from the basal layer of epidermis where, upon in pBp rafts, the K1 signal was visible in all suprabasal activation, causes hemidesmosomal disassembly and basal layers (Fig. 1a, left panels) and increased by FGF7 stimu- 30,31 15 cell migration upward into the suprabasal layers .At lation (Fig. 1a, left panels), as expected . Interestingly, the level of transcriptional control, PKCδ induces the HaCaT pBp-FGFR2b rafts showed, besides the enhance- early differentiation marker desmoglein-1 (DSG1) . ment of K1 staining (Fig. 1a, left panels), its appearance already in the basal layer (Fig. 1a, left panels), suggesting Moreover, recent studies highlighted that PKCδ, through the induction of the transcription factor Kruppel-like the precocious onset of early differentiation. Parallel factor 4 (KLF4), up-modulates p21 and the intermediate evaluation of FIL expression showed that, as expected , differentiation marker involucrin (INV) triggering cell its granular staining appeared confined in the uppermost 28,29 cycle arrest and differentiation, respectively . layers of pBp skin equivalents (Fig. 1a, right panels) and it On the other hand, a role in epidermal late differ- was significantly increased by FGF7 stimulation (Fig. 1a, entiation has been proposed for PKCα. In fact, PKCα right panels). In addition, FGFR2b overexpression expression is restricted to epidermal suprabasal layers induced a general enhancement of this staining and its and its inhibition or depletion represses the expression of appreciable appearance starting from lower layers of the the late differentiation markers filaggrin (FIL) and loricrin rafts (Fig. 1a, right panels), suggesting an accelerated (LOR) , while the early/intermediate differentiation progression also throughout the terminal steps of differ- 32 41 markers keratin 1 (K1), K10, and INV appear sub- entiation. As previously reported , the higher staining of 25,27,33–36 stantially unaffected . Distal-less homeobox 3 the analyzed markers in pBp-FGFR2b untreated organo- (DLX3) could be the molecular player acting downstream typic cultures respect to the corresponding unstimulated PKCα in keratinocyte differentiation: this transcription pBp rafts would be ascribed to the already acquired factor is expressed in the suprabasal layers and is involved enhanced differentiated phenotype of the FGFR2b clones in the induction of FIL and LOR, as well as in the epi- during their growth in complete medium before starva- 37–39 dermal barrier formation . tion. Our observations were also validated at molecular Based on these assumptions, here we propose to clarify level by real time RT-PCR analysis, demonstrating that if FGFR2b could play a general role in the control of the the expression of early (K10 and DSG1), intermediate entire program of keratinocyte differentiation and to (INV, transglutaminase-1: TGM1) and late differentiation assess if the possible function of FGFR2b in the different marker (LOR) genes were significantly increased in steps of the process might be regulated by a signaling response to FGF7 and particularly in cells overexpressing network sequentially involving PKCδ and PKCα iso- FGFR2b (Fig. 1b). Thus, FGFR2b and its signaling appear forms and their respective downstream transcription to play a positive role on the entire program of kerati- factors. nocyte differentiation. Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 3 of 16 HaCaT pBp-FGFR2b HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp K1 FIL DAPI DAPI FGF7 FGF7 FGF7 FGF7 ** 2.5 2.5 ** 2.0 2.0 ** ** 1.5 1.5 1.0 1.0 0.5 0.5 0 0 FGF7 FGF7 - - - - + + + + ** 4 4 10 4 4 *** ** ** * ** 8 * 3 3 3 3 ** 6 * ** ** 2 2 ** 2 2 4 * ** 1 1 1 1 0 0 0 0 0 FGF7 - - - - - - + - - - - + + + + + + + + + Fig. 1 FGFR2b expression and signaling play a positive role on the entire program of keratinocyte differentiation. Organotypic skin equivalents of HaCaT pBp and pBp-FGFR2b clones, prepared as reported in Materials and methods section, were grown in complete medium and left untreated or stimulated with FGF7 for the last 4 days. a Quantitative immunofluorescence analysis shows that, in pBp rafts, K1 staining is visible in all suprabasal layers and it is increased by FGF7 stimulation. In HaCaT pBp-FGFR2b raft K1 staining is enhanced and detectable already in the basal layer. Parallel evaluation of FIL expression shows that the its granular staining appears confined in the uppermost layers of pBp skin equivalents and it is significantly increased by FGF7 stimulation. FGFR2b overexpression induces a general enhancement of the staining and its appreciable appearance starting from lower layers of the rafts. Quantitative analysis of the fluorescence intensity was performed as described in Materials and methods and results sections are expressed as fold increase respect to pBp values ± SE. Student’s t test was performed and significance levels have been defined as p < 0.05: *p < 0.0001 vs the corresponding FGF7-unstimulated cells; **p < 0.0001 vs the corresponding pBp cells. Bar: 25 μm. b Real time RT-PCR analysis shows that mRNA expression of the early (K10 and DSG1), intermediate (TGM1 and INV) and late differentiation (LOR) markers are significantly increased upon FGF7 stimulation, particularly in pBp-FGFR2b rafts. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as p < 0.05: *p < 0.05, and ***p < 0.005 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression fold increase K1 fluorescence intensity DSG1 mRNA fold expression TGM1 mRNA fold expression fold increase FIL fluorescence intensity INV mRNA fold expression LOR mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 4 of 16 a b FGF7 FGF7 - - - - + + + + 140 kDa 140 kDa FGFR2b FGFR2b fold increase 1 1.2 2.3 2.3 fold increase 1 0.8 4.7 4.7 ACTIN 67 kDa K1 67 kDa K1 fold increase 1 1.6 1.9 2.8 fold increase 1 1.2 1.2 1.4 ACTIN 42 kDa ACTIN 42 kDa 165 kDa 165 kDa DSG1 DSG1 fold increase 1 2.2 2.5 3.5 fold increase 1 1.4 1.4 1.6 42 kDa ACTIN 42 kDa ACTIN 68 kDa 68 kDa INV INV fold increase 1 1.7 2.1 2.5 fold increase 1 1.3 1 1.4 ACTIN ACTIN 42 kDa 42 kDa 138 kDa 138 kDa β1-integrin β1-integrin fold increase 1 0.8 0.4 0.2 fold increase 1 1 0.8 0.7 42 kDa 42 kDa ACTIN ACTIN 39 kDa FIL 39 kDa FIL fold increase 1 3.1 3.9 6.2 fold increase 1 1 1 1 TUBULIN 50 kDa 50 kDa TUBULIN 26 kDa 26 kDa LOR LOR fold increase 1 1 1 1.2 fold increase 1 2 2 3.6 42 kDa 42 kDa ACTIN ACTIN Fig. 2 FGFR2b has a sequential role in distinct steps of the keratinocyte differentiation. HaCaT pBp and HaCaT pBp-FGFR2b clones grown up to confluence or post-confluence were left untreated or stimulated with FGF7 as reported in Materials and methods section. a Western blot analysis shows that in confluent conditions the levels of K1, DSG1, and INV are increased upon FGF7 stimulation particularly in pBp-FGFR2b cells, while no appreciable levels of FIL and LOR are visible in these cells. In addition, the expression of β1-integrin appears strongly decreased in HaCaT pBp-FGFR2b in response to ligand stimulation. b In post-confluence conditions all the early/intermediate differentiation markers appear increased making their FGF7-induced modulation less evident compared to the corresponding confluent cells. In contrast, FIL and LOR are detectable and they appear up- regulated in response to FGF7 stimulation, particularly in cells overexpressing FGFR2b. The equal loading was assessed with anti-ACTIN and anti- TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported In order to analyze in detail this role, we “dissected” stimulation (Fig. 2a). Finally, in agreement with their in vitro the process using HaCaT pBp and HaCaT pBp- similarities with basal/differentiating keratinocytes, no FGFR2b cultures grown until confluence, the step that appreciable levels of the late differentiation marker FIL precedes the shift from the basal to suprabasal layer, or up and LOR were found in all confluent HaCaT clones to post-confluence, the step that mimics the late differ- (Fig. 2a). On the other hand, the shift to post-confluence entiation and stratification . Cells were then left strongly increased the expression of all the differentiation untreated or stimulated with FGF7, as reported in Mate- markers (Fig. 2b), making their FGF7-induced modulation rials and methods section, and Western blot analysis was less evident compared to the corresponding confluent performed to estimate the expression of a wide selection cells (Fig. 2b). Moreover, as already observed in 3D of differentiation markers. Results showed that, in con- organotypic cultures, pBp-FGFR2b cells showed a higher fluent conditions, a clear up-regulation of the early dif- differentiated phenotype compared to pBp cultures also in ferentiation markers K1 and DSG1 and of the the absence of FGF7 stimulation, which can be ascribed to intermediate differentiation marker INV was induced by the enhanced differentiation acquired during the growth FGF7, particularly in pBp-FGFR2b cells (Fig. 2a). In in complete medium before starvation. The observed addition, an opposite behavior was observed for the basal progressive modulation in response to FGF7 of early/ marker β1-integrin , whose expression appeared strongly intermediate and LORs in confluent and post-confluent decreased in HaCaT pBp-FGFR2b in response to ligand cultures of HaCaT clones, respectively, was also Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b late markers eraly/intermediate markers Rosato et al. Cell Death and Disease (2018) 9:565 Page 5 of 16 confirmed at the mRNA transcript level (Supplementary These findings were also validated at the mRNA tran- Figure S1). script level by real time RT-PCR analysis, confirming that, in both HaCaT pBp and pBp-FGFR2b confluent cultures, PKCδ and PKCα signaling play sequential roles in FGFR2b- the block of PKCδ activity by PKCδi (Fig. 3c) or protein triggered keratinocyte differentiation depletion by specific siRNA (Fig. 3d) strongly decreased Searching for the possible downstream candidates that FGF7-induced, as well as basal expression, of K10, DSG1, and INV. The results obtained were strengthened by could be alternatively involved in FGFR2b-mediated early or late differentiation, we focused our attention on PKCδ quantitative immunofluorescence analysis (Supplemen- and PKCα isoforms, since they have been proposed to tary Figure S2, left and right panels), in which we intro- 23 15,43,46 exert distinct roles in keratinocyte differentiation . duced also the inhibitor of AKT (AKTi) , in order to Since PKCδ isoform has been proposed to be specifi- interfere with the additional FGFR2b downstream path- 23–29 cally implicated in the onset of the process ,we first way previously identified by us as involved in keratinocyte investigated its contribution in FGFR2b-induced early early differentiation , or the MEK1/2 inhibitor (MEK1/ differentiation taking advantage of the specific inhibitor 2i), as negative control, since we have recently observed rottlerin (PKCδi), previously used to demonstrate the that this pathway did not appear to be involved in the involvement of PKCδ in FGF/FGFR signaling pathways , process (Nanni et al., manuscript in preparation). In fact, as well as in FGFR2b-triggered autophagy . The inhibitor while the role played by AKT signaling appears to be efficiency was confirmed testing its ability to decrease the restricted to some aspects of FGFR2b-induced early steps basal, as well as the FGF7-induced, phosphorylation of of the process, PKCδ emerges as hub signaling down- PKCδ at the autophosphorylation site Serine 645 stream FGFR2b regulating the entire early differentiation. (Fig. 3a, left panel), but not that of PKCα at the autop- Because PKCα isoform has been proposed to be a sig- hosphorylation site Serine 657 (Fig. 3a, left panel). Then naling substrate crucial for the later steps of keratinocyte 23,25,27,33–36 we analyzed the impact of this inhibitor on the modula- differentiation , we investigated its possible tion of early/intermediate differentiation markers induced contribution in FGFR2b-induced terminal steps of the by FGFR2b and its signaling in confluent cultures. Wes- process. To this aim the effects of PKCα inhibition were tern blot analysis clearly showed that PKCδi exerted a analyzed in post-confluent cultures of HaCaT clones strong repressive effect on the K1, DSG1, and INV using the specific inhibitor Go6976 (PKCαi) . The effi- increase induced by FGF7, which was particularly evident ciency of PKCαi was assessed by Western blot analysis, in FGFR2b overexpressing clones (Fig. 3a, right panel). performed in both HaCaT pBp and pBp-FGFR2b cultures, Indeed, in agreement with the proposed general role of showing a decrease of the basal, as well as the FGF7- PKCδ in keratinocyte early differentiation , this inhibi- induced PKCα phosphorylation at the autopho- tory effect, even if less pronounced, was also detectable in sphorylation site Serine 657 (Fig. 4a, left panel), but not cells not stimulated with FGF7 (Fig. 3a, right panel). that of PKCδ at the autophosphorylation site Serine 645 However, to exclude the possibility of the establishment of (Fig. 4a, left panel). Then we investigated if PKCαi could an autocrine signaling due to endogenous production of affect FGFR2b-induced differentiation. Western blot FGF7, which could explain the phenotype displayed by analysis highlighted that PKCαi did not interfere with the unstimulated FGFR2b clones, we performed real time RT- expression of the early differentiation marker K1 (Fig. 4a, PCR analysis: FGF7 mRNA was undetectable in both right panel), but it displayed a strong repressive effect on HaCaT clones also after PKC signaling shut-off (data not the FGF7-induced up-modulation of the late differentia- shown). To unequivocally demonstrate the role of PKCδ tion markers LOR and FIL (Fig. 4a, right panel). More- on early differentiation, we performed its specific deple- over, also the basal level of LOR and FIL markers tion by siRNA transfection. To this aim, HaCaT clones expressed in cells not stimulated with FGF7 appeared were transfected with PKCδ siRNA or with an unrelated weakly dampened by PKCαi, confirming the general role siRNA as control. The efficiency of protein depletion was of PKCα in keratinocyte differentiation . Similar results verified through Western blot analysis (Fig. 3b, left panel). were obtained by specific siRNA approaches. The effi- Transfected cells were grown until confluence and left ciency of protein depletion was first confirmed by Wes- untreated or stimulated with FGF7 as above. Western blot tern blot analysis (Fig. 4b, left panel). Then, the effects of analysis showed that PKCδ silencing strongly counter- PKCα silencing showed repression of the late differ- acted the increase of K1, DSG1, and INV in response to entiation markers LOR and FIL (Fig. 4b, right panel), but FGF7 (Fig. 3b, right panel). In addition, similarly to what not of the early differentiation marker K1 (Fig. 4b, right observed after the pharmacological inhibition, PKCδ panel) in both pBp and pBp-FGFR2b clones. The inhibi- depletion appeared to slightly affect also the basal level of tory effect of either PKCα pharmacological inhibition the markers (Fig. 3b, right panel), confirming its general (Fig. 4c) and protein depletion (Fig. 4d) was also con- role in the control of early differentiation. firmed at the mRNA transcript level by real time RT-PCR Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 6 of 16 - - - - - - - - FGF7 + + + + FGF7 + + + + PKCδi - + - + - + - + PKCδi - + - + - + - + p-PKCδ 67 kDa K1 fold increase 1 0.7 2 1.5 2.3 1.3 2.5 1.5 fold increase 1 0.6 1.6 0.8 1.8 1.4 2.5 1.2 78 kDa PKCδ 165 kDa DSG1 fold increase 1 0.8 2.4 1.5 2.7 2.1 3.2 1.8 42 kDa ACTIN 42 kDa ACTIN 68 kDa INV p-PKCα fold increase 1 0.7 2 1.2 2.3 1.6 2.5 1.5 fold increase 1 1 1.2 1.2 2 1.9 2.3 2.2 42 kDa ACTIN 78 kDa PKCα 42 kDa TUBULIN - - - - - FGF7 + + + + FGF7 - - + + - + + PKCδ siRNA - + - + - + - + PKCδ siRNA - + - + - + - + + + control siRNA - + - + - + control siRNA - + - + - + - 78 kDa PKCδ 67 kDa K1 fold increase 1 0.6 1.1 0.5 1.2 0.6 1.3 0.5 fold increase 1 0.5 2 1.2 3.5 1 4 1.5 42 kDa ACTIN 42 kDa ACTIN 165 kDa DSG1 fold increase 1 0.8 2.3 1.5 2.4 1.5 2.7 1.2 42 kDa ACTIN 68 kDa INV fold increase 1 0.8 2.6 1.9 3.4 2.5 3.7 1 42 kDa ACTIN 4 4 ** 4 ** ** 3 3 3 ** *** * ** 2 2 2 *** ** *** *** *** *** 1 1 1 *** *** *** *** *** *** 0 0 0 - - - - - - - - FGF7 + + + + + + + + - - + + - - + + - + + - - + + - - + + - - + + - PKCdi - + + - - + + - ** 4 4 3 3 ** ** ** * * ** 2 2 ** 2 *** **** **** *** **** 1 1 *** **** **** *** *** *** *** 0 0 0 - - - - - - - - FGF7 - - + + - - + + + + + + + + + + PKCd siRNA - + + - - + + - - + + - - + + - - + + - - + + - + - + - control siRNA + - + - + - + - + - + - + - + - + - + - Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression K10 mRNA fold expression DSG1 mRNA fold expression DSG1 mRNA fold expression INV mRNA fold expression INV mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 7 of 16 (see figure on previous page) Fig. 3 PKCδ controls the onset of FGFR2b-triggered keratinocyte differentiation. Confluent HaCaT pBp and HaCaT pBp-FGFR2b clones were left untreated or stimulated with FGF7 in presence or not of the PKCδ inhibitor as reported in Materials and methods section (a, c). Alternatively, HaCaT clones were transfected with PKCδ siRNA or with an unrelated siRNA as control, and then left untreated or stimulated with FGF7 (b, d). a Western blot analysis confirms that PKCδ inhibitor interferes with the basal, as well as the FGF7-induced, phosphorylation of PKCδ at its autophosphorylation site Ser645 but not with that of PKCα at its autophosphorylation site Ser657 (left panel). PKCδ inhibition induces a repressive effect on the K1, DSG1, and INV increase induced by FGF7, which is particularly evident in FGFR2b overexpressing clones (right panel). b Western blot analysis confirms that PKCδ silencing specifically dampened the basal, as well as the FGF7-induced, PKCδ protein level but not that of PKCα (left panel). PKCδ silencing also strongly counteracts the increase of K1, DSG1, and INV in response to FGF7 (right panel). The equal loading was assessed with anti-PKCδ or anti-PKCα and anti-ACTIN or anti-TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported. c, d Real time RT-PCR analysis confirms that PKCδ inhibition (c) or depletion (d) decreases FGF7-induced, as well as basal mRNA transcript levels, of K10 DSG1 and INV. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: c *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; ***p < 0.05 vs the corresponding PKCδ inhibitor-untreated cells; d *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; ***p < 0.05 and ****p < 0.005 vs the corresponding control siRNA cells analysis. Thus, also PKCα appears a pivotal player in sustained cell proliferation. These findings strongly indi- FGFR2b-triggered keratinocyte differentiation; however, cated that, as a consequence of PKCδ signaling shut-off, differently from PKCδ, its role appears to be specifically keratinocytes are defective in the ability to undergo the restricted to the late stages of the process. differentiation program maintaining a basal/proliferative Then, in order to analyze the effect of PKCδ inhibition phenotype. Real time RT-PCR analysis confirmed that the on the ability of keratinocytes to progress throughout the expression of both early and LORs was significantly entire differentiation program and stratify in response to repressed by PKCδi (Fig. 5b). Finally, the quantitative FGFR2b activation, we again took advantage of the use of evaluation of the raft thickness revealed that, in pBp and the more complete model of organotypic cultures. HaCaT pBp-FGFR2b skin equivalents, the layers were sig- pBp and HaCaT pBp-FGFR2b rafts were lifted to nificantly reduced by PKCδi (Fig. 5c), further indicating air–liquid interface in the presence or not of PKCδi and that inhibition of PKCδ induces impairment of all steps of finally stimulated with FGF7, as above. Quantitative FGFR2b-triggered differentiation, which in turn results in immunofluorescence analysis showed that the K1 and FIL defective stratification. staining, visible in both HaCaT pBp and HaCaT pBp- FGFR2b rafts stimulated with FGF7 (Fig. 5a, upper The transcription factors KLF4 and DLX3, acting panels), appeared overall dampened by the presence of downstream PKCδ and PKCα respectively, are sequentially PKCδi, suggesting that the entire differentiation process up-regulated by FGFR2b signaling induced by FGFR2b signaling was arrested by the sus- Since it has been recently proposed that PKCδ and tained inhibition of PKCδ (Fig. 5a, upper panels). In PKCα control the expression of several genes via the 28,29 36 addition, the immunostaining of K5 revealed that the induction of KLF4 and DLX3 respectively, we canonical distribution in the basal layer of this undiffer- decided to investigate the possible involvement of these entiated/proliferating cell marker , visible in pBp control transcription factors in FGFR2b-triggered keratinocyte rafts (Fig. 5a, lower panels), disappeared in pBp-FGFR2b differentiation. The topic was particularly appealing, since skin equivalents (Fig. 5a, lower panels), confirming the they have been proposed to be involved in an intricate 47–49 precocious onset of differentiation. However, in the pre- network of loops with ΔNp63 , the transcription sence of PKCδi, K5 staining appeared recovered and factor whose down-modulation represents the main distributed all over the rafts (Fig. 5a, lower panels). molecular mechanism known to drive differentiation of Moreover, the parallel evaluation of Ki67 staining in both stratified epithelia . Indeed, we have already described a pBp and pBp-FGFR2b rafts stimulated with FGF7 revealed link between FGFR2b and p63 , but the molecular few positive cells distributed in the basal layer (Fig. 5a, mechanism underlying this interplay remains to be still lower panels); the presence of some stained cells also in clarified. To this aim, we performed Real Time RT-PCR the suprabasal layers (Fig. 5a, lower panels) is in agree- analysis on HaCaT pBp and pBp-FGFR2b 3D organotypic ment with the previously described ability of FGF7 to also rafts, checking for the transcript levels of KLF4 and DLX3 sustain the residual proliferative activity of suprabasal and comparing them with that of p63. The results showed keratinocytes . More interestingly, in pBp-FGFR2b raft a significant increase of both KLF4 and DLX3 and an grown in the presence of PKCδi, several cells intensely opposite decrease of p63 in response to FGF7 stimulation stained for Ki67 marker appeared homogeneously dis- (Fig. 6a), which were more evident in the rafts over- tributed in all the layers (Fig. 5a, lower panels), suggesting expressing FGFR2b (Fig. 6a). Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 8 of 16 - - - - - - - - FGF7 + + + + FGF7 + + + + PKCαi - + - + - + - + PKCαi - + - + - + - + p-PKCα 67 kDa K1 fold increase 1 0.5 2.5 1.5 5 3 6 4 fold increase 1 1 2 2.3 2.3 2.7 3 3.3 26 kDa 78 kDa PKCα LOR fold increase 1 0.5 1.3 0.7 1.8 0.8 2 1.3 42 kDa 42 kDa ACTIN ACTIN 39 kDa p-PKCδ FIL fold increase 1 0.8 2 1.2 1.6 1 2.8 1.2 fold increase 1 1 2 2.3 3.8 4 4 4 50 kDa TUBULIN 78 kDa PKCδ 42 kDa ACTIN - - - - FGF7 + + + + FGF7 - - - - + + + + PKCα siRNA - + - + - + - + PKCα siRNA - - + - + - + + control siRNA - + - + - + - + control siRNA - + - + - + - 78 kDa PKCα 67 kDa K1 fold increase 1 0.6 1.4 0.7 1.6 0.4 1.8 0.6 fold increase 1 1.1 1.8 1.9 2.4 2.2 2.7 2.7 42 kDa ACTIN 26 kDa LOR fold increase 1 0.5 1.4 0.8 2.6 1.8 3 1.9 50 kDa TUBULIN 39 kDa FIL fold increase 1 0.4 1.1 0.7 1.2 0.7 2 0.3 50 kDa TUBULIN 6 6 6 5 5 5 ** 4 4 4 ** NS NS 3 3 3 NS 2 NS 2 ** ** *** *** 1 1 *** *** *** *** 1 *** *** 0 0 - - - - - - - - FGF7 + + + + + + + + - - + + - - + + - + + - - + + - + + + + PKCαi - - - - - + + - - + + - 6 6 5 5 5 ** 4 4 ** NS 3 3 NS ** NS **** 2 2 2 ** NS *** *** 1 1 *** 1 **** **** **** **** 0 0 0 - - - - - - - - - - - - FGF7 + + + + + + + + + + + + + + + + + + + + PKCα siRNA - + + - - + + - - - - - - - - - + + - - + + + - - + + + - control siRNA + - + - - + - - + - - Fig. 4 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression K10 mRNA fold expression LOR mRNA fold expression LOR mRNA fold expression FIL mRNA fold expression FIL mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 9 of 16 (see figure on previous page) Fig. 4 PKCα is required for FGFR2b-induced terminal stages of keratinocyte differentiation. Post-confluent HaCaT pBp and HaCaT pBp-FGFR2b cells were left untreated or stimulated with FGF7 in presence or not of the PKCα inhibitor as reported in Materials and methods section (a, c). Alternatively, HaCaT clones were transfected with PKCα siRNA or with an unrelated siRNA as control, transfected cells were grown until post- confluence and left untreated or stimulated with FGF7 as above (b, d). a Western blot analysis shows that PKCα inhibitor decreases the basal, as well as the FGF7-induced, PKCα phosphorylation, but not that of PKCδ phosphorylation (left panel). PKCα inhibitor does not interfere with the K1 expression, but it displays a repressive effect on the FGF7-induced up-modulation of LOR and FIL (right panel). b Western blot analysis confirms that PKCα silencing specifically dampened the basal, as well as the FGF7-induced, PKCα protein level but not that of PKCδ (left panel). PKCα silencing also strongly counteracts the increase of LOR and FIL but not that of K1 in response to FGF7 (right panel). The equal loading was assessed with anti-PKCδ or anti-PKCα and anti-ACTIN or anti-TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported. c, d Real time RT-PCR analysis confirms that PKCα inhibition (c) or depletion (d) decreases FGF7-induced, as well as basal mRNA transcript levels, of LOR and FIL but it does not affect K10 expression. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; NS and ***p < 0.05 vs the corresponding PKCα inhibitor-untreated cells; d *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; NS, ***p < 0.05 and ****p < 0.005 vs the corresponding control siRNA cells Then, to verify if KLF4 and DLX3 up-regulation could contrasting reports appear to suggest its possible invol- 18–20 display a sequential trend during FGFR2b-induced kera- vement in the later stages of this process . Therefore, tinocyte differentiation, we analyzed their possible mod- here we first analyzed the impact of the receptor signaling ulation in confluent and post-confluent clones stimulates on the induction of terminal differentiation in 3D orga- with FGF7 as above. The results showed that KLF4 up- notypic rafts and 2D cultures of human keratinocytes regulation was mainly appreciable in confluent clones engineered to stably overexpress FGFR2b. The results (Fig. 6b), while DLX3 induction was detectable only in obtained clearly indicated that, particularly when FGFR2b post-confluent cultures (Fig. 6b). As expected, the is overexpressed, the receptor signaling not only induces a induction of KLF4 in confluent cells as well as that of precocious onset of the early differentiation markers, but DLX3 in post-confluent cells were significantly counter- also enhances the expression and accelerates the appear- acted by PKCδi and PKCαi, respectively (Fig. 6b), further ance of the terminal markers. These findings strongly confirming the connection of each PKC signaling with the suggest a key role of FGFR2b in the regulation of the corresponding transcription factor. Moreover, similarly to entire differentiation program. what observed for KLF4 and consistently with its well- Then, in order to analyze the signaling players down- established role in FGFR2b-induced onset of differentia- stream FGFR2b possibly involved in keratinocyte differ- 15,16 tion , p63 repression in response to FGF7 was more entiation, we focused on PKCδ and PKCα isoforms, at the evident in confluent cells and impaired only by PKCδi light of previous evidences showing their sequential role (Fig. 6b). Being p63 a direct negative regulator of KLF4 in in the regulation of the different steps of the process .To normal human keratinocytes , its strong repression the aim to “dissect” in vitro the differentiation steps, cells observed in confluent cells may represent the molecular were grown until confluence or up to post-confluence. mechanism through which FGFR2b-triggered PKCδ sig- Taking advantage of the use of specific substrate inhibi- naling would induce KLF4 and consequently the onset of tors and siRNA interference, we highlighted that PKCδ keratinocyte differentiation. These results suggest that the signaling appears to drive the early molecular events, alternative induction of KLF4 and DLX3 in FGF7- while PKCα regulates the late ones, of FGFR2b-triggered stimulated confluent and post-confluent cells, respec- keratinocyte differentiation. Given the role of PKCδ in the tively, might reflect the sequential activation of their onset of the process, we used the organotypic cultures to upstream activators PKCδ and PKCα during the early and analyze the impact of its inhibition on the ability of ker- late steps of FGFR2b-induced differentiation. atinocytes to terminally differentiate and stratify in response to ligand-mediated activation of FGFR2b: we Discussion demonstrated that PKCδ signaling shut-off was sufficient Because of its hypothesized role as a tumor suppressor to block the entire differentiation program, inducing 9,10 in vitro and in vivo , forced modulation of FGFR2b retention of undifferentiated/basal features and defective could be a winning strategy to revert important aspects of stratification. Interestingly, these rafts also maintained a the malignant phenotype in carcinoma cells, including sustained cell proliferation throughout all the layers that loss of cell differentiation. However, in the physiological could be explained by the previously described ability of context of normal human keratinocytes, despite the FGFR2b to not only promote the differentiation pro- numerous evidences emphasizing the role of FGFR2b in gram , but also sustain the residual proliferative activity the enhancement of early differentiation, only few and of suprabasal, differentiating keratinocytes . Overall, the Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 10 of 16 2.5 HaCaT pBp HaCaT pBp-FGFR2b K1 K1 DAPI DAPI 2.0 ** 1.5 1.0 0.5 FGF7 FGF7 + PKCδi FGF7 FGF7 + PKCδi 2.5 FIL FIL DAPI DAPI 2.0 *p< 0.0001 vs rottlerin- ** 1.5 1.0 0.5 FGF7 FGF7 FGF7 + PKCδi FGF7 + PKCδi FGF7 + + + + PKCδi - + - + HaCaT pBp HaCaT pBp-FGFR2b K5 K5 2.5 DAPI DAPI 2.0 1.5 1.0 0.5 ** FGF7 FGF7 FGF7 + PKCδi Ki67 Ki67 DAPI DAPI NS FGF7 FGF7 FGF7 + PKCδi FGF7 + + + PKCδi - - + b c ** 2.5 2.5 2.5 ** ** 2.0 2.0 2.0 200 1.5 1.5 1.5 1.0 1.0 1.0 * 100 0.5 * 0.5 0.5 50 0 0 0 FGF7 FGF7 FGF7 FGF7 + + + + + + + + + + + + + + + + PKCδi PKCδi PKCδi PKCδi - + - + - + - + - + - + - + - + Fig. 5 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression DSG1 mRNA fold expression LOR mRNA fold expression Organotypic culture fold increase thickness (μm) K5 fluorescence intensity % of Ki67 positive cells fold increase fold increase FIL fluorescence intensity K1 fluorescence intensity Rosato et al. Cell Death and Disease (2018) 9:565 Page 11 of 16 (see figure on previous page) Fig. 5 PKCδ signaling shut-off blocks the entire differentiation program and induces defective stratification in keratinocytes. Organotypic skin equivalents of HaCaT pBp and pBp-FGFR2b clones, prepared as reported in Materials and methods section, were lifted to air–liquid interface in the presence or not of PKCδ inhibitor and finally stimulated with FGF7 as above. a Quantitative immunofluorescence analysis shows that K1 and FIL staining, visible in both HaCaT pBp and HaCaT pBp-FGFR2b rafts stimulated with FGF7, appears overall impaired by the presence of PKCδ inhibitor (upper panels). In addition, K5 signal, visible in the basal layer of pBp rafts, disappears in pBp-FGFR2b skin equivalents while it is recovered and distributed all over the pBp-FGFR2b rafts by the presence of PKCδ inhibitor (lower panels). Parallel evaluation of the nuclear proliferation marker Ki67 reveals the presence of few positive cells distributed in the basal layer and randomly throughout the suprabasal layers in pBp rafts and FGFR2b rafts stimulated with FGF7 (lower panels). In pBp-FGFR2b raft grown in the presence of PKCδi, several cells intensely stained for Ki67 marker are visible in all the layers (lower panels). Quantitative analysis of the fluorescence intensity and of the percentage of Ki67 positive cells were performed as described in Materials and methods and results sections are expressed as fold increase respect to pBp values ± SE. Student’s t test was performed and significance levels have been defined as above: *p < 0.0001 vs the corresponding PKCδ inhibitor-untreated cells; NS and **p < 0.0001 vs the corresponding pBp cells. Bar: 25 μm. b Real time RT-PCR analysis confirms that, in pBp and pBp-FGFR2b rafts stimulated with FGF7, the expression of K10, DSG1, and LOR is significantly repressed by PKCδ inhibitor. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: *p < 0.05 vs PKCδ inhibitor-untreated cells; **p < 0.05 vs the corresponding pBp cells. c Quantitative evaluation of the raft thickness shows that the layers of pBp and pBp-FGFR2b skin equivalents are significantly reduced by the presence of PKCδ inhibitor. Quantitative analysis of the raft thickness was expressed as µm mean ± SD from three independent experiments. Student’s t test was performed and significance levels have been defined as p < 0.05: *p < 0.0001 vs the corresponding PKCδ inhibitor-untreated cells 16,41 phenotypic features derived by PKCδ signaling shut-off and induced by FGFR2b signaling . In fact, the indicate that the inhibition of this signaling pathway alone FGFR2b/p63 crosstalk appears to be central in the is sufficient to impair the balance between cell prolifera- homeostatic balance between keratinocyte proliferation tion and differentiation controlled by FGFR2b. On the and differentiation and its impairment, due to out-of- other hand, we can not exclude the contribution of the context appearance of FGFR2c, leads to loss of the dif- epidermal growth factor receptor (EGFR) in such ferentiated phenotype and acquisition of tumorigenic increased cell proliferation: in fact, the antagonistic role of features . For this reason, to identify the molecular FGFR2b and EGFR in epithelial cells has been previously players contributing to the regulation of this crosstalk described by our group . While FGFR2b is up-regulated seems to represent a very urgent and important goal. Indeed, both KLF4 and DLX3 have been proposed to be during the shift from the basal to the suprabasal layers, 48,49 EGFR mainly displays a basal distribution and its ligand- involved in negative loops with p63: consistent with dependent activation triggers cell proliferation. In addi- these assumptions, we observed that they displayed tion, it has been also proposed that FGF7/KGF treatment opposite modulations compared to p63. Interestingly, the may induce endogenous synthesis of the EGFR ligand up-regulation of KLF4 appeared more appreciable in TGFα. In any case, these results are in agreement with confluent cells, the same culture condition in which the the widely described role of PKCδ as tumor suppressor, down-regulation of p63 was more evident: this simulta- which is down-regulated in squamous cell carcinomas neous detection of p63 down-regulation and KLF4 52,53 (SCCs) . induction mainly in confluent cells strongly suggests that Concerning the molecular effectors acting downstream they take place in the same step of the differentiation PKC isoforms, our results showed that, in agreement with program, and possibly during the shift between the basal 28,29,36 previous reports , PKCδ and PKCα isoforms act via to the suprabasal layer. These observations, together with the induction of the downstream transcription factors the recent evidence of a possible direct role of PKCδ in KLF4 and DLX3, respectively. In fact, the expression of p63 down-regulation , allowed us to speculate that the both KLF4 and DLX3 was clearly induced in response to down-regulation of p63 could represent the molecular FGF7, particularly in rafts or cell cultures overexpressing mechanism through which FGFR2b and its downstream FGFR2b. Interestingly, in cultures grown at different cell PKCδ signaling could induce the up-regulation of KLF4 densities to mimic the various steps of differentiation, we and consequently the onset of keratinocyte differentiation. found that KLF4 up-regulation was mainly appreciable in confluent cells, while DLX3 induction was detectable only Materials and methods in post-confluent cultures. These sequential trends of Cells and treatments induction could reflect the progressive involvement of The human keratinocyte cell line HaCaT , stably their upstream inductors PKCδ and PKCα in the distinct overexpressing FGFR2b (pBp-FGFR2b) or the empty stages (early and late, respectively) of differentiation. vector (pBp) and generated as previously described , were Finally, we also decided to compare the modulation of cultured in Dulbecco’s modified Eagle’s medium KLF4 and DLX3 with that previously described for p63 (DMEM), supplemented with 10% fetal bovine serum Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 12 of 16 3D organotypic cultures *** 4 4 1.2 3 3 0.8 ** ** * 2 2 0.6 ** 0.4 ** 1 1 0.2 0 0 - - + + FGF7 - + - + - - + + *** *** 6 12 * 1.2 *** *** 5 10 1.0 0.8 4 8 ** 0.6 3 6 *** ** 4 0.4 *** *** ** NS NS NS *** 0.2 1 2 NS NS 0 0 - - - - - - - - - - - - FGF7 + + + + + + + + + + + + - + + - - + + - - + + - - + + - - + + - - + + - PKCδi 6 12 1.2 NS 5 10 ** 1.0 ** NS * NS 8 0.8 ** ** * NS NS 0.6 3 6 ** * NS NS 4 0.4 2 ^ NS NS 2 0.2 0 0 0 FGF7 - - - - - - - - + + + + + + + + - - + + - - + + - + + - - + + - - + + - - + + - PKCαi - + + - - + + - Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b KLF4 mRNA fold expression KLF4 mRNA fold expression KLF4 mRNA fold expression DLX3 mRNA fold expression DLX3 mRNA fold expression DLX3 mRNA fold expression p63 mRNA fold expression p63 mRNA fold expression p63 mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 13 of 16 (see figure on previous page) Fig. 6 PKCδ and PKCα signaling are responsible for FGF7-mediated sequential induction of KLF4 and DLX3, respectively. a Organotypic skin equivalents of HaCaT pBp and pBp-FGFR2b clones, prepared as reported in Materials and methods section, were grown in complete medium and left untreated or stimulated with FGF7 as above. Real time RT-PCR analysis shows a significant increase of both KLF4 and DLX3 expression upon FGF7 stimulation, while p63 displays an opposite trend. b HaCaT pBp and HaCaT pBp-FGFR2b cells were grown up to confluence or post-confluence in the presence or not of PKCδ and PKCα inhibitors, respectively, and stimulated with FGF7 as above. Real time RT-PCR analysis shows that KLF4 up- regulation is mainly appreciable in confluent clones, while DLX3 induction is detectable only in post-confluent cultures. The increased expression of KLF4 in confluent cells and that of DLX3 in post-confluent cultures are significantly dampened by PKCδi and PKCαi, respectively. Similarly to KLF4, p63 down-modulation in response to FGF7 is more evident in confluent cells and it appears impaired only by PKCδi. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: a *p < 0.05 and ***p < 0.001 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; b NS and *p < 0.05 vs the corresponding FGF7-unstimulated cells; NS and **p < 0.05 vs the corresponding pBp cells; NS and ***p < 0.05 vs the corresponding PKCδ inhibitor-untreated cells; NS and ^p < 0.005 vs the corresponding PKCα inhibitor-untreated cells (FBS) plus antibiotics. Primary cultures of human fibro- mixture was left to polymerize for 30 min at 37 °C. After blasts derived from healthy skin (HFs) were obtained from 24 h 2 × 10 HaCaT pBp or pBp-FGFR2b cells were see- patients attending the Dermatology Unit of the Sant’An- ded on the collagen gel and left to grow for a week in drea Hospital of Rome; all patients were extensively complete medium added in both the top and the bottom informed and their consent for the investigation was given wells. Then, the organotypic cultures were lifted to the and collected in written form in accordance with guide- air–liquid interface and cultured for further two weeks in lines approved by the management of the Sant’Andrea complete medium added or not with the specific PKCδ Hospital. HFs were isolated and cultured as previously inhibitor rottlerin (5 μM; Calbiochem, 557370). Then described . cultures were left untreated or stimulated with FGF7 For growth factors stimulation, cells were left untreated (Upstate Biotechnology) 100 ng/ml for the last 4 days. or incubated with FGF7 (Upstate Biotechnology, Lake Rafts were finally fixed in 10% formalin, embedded in Placid, NY, USA, 01–118) 100 ng/ml for 24 or 48 h at 37 ° paraffin and 4 µm slices were obtained. To evaluate C. To inhibit AKT or MEK1/2 or PKCδ or PKCα cells organotypic thickness slices were stained with hematox- were respectively incubated with the specific AKT inhi- ylin and eosin using standard procedures then bright field bitor 1L-6-hydroxy-methyl-chiro-inositol 2-(R)-2-O- images were taken with an Axiocam ICc 5 (Zeiss, Ober- methyl-3-O-octadecylcarbonate (1 μM; Calbiochem, kochen, Germany) connected with an Axioplan 100 Nottingham, UK, 124005) or with the specific MEK1/2 microscope (Zeiss). The organotypic culture thickness inhibitor PD0325901 (1 μM; Sigma-Aldrich, Saint Louis, was measured using the Axiovision software (Zeiss) and MO, USA, PZ0162) or with the specific PKCδ inhibitor expressed as mean µm ± standard deviation (SD). Stu- rottlerin (5 μM; Calbiochem, 557370) or with the specific dent’s t test was performed and significance levels have PKCα inhibitor Go6976 (3 μM; Calbiochem, 365250) for been defined as p < 0.05. 1 h at 37 °C before treatment with FGF7 in the presence of each inhibitor. Immunofluorescence For RNA interference and PKCδ or PKCα silencing, HaCaT clones, grown on coverslips, were fixed with 4% cells were transfected with PKCδ small interfering RNA paraformaldehyde in PBS for 30 min at 25 °C followed by (PKCδ siRNA) (Santa Cruz Biotechnology, Santa Cruz, treatment with 0.1 M glycine for 20 min at 25 °C and with CA, USA), or PKCα small interfering RNA (PKCα siRNA) 0.1% Triton X-100 for additional 5 min at 25 °C to allow (Santa Cruz Biotechnology), or with an unrelated siRNA permeabilization. Cells were then incubated for 1 h at 25 ° as a control (control siRNA), using Lipofectamine 2000 C with the following primary antibodies: rabbit polyclonal Transfection Reagent (Invitrogen, Carlsbad, CA, USA) anti-K1 (1:50 in PBS, AF 87, Covance, Princeton, NJ, according to the manufacturer’s protocol. USA) or mouse monoclonal anti-β1 integrin (1:1000 in PBS; ST2–16, Santa Cruz Biotechnology). The primary Organotypic cultures antibodies were visualized using goat anti-rabbit IgG- For three-dimensional (3D) organotypic cultures, col- Texas Red (1:200 in PBS; Jackson Immunoresearch lagen rafts were prepared adding 5 mg/ml rat tail type I Laboratories, West Grove, PA, USA) or goat anti-mouse collagen (Corning, Lowell, MA, USA) to DMEM and IgG-Alexa Fluor 488 (1:200 in PBS; Life Technologies, Reconstitution buffer (8:1:1) as previously described . Carlsbad, CA, USA) antibodies for 30 min at 25 °C. Nuclei HFs (1 × 10 ) were added to 2 ml of the collagen mixture were stained with DAPI (1:1000 in PBS; Sigma). Cover- in polycarbonate micron inserts (23 mm diameter, pore slips were finally mounted with mowiol (Sigma) for size 0.3 µm; Corning) in 6-deep well plates (Corning). The observation. Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 14 of 16 Organotypic raft sections, obtained as above, were Covance), anti-Bek (C-17, Santa Cruz Biotechnology), deparaffinized in xylene and re-hydrated through graded anti-loricrin (Covance), anti-p-PKCδ (Ser645, Santa Cruz ethanols to PBS, pH 7.4. Antigen retrieval was achieved by Biotechnology) polyclonal antibodies, all followed by heating sections in target retrieval solution low pH (Dako, enhanced chemiluminescence detection (ECL, Amer- Carpinteria, CA, USA) for 15 min at 97 °C. Sections were sham, Alington Heights, IL, USA). The membranes were then washed with PBS and blocked using 10% bovine calf rehydrated by being washed in PBS-Tween 20, stripped with 100 mM mercaptoethanol and 2% SDS for 30 min at serum and 0.2% Triton X-100 for 30 min at 25 °C before staining with rabbit polyclonal anti-K1 (1:500 in PBS, AF 55 °C, and probed again with anti-PKCδ (C-20, Santa 87, Covance), mouse monoclonal anti-filaggrin (1:1000 in Cruz Biotechnology) polyclonal antibodies, anti-PKCα PBS, 15C10, Monosan Sanbio, Uden, The Netherland), monoclonal antibody (Y124, Abcam) or with anti-β- rabbit polyclonal anti-cytokeratin 5 (1:400 in PBS, Abcam, ACTIN (AC-15, Sigma) monoclonal antibody or anti-α- Cambridge, UK) or mouse monoclonal anti-Ki67 (1:100 in TUBULIN (2148S, Cell Signaling) polyclonal antibodies to PBS, MIB-1, Dako) antibodies for 1 h in a humidified estimate the protein equal loading. Densitometric analysis chamber. The primary antibodies were visualized using was performed using Quantity One Program (Bio-Rad goat anti-mouse IgG-Texas Red (1:200 in PBS; Jackson Laboratories, Hercules, CA, USA). Results from three Immunoresearch Laboratories) or goat anti-rabbit IgG- different experiments were normalized and expressed as Texas Red (1:200 in PBS; Jackson Immunoresearch fold increase respect to the control value. Values from a Laboratories) antibodies for 30 min at 25 °C. Nuclei were representative of three independent experiments were stained with DAPI (1:1000 in PBS; Sigma). Sections were reported in each figure. permanently mounted under a coverslip. Fluorescence signals were analyzed by conventional fluorescence or by Primers scanning cells in a series of sequential sections with an Oligonucleotide primers necessary for target genes and ApoTome System (Zeiss) connected with an Axiovert 200 the housekeeping gene were chosen utilizing the online inverted microscope (Zeiss); image analysis was per- tool Primer-BLAST and purchased from Invitrogen. Pri- formed by the Axiovision software (Zeiss) and images mers list and characteristics are reported in Supplemen- were obtained by 3D reconstruction of the total number tary Table S1. For p63 target gene, the reported primers of the serial optical sections. Quantitative analysis of the were designed to recognize both ΔNp63 and TAp63 α and fluorescence intensity was performed by the Axiovision β isoforms. For each primer pair, we performed no- software (Zeiss), analyzing 10 different fields randomly template control and no-reverse-transcriptase control taken from three independent experiments. Quantitative (RT negative) assays, which produced negligible signals. analysis of the percentage of Ki67 positive cells was assessed counting for each sample a total of 50 cells, RNA extraction and cDNA synthesis randomly observed in 10 microscopic fields from three Organotypic cultures were deparaffinized and RNA was different experiments. Results are shown as means ± extracted using the TRIzol method (Invitrogen) according standard error (SE). Student’s t test was performed and to manufacturer’s instructions and eluted with 0.1% die- significance levels have been defined as p < 0.05. thylpyrocarbonate (DEPC)-treated water. Each sample was treated with DNAase I (Invitrogen). Total RNA Western blot analysis concentration was quantitated by spectrophotometry; 1 Cells were lysed in a buffer containing 50 mM HEPES, μg of total RNA was used to reverse transcription using pH 7.5, 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 iScriptTM cDNA synthesis kit (Bio-Rad) according to mM MgCl , 5 mM EGTA, supplemented with protease manufacturer’s instructions. inhibitors (10 μg/ml aprotinin, 1 mM PMSF, 10 μg/ml leupeptin), and phosphatase inhibitors (1 mM sodium PCR amplification and real time quantitation orthovanadate, 20 mM sodium pyrophosphate, 0.5 M Real time RT-PCR was performed using the iCycler Real NaF). A range between 20 and 50 μg of total protein was Time Detection System (iQ5 Bio-Rad) with optimized resolved under reducing conditions by 8 or 12% SDS- PCR conditions. The reaction was carried out in 96-well PAGE and transferred to reinforced nitrocellulose (BA-S plate using iQ SYBR Green Supermix (Bio- Rad) adding 83, Schleider and Schuell, Keene, NH, USA). The mem- forward and reverse primers for each gene and 1 μlof branes were blocked with 5% nonfat dry milk in PBS 0.1% diluted template cDNA to a final reaction volume of 15 μl. Tween 20 and incubated with anti-DSG1 (27B2, Life All assays included a negative control and were replicated Technologies), anti-β1 integrin (ST2–16, Santa Cruz three times. The thermal cycling program was performed Biotechnology), anti-involucrin (SY5, Abcam), anti- as described . Real time quantitation was performed with filaggrin (15C10, Monosan), anti-p-PKCα (Ser657, the help of the iCycler IQ optical system software version Abcam) monoclonal antibody or with anti-K1 (AF 87, 3.0a (Bio-Rad), according to the manufacturer’s manual. Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 15 of 16 Results are reported as mean ± SD from three different 13. Yang, J. et al. Fibroblast growth factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous homeostasis. J. Cell Biol. 188, experiments in triplicate. Student’s t test was performed 935–952 (2010). and significance levels have been defined as p < 0.05. 14. Capone, A. et al. Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human kerati- nocyte HaCaT cell line. Cell Growth Differ. 11,607–614 (2000). Statistical analysis 15. Belleudi, F., Purpura, V. & Torrisi, M. R. The receptor tyrosine kinase FGFR2b/ Data were statistically analyzed with unpaired two-tailed KGFR controls early differentiation of human keratinocytes. PLoS ONE 6, Student’s t test. Differences were considered significant at e24194 (2011). 16. Purpura, V., Belleudi, F.,Caputo, S. &Torrisi,M.R.HPV16 E5 andKGFR/FGFR2b the level of p < 0.05. Statistical analysis was performed by interplay in differentiating epithelial cells. 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Role of FGFR2b expression and signaling in keratinocyte differentiation: sequential involvement of PKCδ and PKCα

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Abstract

The tumor suppressor epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) induces human keratinocyte early differentiation. Moreover, protein kinases C (PKCs) are known to regulate the differentiation program in several cellular contexts, including keratinocytes. Therefore, in this paper we propose to clarify if FGFR2b could play a role also in the late steps of keratinocyte differentiation and to assess if this receptor-induced process would sequentially involve PKCδ and PKCα isoforms. Immunofluorescence, biochemical, and molecular approaches, performed on 2D cultures or 3D organotypic rafts of human keratinocytes overexpressing FGFR2b by stable transduction, showed that receptor signaling induced the precocious onset and an accelerated progression of keratinocyte differentiation, indicating that FGFR2b is a crucial regulator of the entire program of keratinocyte differentiation. In addition, the use of specific inhibitors and gene silencing approaches through specific siRNA demonstrated that PKCδ controls the onset of FGFR2b-triggered differentiation, while PKCα plays a role restricted to the terminal stages of the process. Molecular analysis revealed that the two PKC isoforms sequentially act via induction of KLF4 and DLX3, two transcription factors linked by negative loops to p63, suggesting that p63 would represent the hub molecule at the crossroad of an intricate signaling network downstream FGFR2b, involving multiple PKC-induced transcription factors. 3,4 Introduction roles . In the case of FGFR2, altered isoform switching The family of fibroblast growth factor receptors and aberrant expression of the mesenchymal FGFR2c (FGFRs) includes four transmembrane receptor tyrosine isoform in epithelial cells induce epithelial-mesenchymal 5,6 7,8 kinases (FGFR1–4) involved in the regulation of crucial transition (EMT) and are involved in cancerogenesis , biological processes, such as proliferation, migration, while the epithelial FGFR2b variant appears to exert 1,2 9,10 survival, and differentiation . Alternative splicing of the tumor suppressive functions . extracellular IgIII loop in FGFR1-3 generates epithelial In agreement with the knowledge of the key role played 11–13 FGFRb and mesenchymal FGFRc isoforms, determining by FGFR2b in the regulation of skin homeostasis , ligand specificity . Deregulation of the expression and studies from our group have demonstrated that FGFR2b signaling of FGFRs is well known to play oncogenic is up-regulated in epidermal suprabasal layers and is able to induce early differentiation in normal human keratinocytes . We have also suggested that FGFR2b Correspondence: Francesca Belleudi (francesca.belleudi@uniroma1.it) exerts its differentiative function via the repression of Department of Clinical and Molecular Medicine,Laboratory affiliated to Istituto p63 , a transcription factor whose down-modulation Pasteur Italia—Fondazione Cenci Bolognetti, Sapienza University of Rome, Roma, Italy represents the main molecular mechanism known to drive S. Andrea University Hospital, Rome, Italy These authors contributed equally: Benedetta Rosato, Danilo Ranieri. These authors jointly supervised this work: Maria Rosaria Torrisi, Francesca Belleudi. Edited by E Candi. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Rosato et al. Cell Death and Disease (2018) 9:565 Page 2 of 16 early differentiation of stratified epithelia . Although the Results role of FGFR2b in early stages of keratinocyte differ- FGFR2b controls not only the early, but also the late steps entiation has been deeply investigated, its involvement in of keratinocyte differentiation 18–20 the later steps of the process still remains debated Studies from our group have demonstrated that and to be clarified. FGFR2b expression and activation promote keratinocyte 15,16 With the aim to analyze the possible signaling candi- early differentiation . Here, in order to investigate if dates for the regulation of FGFR2b-mediated early dif- FGFR2b might also control the later stages of differ- ferentiation,wehavepreviously demonstratedthe entiation, we first analyzed the impact of the receptor involvement of the PI3K/AKT pathway . However, the signaling on the expression and distribution throughout keratinocyte differentiation is a complex program the epidermal layers of two distinct markers of the most implying several steps of coordinated molecular events early or the most late differentiation steps: the early driven by an intricate signaling network; therefore, to cytokeratin K1 and the terminal differentiation FIL. For verify if additional pathways downstream FGFR2b could this purpose we reproduced in vitro the keratinocyte participate to such network, we focused our attention on differentiation program occurring in vivo, using 3D protein kinases C (PKCs) and in particular to the PKCδ organotypic skin equivalents prepared with human and PKCα isoforms. In fact, these signaling substrates HaCaT keratinocytes stably transduced with pBp- are involved in FGFR2-mediated differentiation in the FGFR2b retroviral constructs or with empty pBp vector 21,22 6 bone context and it has been proposed that PKCδ as negative control . In fact, through this model of and PKCα could regulate keratinocyte differentiation , receptor-overexpressing cells we are able to force the during which they appear to exert temporally and spa- system toward the differentiated phenotype. The orga- tially distinct roles. notypic cultures were left untreated or stimulated with A function in the onset of keratinocyte differentiation FGF7 as reported in Materials and methods section. 23–29 has been proposed for PKCδ , which is expressed Quantitative immunofluorescence analysis showed that, starting from the basal layer of epidermis where, upon in pBp rafts, the K1 signal was visible in all suprabasal activation, causes hemidesmosomal disassembly and basal layers (Fig. 1a, left panels) and increased by FGF7 stimu- 30,31 15 cell migration upward into the suprabasal layers .At lation (Fig. 1a, left panels), as expected . Interestingly, the level of transcriptional control, PKCδ induces the HaCaT pBp-FGFR2b rafts showed, besides the enhance- early differentiation marker desmoglein-1 (DSG1) . ment of K1 staining (Fig. 1a, left panels), its appearance already in the basal layer (Fig. 1a, left panels), suggesting Moreover, recent studies highlighted that PKCδ, through the induction of the transcription factor Kruppel-like the precocious onset of early differentiation. Parallel factor 4 (KLF4), up-modulates p21 and the intermediate evaluation of FIL expression showed that, as expected , differentiation marker involucrin (INV) triggering cell its granular staining appeared confined in the uppermost 28,29 cycle arrest and differentiation, respectively . layers of pBp skin equivalents (Fig. 1a, right panels) and it On the other hand, a role in epidermal late differ- was significantly increased by FGF7 stimulation (Fig. 1a, entiation has been proposed for PKCα. In fact, PKCα right panels). In addition, FGFR2b overexpression expression is restricted to epidermal suprabasal layers induced a general enhancement of this staining and its and its inhibition or depletion represses the expression of appreciable appearance starting from lower layers of the the late differentiation markers filaggrin (FIL) and loricrin rafts (Fig. 1a, right panels), suggesting an accelerated (LOR) , while the early/intermediate differentiation progression also throughout the terminal steps of differ- 32 41 markers keratin 1 (K1), K10, and INV appear sub- entiation. As previously reported , the higher staining of 25,27,33–36 stantially unaffected . Distal-less homeobox 3 the analyzed markers in pBp-FGFR2b untreated organo- (DLX3) could be the molecular player acting downstream typic cultures respect to the corresponding unstimulated PKCα in keratinocyte differentiation: this transcription pBp rafts would be ascribed to the already acquired factor is expressed in the suprabasal layers and is involved enhanced differentiated phenotype of the FGFR2b clones in the induction of FIL and LOR, as well as in the epi- during their growth in complete medium before starva- 37–39 dermal barrier formation . tion. Our observations were also validated at molecular Based on these assumptions, here we propose to clarify level by real time RT-PCR analysis, demonstrating that if FGFR2b could play a general role in the control of the the expression of early (K10 and DSG1), intermediate entire program of keratinocyte differentiation and to (INV, transglutaminase-1: TGM1) and late differentiation assess if the possible function of FGFR2b in the different marker (LOR) genes were significantly increased in steps of the process might be regulated by a signaling response to FGF7 and particularly in cells overexpressing network sequentially involving PKCδ and PKCα iso- FGFR2b (Fig. 1b). Thus, FGFR2b and its signaling appear forms and their respective downstream transcription to play a positive role on the entire program of kerati- factors. nocyte differentiation. Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 3 of 16 HaCaT pBp-FGFR2b HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp K1 FIL DAPI DAPI FGF7 FGF7 FGF7 FGF7 ** 2.5 2.5 ** 2.0 2.0 ** ** 1.5 1.5 1.0 1.0 0.5 0.5 0 0 FGF7 FGF7 - - - - + + + + ** 4 4 10 4 4 *** ** ** * ** 8 * 3 3 3 3 ** 6 * ** ** 2 2 ** 2 2 4 * ** 1 1 1 1 0 0 0 0 0 FGF7 - - - - - - + - - - - + + + + + + + + + Fig. 1 FGFR2b expression and signaling play a positive role on the entire program of keratinocyte differentiation. Organotypic skin equivalents of HaCaT pBp and pBp-FGFR2b clones, prepared as reported in Materials and methods section, were grown in complete medium and left untreated or stimulated with FGF7 for the last 4 days. a Quantitative immunofluorescence analysis shows that, in pBp rafts, K1 staining is visible in all suprabasal layers and it is increased by FGF7 stimulation. In HaCaT pBp-FGFR2b raft K1 staining is enhanced and detectable already in the basal layer. Parallel evaluation of FIL expression shows that the its granular staining appears confined in the uppermost layers of pBp skin equivalents and it is significantly increased by FGF7 stimulation. FGFR2b overexpression induces a general enhancement of the staining and its appreciable appearance starting from lower layers of the rafts. Quantitative analysis of the fluorescence intensity was performed as described in Materials and methods and results sections are expressed as fold increase respect to pBp values ± SE. Student’s t test was performed and significance levels have been defined as p < 0.05: *p < 0.0001 vs the corresponding FGF7-unstimulated cells; **p < 0.0001 vs the corresponding pBp cells. Bar: 25 μm. b Real time RT-PCR analysis shows that mRNA expression of the early (K10 and DSG1), intermediate (TGM1 and INV) and late differentiation (LOR) markers are significantly increased upon FGF7 stimulation, particularly in pBp-FGFR2b rafts. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as p < 0.05: *p < 0.05, and ***p < 0.005 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression fold increase K1 fluorescence intensity DSG1 mRNA fold expression TGM1 mRNA fold expression fold increase FIL fluorescence intensity INV mRNA fold expression LOR mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 4 of 16 a b FGF7 FGF7 - - - - + + + + 140 kDa 140 kDa FGFR2b FGFR2b fold increase 1 1.2 2.3 2.3 fold increase 1 0.8 4.7 4.7 ACTIN 67 kDa K1 67 kDa K1 fold increase 1 1.6 1.9 2.8 fold increase 1 1.2 1.2 1.4 ACTIN 42 kDa ACTIN 42 kDa 165 kDa 165 kDa DSG1 DSG1 fold increase 1 2.2 2.5 3.5 fold increase 1 1.4 1.4 1.6 42 kDa ACTIN 42 kDa ACTIN 68 kDa 68 kDa INV INV fold increase 1 1.7 2.1 2.5 fold increase 1 1.3 1 1.4 ACTIN ACTIN 42 kDa 42 kDa 138 kDa 138 kDa β1-integrin β1-integrin fold increase 1 0.8 0.4 0.2 fold increase 1 1 0.8 0.7 42 kDa 42 kDa ACTIN ACTIN 39 kDa FIL 39 kDa FIL fold increase 1 3.1 3.9 6.2 fold increase 1 1 1 1 TUBULIN 50 kDa 50 kDa TUBULIN 26 kDa 26 kDa LOR LOR fold increase 1 1 1 1.2 fold increase 1 2 2 3.6 42 kDa 42 kDa ACTIN ACTIN Fig. 2 FGFR2b has a sequential role in distinct steps of the keratinocyte differentiation. HaCaT pBp and HaCaT pBp-FGFR2b clones grown up to confluence or post-confluence were left untreated or stimulated with FGF7 as reported in Materials and methods section. a Western blot analysis shows that in confluent conditions the levels of K1, DSG1, and INV are increased upon FGF7 stimulation particularly in pBp-FGFR2b cells, while no appreciable levels of FIL and LOR are visible in these cells. In addition, the expression of β1-integrin appears strongly decreased in HaCaT pBp-FGFR2b in response to ligand stimulation. b In post-confluence conditions all the early/intermediate differentiation markers appear increased making their FGF7-induced modulation less evident compared to the corresponding confluent cells. In contrast, FIL and LOR are detectable and they appear up- regulated in response to FGF7 stimulation, particularly in cells overexpressing FGFR2b. The equal loading was assessed with anti-ACTIN and anti- TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported In order to analyze in detail this role, we “dissected” stimulation (Fig. 2a). Finally, in agreement with their in vitro the process using HaCaT pBp and HaCaT pBp- similarities with basal/differentiating keratinocytes, no FGFR2b cultures grown until confluence, the step that appreciable levels of the late differentiation marker FIL precedes the shift from the basal to suprabasal layer, or up and LOR were found in all confluent HaCaT clones to post-confluence, the step that mimics the late differ- (Fig. 2a). On the other hand, the shift to post-confluence entiation and stratification . Cells were then left strongly increased the expression of all the differentiation untreated or stimulated with FGF7, as reported in Mate- markers (Fig. 2b), making their FGF7-induced modulation rials and methods section, and Western blot analysis was less evident compared to the corresponding confluent performed to estimate the expression of a wide selection cells (Fig. 2b). Moreover, as already observed in 3D of differentiation markers. Results showed that, in con- organotypic cultures, pBp-FGFR2b cells showed a higher fluent conditions, a clear up-regulation of the early dif- differentiated phenotype compared to pBp cultures also in ferentiation markers K1 and DSG1 and of the the absence of FGF7 stimulation, which can be ascribed to intermediate differentiation marker INV was induced by the enhanced differentiation acquired during the growth FGF7, particularly in pBp-FGFR2b cells (Fig. 2a). In in complete medium before starvation. The observed addition, an opposite behavior was observed for the basal progressive modulation in response to FGF7 of early/ marker β1-integrin , whose expression appeared strongly intermediate and LORs in confluent and post-confluent decreased in HaCaT pBp-FGFR2b in response to ligand cultures of HaCaT clones, respectively, was also Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b late markers eraly/intermediate markers Rosato et al. Cell Death and Disease (2018) 9:565 Page 5 of 16 confirmed at the mRNA transcript level (Supplementary These findings were also validated at the mRNA tran- Figure S1). script level by real time RT-PCR analysis, confirming that, in both HaCaT pBp and pBp-FGFR2b confluent cultures, PKCδ and PKCα signaling play sequential roles in FGFR2b- the block of PKCδ activity by PKCδi (Fig. 3c) or protein triggered keratinocyte differentiation depletion by specific siRNA (Fig. 3d) strongly decreased Searching for the possible downstream candidates that FGF7-induced, as well as basal expression, of K10, DSG1, and INV. The results obtained were strengthened by could be alternatively involved in FGFR2b-mediated early or late differentiation, we focused our attention on PKCδ quantitative immunofluorescence analysis (Supplemen- and PKCα isoforms, since they have been proposed to tary Figure S2, left and right panels), in which we intro- 23 15,43,46 exert distinct roles in keratinocyte differentiation . duced also the inhibitor of AKT (AKTi) , in order to Since PKCδ isoform has been proposed to be specifi- interfere with the additional FGFR2b downstream path- 23–29 cally implicated in the onset of the process ,we first way previously identified by us as involved in keratinocyte investigated its contribution in FGFR2b-induced early early differentiation , or the MEK1/2 inhibitor (MEK1/ differentiation taking advantage of the specific inhibitor 2i), as negative control, since we have recently observed rottlerin (PKCδi), previously used to demonstrate the that this pathway did not appear to be involved in the involvement of PKCδ in FGF/FGFR signaling pathways , process (Nanni et al., manuscript in preparation). In fact, as well as in FGFR2b-triggered autophagy . The inhibitor while the role played by AKT signaling appears to be efficiency was confirmed testing its ability to decrease the restricted to some aspects of FGFR2b-induced early steps basal, as well as the FGF7-induced, phosphorylation of of the process, PKCδ emerges as hub signaling down- PKCδ at the autophosphorylation site Serine 645 stream FGFR2b regulating the entire early differentiation. (Fig. 3a, left panel), but not that of PKCα at the autop- Because PKCα isoform has been proposed to be a sig- hosphorylation site Serine 657 (Fig. 3a, left panel). Then naling substrate crucial for the later steps of keratinocyte 23,25,27,33–36 we analyzed the impact of this inhibitor on the modula- differentiation , we investigated its possible tion of early/intermediate differentiation markers induced contribution in FGFR2b-induced terminal steps of the by FGFR2b and its signaling in confluent cultures. Wes- process. To this aim the effects of PKCα inhibition were tern blot analysis clearly showed that PKCδi exerted a analyzed in post-confluent cultures of HaCaT clones strong repressive effect on the K1, DSG1, and INV using the specific inhibitor Go6976 (PKCαi) . The effi- increase induced by FGF7, which was particularly evident ciency of PKCαi was assessed by Western blot analysis, in FGFR2b overexpressing clones (Fig. 3a, right panel). performed in both HaCaT pBp and pBp-FGFR2b cultures, Indeed, in agreement with the proposed general role of showing a decrease of the basal, as well as the FGF7- PKCδ in keratinocyte early differentiation , this inhibi- induced PKCα phosphorylation at the autopho- tory effect, even if less pronounced, was also detectable in sphorylation site Serine 657 (Fig. 4a, left panel), but not cells not stimulated with FGF7 (Fig. 3a, right panel). that of PKCδ at the autophosphorylation site Serine 645 However, to exclude the possibility of the establishment of (Fig. 4a, left panel). Then we investigated if PKCαi could an autocrine signaling due to endogenous production of affect FGFR2b-induced differentiation. Western blot FGF7, which could explain the phenotype displayed by analysis highlighted that PKCαi did not interfere with the unstimulated FGFR2b clones, we performed real time RT- expression of the early differentiation marker K1 (Fig. 4a, PCR analysis: FGF7 mRNA was undetectable in both right panel), but it displayed a strong repressive effect on HaCaT clones also after PKC signaling shut-off (data not the FGF7-induced up-modulation of the late differentia- shown). To unequivocally demonstrate the role of PKCδ tion markers LOR and FIL (Fig. 4a, right panel). More- on early differentiation, we performed its specific deple- over, also the basal level of LOR and FIL markers tion by siRNA transfection. To this aim, HaCaT clones expressed in cells not stimulated with FGF7 appeared were transfected with PKCδ siRNA or with an unrelated weakly dampened by PKCαi, confirming the general role siRNA as control. The efficiency of protein depletion was of PKCα in keratinocyte differentiation . Similar results verified through Western blot analysis (Fig. 3b, left panel). were obtained by specific siRNA approaches. The effi- Transfected cells were grown until confluence and left ciency of protein depletion was first confirmed by Wes- untreated or stimulated with FGF7 as above. Western blot tern blot analysis (Fig. 4b, left panel). Then, the effects of analysis showed that PKCδ silencing strongly counter- PKCα silencing showed repression of the late differ- acted the increase of K1, DSG1, and INV in response to entiation markers LOR and FIL (Fig. 4b, right panel), but FGF7 (Fig. 3b, right panel). In addition, similarly to what not of the early differentiation marker K1 (Fig. 4b, right observed after the pharmacological inhibition, PKCδ panel) in both pBp and pBp-FGFR2b clones. The inhibi- depletion appeared to slightly affect also the basal level of tory effect of either PKCα pharmacological inhibition the markers (Fig. 3b, right panel), confirming its general (Fig. 4c) and protein depletion (Fig. 4d) was also con- role in the control of early differentiation. firmed at the mRNA transcript level by real time RT-PCR Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 6 of 16 - - - - - - - - FGF7 + + + + FGF7 + + + + PKCδi - + - + - + - + PKCδi - + - + - + - + p-PKCδ 67 kDa K1 fold increase 1 0.7 2 1.5 2.3 1.3 2.5 1.5 fold increase 1 0.6 1.6 0.8 1.8 1.4 2.5 1.2 78 kDa PKCδ 165 kDa DSG1 fold increase 1 0.8 2.4 1.5 2.7 2.1 3.2 1.8 42 kDa ACTIN 42 kDa ACTIN 68 kDa INV p-PKCα fold increase 1 0.7 2 1.2 2.3 1.6 2.5 1.5 fold increase 1 1 1.2 1.2 2 1.9 2.3 2.2 42 kDa ACTIN 78 kDa PKCα 42 kDa TUBULIN - - - - - FGF7 + + + + FGF7 - - + + - + + PKCδ siRNA - + - + - + - + PKCδ siRNA - + - + - + - + + + control siRNA - + - + - + control siRNA - + - + - + - 78 kDa PKCδ 67 kDa K1 fold increase 1 0.6 1.1 0.5 1.2 0.6 1.3 0.5 fold increase 1 0.5 2 1.2 3.5 1 4 1.5 42 kDa ACTIN 42 kDa ACTIN 165 kDa DSG1 fold increase 1 0.8 2.3 1.5 2.4 1.5 2.7 1.2 42 kDa ACTIN 68 kDa INV fold increase 1 0.8 2.6 1.9 3.4 2.5 3.7 1 42 kDa ACTIN 4 4 ** 4 ** ** 3 3 3 ** *** * ** 2 2 2 *** ** *** *** *** *** 1 1 1 *** *** *** *** *** *** 0 0 0 - - - - - - - - FGF7 + + + + + + + + - - + + - - + + - + + - - + + - - + + - - + + - PKCdi - + + - - + + - ** 4 4 3 3 ** ** ** * * ** 2 2 ** 2 *** **** **** *** **** 1 1 *** **** **** *** *** *** *** 0 0 0 - - - - - - - - FGF7 - - + + - - + + + + + + + + + + PKCd siRNA - + + - - + + - - + + - - + + - - + + - - + + - + - + - control siRNA + - + - + - + - + - + - + - + - + - + - Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression K10 mRNA fold expression DSG1 mRNA fold expression DSG1 mRNA fold expression INV mRNA fold expression INV mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 7 of 16 (see figure on previous page) Fig. 3 PKCδ controls the onset of FGFR2b-triggered keratinocyte differentiation. Confluent HaCaT pBp and HaCaT pBp-FGFR2b clones were left untreated or stimulated with FGF7 in presence or not of the PKCδ inhibitor as reported in Materials and methods section (a, c). Alternatively, HaCaT clones were transfected with PKCδ siRNA or with an unrelated siRNA as control, and then left untreated or stimulated with FGF7 (b, d). a Western blot analysis confirms that PKCδ inhibitor interferes with the basal, as well as the FGF7-induced, phosphorylation of PKCδ at its autophosphorylation site Ser645 but not with that of PKCα at its autophosphorylation site Ser657 (left panel). PKCδ inhibition induces a repressive effect on the K1, DSG1, and INV increase induced by FGF7, which is particularly evident in FGFR2b overexpressing clones (right panel). b Western blot analysis confirms that PKCδ silencing specifically dampened the basal, as well as the FGF7-induced, PKCδ protein level but not that of PKCα (left panel). PKCδ silencing also strongly counteracts the increase of K1, DSG1, and INV in response to FGF7 (right panel). The equal loading was assessed with anti-PKCδ or anti-PKCα and anti-ACTIN or anti-TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported. c, d Real time RT-PCR analysis confirms that PKCδ inhibition (c) or depletion (d) decreases FGF7-induced, as well as basal mRNA transcript levels, of K10 DSG1 and INV. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: c *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; ***p < 0.05 vs the corresponding PKCδ inhibitor-untreated cells; d *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; ***p < 0.05 and ****p < 0.005 vs the corresponding control siRNA cells analysis. Thus, also PKCα appears a pivotal player in sustained cell proliferation. These findings strongly indi- FGFR2b-triggered keratinocyte differentiation; however, cated that, as a consequence of PKCδ signaling shut-off, differently from PKCδ, its role appears to be specifically keratinocytes are defective in the ability to undergo the restricted to the late stages of the process. differentiation program maintaining a basal/proliferative Then, in order to analyze the effect of PKCδ inhibition phenotype. Real time RT-PCR analysis confirmed that the on the ability of keratinocytes to progress throughout the expression of both early and LORs was significantly entire differentiation program and stratify in response to repressed by PKCδi (Fig. 5b). Finally, the quantitative FGFR2b activation, we again took advantage of the use of evaluation of the raft thickness revealed that, in pBp and the more complete model of organotypic cultures. HaCaT pBp-FGFR2b skin equivalents, the layers were sig- pBp and HaCaT pBp-FGFR2b rafts were lifted to nificantly reduced by PKCδi (Fig. 5c), further indicating air–liquid interface in the presence or not of PKCδi and that inhibition of PKCδ induces impairment of all steps of finally stimulated with FGF7, as above. Quantitative FGFR2b-triggered differentiation, which in turn results in immunofluorescence analysis showed that the K1 and FIL defective stratification. staining, visible in both HaCaT pBp and HaCaT pBp- FGFR2b rafts stimulated with FGF7 (Fig. 5a, upper The transcription factors KLF4 and DLX3, acting panels), appeared overall dampened by the presence of downstream PKCδ and PKCα respectively, are sequentially PKCδi, suggesting that the entire differentiation process up-regulated by FGFR2b signaling induced by FGFR2b signaling was arrested by the sus- Since it has been recently proposed that PKCδ and tained inhibition of PKCδ (Fig. 5a, upper panels). In PKCα control the expression of several genes via the 28,29 36 addition, the immunostaining of K5 revealed that the induction of KLF4 and DLX3 respectively, we canonical distribution in the basal layer of this undiffer- decided to investigate the possible involvement of these entiated/proliferating cell marker , visible in pBp control transcription factors in FGFR2b-triggered keratinocyte rafts (Fig. 5a, lower panels), disappeared in pBp-FGFR2b differentiation. The topic was particularly appealing, since skin equivalents (Fig. 5a, lower panels), confirming the they have been proposed to be involved in an intricate 47–49 precocious onset of differentiation. However, in the pre- network of loops with ΔNp63 , the transcription sence of PKCδi, K5 staining appeared recovered and factor whose down-modulation represents the main distributed all over the rafts (Fig. 5a, lower panels). molecular mechanism known to drive differentiation of Moreover, the parallel evaluation of Ki67 staining in both stratified epithelia . Indeed, we have already described a pBp and pBp-FGFR2b rafts stimulated with FGF7 revealed link between FGFR2b and p63 , but the molecular few positive cells distributed in the basal layer (Fig. 5a, mechanism underlying this interplay remains to be still lower panels); the presence of some stained cells also in clarified. To this aim, we performed Real Time RT-PCR the suprabasal layers (Fig. 5a, lower panels) is in agree- analysis on HaCaT pBp and pBp-FGFR2b 3D organotypic ment with the previously described ability of FGF7 to also rafts, checking for the transcript levels of KLF4 and DLX3 sustain the residual proliferative activity of suprabasal and comparing them with that of p63. The results showed keratinocytes . More interestingly, in pBp-FGFR2b raft a significant increase of both KLF4 and DLX3 and an grown in the presence of PKCδi, several cells intensely opposite decrease of p63 in response to FGF7 stimulation stained for Ki67 marker appeared homogeneously dis- (Fig. 6a), which were more evident in the rafts over- tributed in all the layers (Fig. 5a, lower panels), suggesting expressing FGFR2b (Fig. 6a). Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 8 of 16 - - - - - - - - FGF7 + + + + FGF7 + + + + PKCαi - + - + - + - + PKCαi - + - + - + - + p-PKCα 67 kDa K1 fold increase 1 0.5 2.5 1.5 5 3 6 4 fold increase 1 1 2 2.3 2.3 2.7 3 3.3 26 kDa 78 kDa PKCα LOR fold increase 1 0.5 1.3 0.7 1.8 0.8 2 1.3 42 kDa 42 kDa ACTIN ACTIN 39 kDa p-PKCδ FIL fold increase 1 0.8 2 1.2 1.6 1 2.8 1.2 fold increase 1 1 2 2.3 3.8 4 4 4 50 kDa TUBULIN 78 kDa PKCδ 42 kDa ACTIN - - - - FGF7 + + + + FGF7 - - - - + + + + PKCα siRNA - + - + - + - + PKCα siRNA - - + - + - + + control siRNA - + - + - + - + control siRNA - + - + - + - 78 kDa PKCα 67 kDa K1 fold increase 1 0.6 1.4 0.7 1.6 0.4 1.8 0.6 fold increase 1 1.1 1.8 1.9 2.4 2.2 2.7 2.7 42 kDa ACTIN 26 kDa LOR fold increase 1 0.5 1.4 0.8 2.6 1.8 3 1.9 50 kDa TUBULIN 39 kDa FIL fold increase 1 0.4 1.1 0.7 1.2 0.7 2 0.3 50 kDa TUBULIN 6 6 6 5 5 5 ** 4 4 4 ** NS NS 3 3 3 NS 2 NS 2 ** ** *** *** 1 1 *** *** *** *** 1 *** *** 0 0 - - - - - - - - FGF7 + + + + + + + + - - + + - - + + - + + - - + + - + + + + PKCαi - - - - - + + - - + + - 6 6 5 5 5 ** 4 4 ** NS 3 3 NS ** NS **** 2 2 2 ** NS *** *** 1 1 *** 1 **** **** **** **** 0 0 0 - - - - - - - - - - - - FGF7 + + + + + + + + + + + + + + + + + + + + PKCα siRNA - + + - - + + - - - - - - - - - + + - - + + + - - + + + - control siRNA + - + - - + - - + - - Fig. 4 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression K10 mRNA fold expression LOR mRNA fold expression LOR mRNA fold expression FIL mRNA fold expression FIL mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 9 of 16 (see figure on previous page) Fig. 4 PKCα is required for FGFR2b-induced terminal stages of keratinocyte differentiation. Post-confluent HaCaT pBp and HaCaT pBp-FGFR2b cells were left untreated or stimulated with FGF7 in presence or not of the PKCα inhibitor as reported in Materials and methods section (a, c). Alternatively, HaCaT clones were transfected with PKCα siRNA or with an unrelated siRNA as control, transfected cells were grown until post- confluence and left untreated or stimulated with FGF7 as above (b, d). a Western blot analysis shows that PKCα inhibitor decreases the basal, as well as the FGF7-induced, PKCα phosphorylation, but not that of PKCδ phosphorylation (left panel). PKCα inhibitor does not interfere with the K1 expression, but it displays a repressive effect on the FGF7-induced up-modulation of LOR and FIL (right panel). b Western blot analysis confirms that PKCα silencing specifically dampened the basal, as well as the FGF7-induced, PKCα protein level but not that of PKCδ (left panel). PKCα silencing also strongly counteracts the increase of LOR and FIL but not that of K1 in response to FGF7 (right panel). The equal loading was assessed with anti-PKCδ or anti-PKCα and anti-ACTIN or anti-TUBULIN antibodies. For densitometric analysis the values from a representative of three independent experiments are reported. c, d Real time RT-PCR analysis confirms that PKCα inhibition (c) or depletion (d) decreases FGF7-induced, as well as basal mRNA transcript levels, of LOR and FIL but it does not affect K10 expression. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; NS and ***p < 0.05 vs the corresponding PKCα inhibitor-untreated cells; d *p < 0.05 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; NS, ***p < 0.05 and ****p < 0.005 vs the corresponding control siRNA cells Then, to verify if KLF4 and DLX3 up-regulation could contrasting reports appear to suggest its possible invol- 18–20 display a sequential trend during FGFR2b-induced kera- vement in the later stages of this process . Therefore, tinocyte differentiation, we analyzed their possible mod- here we first analyzed the impact of the receptor signaling ulation in confluent and post-confluent clones stimulates on the induction of terminal differentiation in 3D orga- with FGF7 as above. The results showed that KLF4 up- notypic rafts and 2D cultures of human keratinocytes regulation was mainly appreciable in confluent clones engineered to stably overexpress FGFR2b. The results (Fig. 6b), while DLX3 induction was detectable only in obtained clearly indicated that, particularly when FGFR2b post-confluent cultures (Fig. 6b). As expected, the is overexpressed, the receptor signaling not only induces a induction of KLF4 in confluent cells as well as that of precocious onset of the early differentiation markers, but DLX3 in post-confluent cells were significantly counter- also enhances the expression and accelerates the appear- acted by PKCδi and PKCαi, respectively (Fig. 6b), further ance of the terminal markers. These findings strongly confirming the connection of each PKC signaling with the suggest a key role of FGFR2b in the regulation of the corresponding transcription factor. Moreover, similarly to entire differentiation program. what observed for KLF4 and consistently with its well- Then, in order to analyze the signaling players down- established role in FGFR2b-induced onset of differentia- stream FGFR2b possibly involved in keratinocyte differ- 15,16 tion , p63 repression in response to FGF7 was more entiation, we focused on PKCδ and PKCα isoforms, at the evident in confluent cells and impaired only by PKCδi light of previous evidences showing their sequential role (Fig. 6b). Being p63 a direct negative regulator of KLF4 in in the regulation of the different steps of the process .To normal human keratinocytes , its strong repression the aim to “dissect” in vitro the differentiation steps, cells observed in confluent cells may represent the molecular were grown until confluence or up to post-confluence. mechanism through which FGFR2b-triggered PKCδ sig- Taking advantage of the use of specific substrate inhibi- naling would induce KLF4 and consequently the onset of tors and siRNA interference, we highlighted that PKCδ keratinocyte differentiation. These results suggest that the signaling appears to drive the early molecular events, alternative induction of KLF4 and DLX3 in FGF7- while PKCα regulates the late ones, of FGFR2b-triggered stimulated confluent and post-confluent cells, respec- keratinocyte differentiation. Given the role of PKCδ in the tively, might reflect the sequential activation of their onset of the process, we used the organotypic cultures to upstream activators PKCδ and PKCα during the early and analyze the impact of its inhibition on the ability of ker- late steps of FGFR2b-induced differentiation. atinocytes to terminally differentiate and stratify in response to ligand-mediated activation of FGFR2b: we Discussion demonstrated that PKCδ signaling shut-off was sufficient Because of its hypothesized role as a tumor suppressor to block the entire differentiation program, inducing 9,10 in vitro and in vivo , forced modulation of FGFR2b retention of undifferentiated/basal features and defective could be a winning strategy to revert important aspects of stratification. Interestingly, these rafts also maintained a the malignant phenotype in carcinoma cells, including sustained cell proliferation throughout all the layers that loss of cell differentiation. However, in the physiological could be explained by the previously described ability of context of normal human keratinocytes, despite the FGFR2b to not only promote the differentiation pro- numerous evidences emphasizing the role of FGFR2b in gram , but also sustain the residual proliferative activity the enhancement of early differentiation, only few and of suprabasal, differentiating keratinocytes . Overall, the Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 10 of 16 2.5 HaCaT pBp HaCaT pBp-FGFR2b K1 K1 DAPI DAPI 2.0 ** 1.5 1.0 0.5 FGF7 FGF7 + PKCδi FGF7 FGF7 + PKCδi 2.5 FIL FIL DAPI DAPI 2.0 *p< 0.0001 vs rottlerin- ** 1.5 1.0 0.5 FGF7 FGF7 FGF7 + PKCδi FGF7 + PKCδi FGF7 + + + + PKCδi - + - + HaCaT pBp HaCaT pBp-FGFR2b K5 K5 2.5 DAPI DAPI 2.0 1.5 1.0 0.5 ** FGF7 FGF7 FGF7 + PKCδi Ki67 Ki67 DAPI DAPI NS FGF7 FGF7 FGF7 + PKCδi FGF7 + + + PKCδi - - + b c ** 2.5 2.5 2.5 ** ** 2.0 2.0 2.0 200 1.5 1.5 1.5 1.0 1.0 1.0 * 100 0.5 * 0.5 0.5 50 0 0 0 FGF7 FGF7 FGF7 FGF7 + + + + + + + + + + + + + + + + PKCδi PKCδi PKCδi PKCδi - + - + - + - + - + - + - + - + Fig. 5 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b K10 mRNA fold expression DSG1 mRNA fold expression LOR mRNA fold expression Organotypic culture fold increase thickness (μm) K5 fluorescence intensity % of Ki67 positive cells fold increase fold increase FIL fluorescence intensity K1 fluorescence intensity Rosato et al. Cell Death and Disease (2018) 9:565 Page 11 of 16 (see figure on previous page) Fig. 5 PKCδ signaling shut-off blocks the entire differentiation program and induces defective stratification in keratinocytes. Organotypic skin equivalents of HaCaT pBp and pBp-FGFR2b clones, prepared as reported in Materials and methods section, were lifted to air–liquid interface in the presence or not of PKCδ inhibitor and finally stimulated with FGF7 as above. a Quantitative immunofluorescence analysis shows that K1 and FIL staining, visible in both HaCaT pBp and HaCaT pBp-FGFR2b rafts stimulated with FGF7, appears overall impaired by the presence of PKCδ inhibitor (upper panels). In addition, K5 signal, visible in the basal layer of pBp rafts, disappears in pBp-FGFR2b skin equivalents while it is recovered and distributed all over the pBp-FGFR2b rafts by the presence of PKCδ inhibitor (lower panels). Parallel evaluation of the nuclear proliferation marker Ki67 reveals the presence of few positive cells distributed in the basal layer and randomly throughout the suprabasal layers in pBp rafts and FGFR2b rafts stimulated with FGF7 (lower panels). In pBp-FGFR2b raft grown in the presence of PKCδi, several cells intensely stained for Ki67 marker are visible in all the layers (lower panels). Quantitative analysis of the fluorescence intensity and of the percentage of Ki67 positive cells were performed as described in Materials and methods and results sections are expressed as fold increase respect to pBp values ± SE. Student’s t test was performed and significance levels have been defined as above: *p < 0.0001 vs the corresponding PKCδ inhibitor-untreated cells; NS and **p < 0.0001 vs the corresponding pBp cells. Bar: 25 μm. b Real time RT-PCR analysis confirms that, in pBp and pBp-FGFR2b rafts stimulated with FGF7, the expression of K10, DSG1, and LOR is significantly repressed by PKCδ inhibitor. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: *p < 0.05 vs PKCδ inhibitor-untreated cells; **p < 0.05 vs the corresponding pBp cells. c Quantitative evaluation of the raft thickness shows that the layers of pBp and pBp-FGFR2b skin equivalents are significantly reduced by the presence of PKCδ inhibitor. Quantitative analysis of the raft thickness was expressed as µm mean ± SD from three independent experiments. Student’s t test was performed and significance levels have been defined as p < 0.05: *p < 0.0001 vs the corresponding PKCδ inhibitor-untreated cells 16,41 phenotypic features derived by PKCδ signaling shut-off and induced by FGFR2b signaling . In fact, the indicate that the inhibition of this signaling pathway alone FGFR2b/p63 crosstalk appears to be central in the is sufficient to impair the balance between cell prolifera- homeostatic balance between keratinocyte proliferation tion and differentiation controlled by FGFR2b. On the and differentiation and its impairment, due to out-of- other hand, we can not exclude the contribution of the context appearance of FGFR2c, leads to loss of the dif- epidermal growth factor receptor (EGFR) in such ferentiated phenotype and acquisition of tumorigenic increased cell proliferation: in fact, the antagonistic role of features . For this reason, to identify the molecular FGFR2b and EGFR in epithelial cells has been previously players contributing to the regulation of this crosstalk described by our group . While FGFR2b is up-regulated seems to represent a very urgent and important goal. Indeed, both KLF4 and DLX3 have been proposed to be during the shift from the basal to the suprabasal layers, 48,49 EGFR mainly displays a basal distribution and its ligand- involved in negative loops with p63: consistent with dependent activation triggers cell proliferation. In addi- these assumptions, we observed that they displayed tion, it has been also proposed that FGF7/KGF treatment opposite modulations compared to p63. Interestingly, the may induce endogenous synthesis of the EGFR ligand up-regulation of KLF4 appeared more appreciable in TGFα. In any case, these results are in agreement with confluent cells, the same culture condition in which the the widely described role of PKCδ as tumor suppressor, down-regulation of p63 was more evident: this simulta- which is down-regulated in squamous cell carcinomas neous detection of p63 down-regulation and KLF4 52,53 (SCCs) . induction mainly in confluent cells strongly suggests that Concerning the molecular effectors acting downstream they take place in the same step of the differentiation PKC isoforms, our results showed that, in agreement with program, and possibly during the shift between the basal 28,29,36 previous reports , PKCδ and PKCα isoforms act via to the suprabasal layer. These observations, together with the induction of the downstream transcription factors the recent evidence of a possible direct role of PKCδ in KLF4 and DLX3, respectively. In fact, the expression of p63 down-regulation , allowed us to speculate that the both KLF4 and DLX3 was clearly induced in response to down-regulation of p63 could represent the molecular FGF7, particularly in rafts or cell cultures overexpressing mechanism through which FGFR2b and its downstream FGFR2b. Interestingly, in cultures grown at different cell PKCδ signaling could induce the up-regulation of KLF4 densities to mimic the various steps of differentiation, we and consequently the onset of keratinocyte differentiation. found that KLF4 up-regulation was mainly appreciable in confluent cells, while DLX3 induction was detectable only Materials and methods in post-confluent cultures. These sequential trends of Cells and treatments induction could reflect the progressive involvement of The human keratinocyte cell line HaCaT , stably their upstream inductors PKCδ and PKCα in the distinct overexpressing FGFR2b (pBp-FGFR2b) or the empty stages (early and late, respectively) of differentiation. vector (pBp) and generated as previously described , were Finally, we also decided to compare the modulation of cultured in Dulbecco’s modified Eagle’s medium KLF4 and DLX3 with that previously described for p63 (DMEM), supplemented with 10% fetal bovine serum Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 12 of 16 3D organotypic cultures *** 4 4 1.2 3 3 0.8 ** ** * 2 2 0.6 ** 0.4 ** 1 1 0.2 0 0 - - + + FGF7 - + - + - - + + *** *** 6 12 * 1.2 *** *** 5 10 1.0 0.8 4 8 ** 0.6 3 6 *** ** 4 0.4 *** *** ** NS NS NS *** 0.2 1 2 NS NS 0 0 - - - - - - - - - - - - FGF7 + + + + + + + + + + + + - + + - - + + - - + + - - + + - - + + - - + + - PKCδi 6 12 1.2 NS 5 10 ** 1.0 ** NS * NS 8 0.8 ** ** * NS NS 0.6 3 6 ** * NS NS 4 0.4 2 ^ NS NS 2 0.2 0 0 0 FGF7 - - - - - - - - + + + + + + + + - - + + - - + + - + + - - + + - - + + - - + + - PKCαi - + + - - + + - Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b HaCaT pBp HaCaT pBp-FGFR2b KLF4 mRNA fold expression KLF4 mRNA fold expression KLF4 mRNA fold expression DLX3 mRNA fold expression DLX3 mRNA fold expression DLX3 mRNA fold expression p63 mRNA fold expression p63 mRNA fold expression p63 mRNA fold expression Rosato et al. Cell Death and Disease (2018) 9:565 Page 13 of 16 (see figure on previous page) Fig. 6 PKCδ and PKCα signaling are responsible for FGF7-mediated sequential induction of KLF4 and DLX3, respectively. a Organotypic skin equivalents of HaCaT pBp and pBp-FGFR2b clones, prepared as reported in Materials and methods section, were grown in complete medium and left untreated or stimulated with FGF7 as above. Real time RT-PCR analysis shows a significant increase of both KLF4 and DLX3 expression upon FGF7 stimulation, while p63 displays an opposite trend. b HaCaT pBp and HaCaT pBp-FGFR2b cells were grown up to confluence or post-confluence in the presence or not of PKCδ and PKCα inhibitors, respectively, and stimulated with FGF7 as above. Real time RT-PCR analysis shows that KLF4 up- regulation is mainly appreciable in confluent clones, while DLX3 induction is detectable only in post-confluent cultures. The increased expression of KLF4 in confluent cells and that of DLX3 in post-confluent cultures are significantly dampened by PKCδi and PKCαi, respectively. Similarly to KLF4, p63 down-modulation in response to FGF7 is more evident in confluent cells and it appears impaired only by PKCδi. Results are expressed as mean values ± SD. Student’s t test was performed and significance levels have been defined as above: a *p < 0.05 and ***p < 0.001 vs the corresponding FGF7-unstimulated cells; **p < 0.05 vs the corresponding pBp cells; b NS and *p < 0.05 vs the corresponding FGF7-unstimulated cells; NS and **p < 0.05 vs the corresponding pBp cells; NS and ***p < 0.05 vs the corresponding PKCδ inhibitor-untreated cells; NS and ^p < 0.005 vs the corresponding PKCα inhibitor-untreated cells (FBS) plus antibiotics. Primary cultures of human fibro- mixture was left to polymerize for 30 min at 37 °C. After blasts derived from healthy skin (HFs) were obtained from 24 h 2 × 10 HaCaT pBp or pBp-FGFR2b cells were see- patients attending the Dermatology Unit of the Sant’An- ded on the collagen gel and left to grow for a week in drea Hospital of Rome; all patients were extensively complete medium added in both the top and the bottom informed and their consent for the investigation was given wells. Then, the organotypic cultures were lifted to the and collected in written form in accordance with guide- air–liquid interface and cultured for further two weeks in lines approved by the management of the Sant’Andrea complete medium added or not with the specific PKCδ Hospital. HFs were isolated and cultured as previously inhibitor rottlerin (5 μM; Calbiochem, 557370). Then described . cultures were left untreated or stimulated with FGF7 For growth factors stimulation, cells were left untreated (Upstate Biotechnology) 100 ng/ml for the last 4 days. or incubated with FGF7 (Upstate Biotechnology, Lake Rafts were finally fixed in 10% formalin, embedded in Placid, NY, USA, 01–118) 100 ng/ml for 24 or 48 h at 37 ° paraffin and 4 µm slices were obtained. To evaluate C. To inhibit AKT or MEK1/2 or PKCδ or PKCα cells organotypic thickness slices were stained with hematox- were respectively incubated with the specific AKT inhi- ylin and eosin using standard procedures then bright field bitor 1L-6-hydroxy-methyl-chiro-inositol 2-(R)-2-O- images were taken with an Axiocam ICc 5 (Zeiss, Ober- methyl-3-O-octadecylcarbonate (1 μM; Calbiochem, kochen, Germany) connected with an Axioplan 100 Nottingham, UK, 124005) or with the specific MEK1/2 microscope (Zeiss). The organotypic culture thickness inhibitor PD0325901 (1 μM; Sigma-Aldrich, Saint Louis, was measured using the Axiovision software (Zeiss) and MO, USA, PZ0162) or with the specific PKCδ inhibitor expressed as mean µm ± standard deviation (SD). Stu- rottlerin (5 μM; Calbiochem, 557370) or with the specific dent’s t test was performed and significance levels have PKCα inhibitor Go6976 (3 μM; Calbiochem, 365250) for been defined as p < 0.05. 1 h at 37 °C before treatment with FGF7 in the presence of each inhibitor. Immunofluorescence For RNA interference and PKCδ or PKCα silencing, HaCaT clones, grown on coverslips, were fixed with 4% cells were transfected with PKCδ small interfering RNA paraformaldehyde in PBS for 30 min at 25 °C followed by (PKCδ siRNA) (Santa Cruz Biotechnology, Santa Cruz, treatment with 0.1 M glycine for 20 min at 25 °C and with CA, USA), or PKCα small interfering RNA (PKCα siRNA) 0.1% Triton X-100 for additional 5 min at 25 °C to allow (Santa Cruz Biotechnology), or with an unrelated siRNA permeabilization. Cells were then incubated for 1 h at 25 ° as a control (control siRNA), using Lipofectamine 2000 C with the following primary antibodies: rabbit polyclonal Transfection Reagent (Invitrogen, Carlsbad, CA, USA) anti-K1 (1:50 in PBS, AF 87, Covance, Princeton, NJ, according to the manufacturer’s protocol. USA) or mouse monoclonal anti-β1 integrin (1:1000 in PBS; ST2–16, Santa Cruz Biotechnology). The primary Organotypic cultures antibodies were visualized using goat anti-rabbit IgG- For three-dimensional (3D) organotypic cultures, col- Texas Red (1:200 in PBS; Jackson Immunoresearch lagen rafts were prepared adding 5 mg/ml rat tail type I Laboratories, West Grove, PA, USA) or goat anti-mouse collagen (Corning, Lowell, MA, USA) to DMEM and IgG-Alexa Fluor 488 (1:200 in PBS; Life Technologies, Reconstitution buffer (8:1:1) as previously described . Carlsbad, CA, USA) antibodies for 30 min at 25 °C. Nuclei HFs (1 × 10 ) were added to 2 ml of the collagen mixture were stained with DAPI (1:1000 in PBS; Sigma). Cover- in polycarbonate micron inserts (23 mm diameter, pore slips were finally mounted with mowiol (Sigma) for size 0.3 µm; Corning) in 6-deep well plates (Corning). The observation. Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 14 of 16 Organotypic raft sections, obtained as above, were Covance), anti-Bek (C-17, Santa Cruz Biotechnology), deparaffinized in xylene and re-hydrated through graded anti-loricrin (Covance), anti-p-PKCδ (Ser645, Santa Cruz ethanols to PBS, pH 7.4. Antigen retrieval was achieved by Biotechnology) polyclonal antibodies, all followed by heating sections in target retrieval solution low pH (Dako, enhanced chemiluminescence detection (ECL, Amer- Carpinteria, CA, USA) for 15 min at 97 °C. Sections were sham, Alington Heights, IL, USA). The membranes were then washed with PBS and blocked using 10% bovine calf rehydrated by being washed in PBS-Tween 20, stripped with 100 mM mercaptoethanol and 2% SDS for 30 min at serum and 0.2% Triton X-100 for 30 min at 25 °C before staining with rabbit polyclonal anti-K1 (1:500 in PBS, AF 55 °C, and probed again with anti-PKCδ (C-20, Santa 87, Covance), mouse monoclonal anti-filaggrin (1:1000 in Cruz Biotechnology) polyclonal antibodies, anti-PKCα PBS, 15C10, Monosan Sanbio, Uden, The Netherland), monoclonal antibody (Y124, Abcam) or with anti-β- rabbit polyclonal anti-cytokeratin 5 (1:400 in PBS, Abcam, ACTIN (AC-15, Sigma) monoclonal antibody or anti-α- Cambridge, UK) or mouse monoclonal anti-Ki67 (1:100 in TUBULIN (2148S, Cell Signaling) polyclonal antibodies to PBS, MIB-1, Dako) antibodies for 1 h in a humidified estimate the protein equal loading. Densitometric analysis chamber. The primary antibodies were visualized using was performed using Quantity One Program (Bio-Rad goat anti-mouse IgG-Texas Red (1:200 in PBS; Jackson Laboratories, Hercules, CA, USA). Results from three Immunoresearch Laboratories) or goat anti-rabbit IgG- different experiments were normalized and expressed as Texas Red (1:200 in PBS; Jackson Immunoresearch fold increase respect to the control value. Values from a Laboratories) antibodies for 30 min at 25 °C. Nuclei were representative of three independent experiments were stained with DAPI (1:1000 in PBS; Sigma). Sections were reported in each figure. permanently mounted under a coverslip. Fluorescence signals were analyzed by conventional fluorescence or by Primers scanning cells in a series of sequential sections with an Oligonucleotide primers necessary for target genes and ApoTome System (Zeiss) connected with an Axiovert 200 the housekeeping gene were chosen utilizing the online inverted microscope (Zeiss); image analysis was per- tool Primer-BLAST and purchased from Invitrogen. Pri- formed by the Axiovision software (Zeiss) and images mers list and characteristics are reported in Supplemen- were obtained by 3D reconstruction of the total number tary Table S1. For p63 target gene, the reported primers of the serial optical sections. Quantitative analysis of the were designed to recognize both ΔNp63 and TAp63 α and fluorescence intensity was performed by the Axiovision β isoforms. For each primer pair, we performed no- software (Zeiss), analyzing 10 different fields randomly template control and no-reverse-transcriptase control taken from three independent experiments. Quantitative (RT negative) assays, which produced negligible signals. analysis of the percentage of Ki67 positive cells was assessed counting for each sample a total of 50 cells, RNA extraction and cDNA synthesis randomly observed in 10 microscopic fields from three Organotypic cultures were deparaffinized and RNA was different experiments. Results are shown as means ± extracted using the TRIzol method (Invitrogen) according standard error (SE). Student’s t test was performed and to manufacturer’s instructions and eluted with 0.1% die- significance levels have been defined as p < 0.05. thylpyrocarbonate (DEPC)-treated water. Each sample was treated with DNAase I (Invitrogen). Total RNA Western blot analysis concentration was quantitated by spectrophotometry; 1 Cells were lysed in a buffer containing 50 mM HEPES, μg of total RNA was used to reverse transcription using pH 7.5, 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 iScriptTM cDNA synthesis kit (Bio-Rad) according to mM MgCl , 5 mM EGTA, supplemented with protease manufacturer’s instructions. inhibitors (10 μg/ml aprotinin, 1 mM PMSF, 10 μg/ml leupeptin), and phosphatase inhibitors (1 mM sodium PCR amplification and real time quantitation orthovanadate, 20 mM sodium pyrophosphate, 0.5 M Real time RT-PCR was performed using the iCycler Real NaF). A range between 20 and 50 μg of total protein was Time Detection System (iQ5 Bio-Rad) with optimized resolved under reducing conditions by 8 or 12% SDS- PCR conditions. The reaction was carried out in 96-well PAGE and transferred to reinforced nitrocellulose (BA-S plate using iQ SYBR Green Supermix (Bio- Rad) adding 83, Schleider and Schuell, Keene, NH, USA). The mem- forward and reverse primers for each gene and 1 μlof branes were blocked with 5% nonfat dry milk in PBS 0.1% diluted template cDNA to a final reaction volume of 15 μl. Tween 20 and incubated with anti-DSG1 (27B2, Life All assays included a negative control and were replicated Technologies), anti-β1 integrin (ST2–16, Santa Cruz three times. The thermal cycling program was performed Biotechnology), anti-involucrin (SY5, Abcam), anti- as described . Real time quantitation was performed with filaggrin (15C10, Monosan), anti-p-PKCα (Ser657, the help of the iCycler IQ optical system software version Abcam) monoclonal antibody or with anti-K1 (AF 87, 3.0a (Bio-Rad), according to the manufacturer’s manual. Official journal of the Cell Death Differentiation Association Rosato et al. Cell Death and Disease (2018) 9:565 Page 15 of 16 Results are reported as mean ± SD from three different 13. Yang, J. et al. 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Official journal of the Cell Death Differentiation Association

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Cell Death & DiseaseSpringer Journals

Published: May 11, 2018

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