TY - JOUR
AU - Han, Ho Jae
AB - Abstract Bioactive molecules and stem cell-based regenerative engineering is emerging a promising approach for regenerating tissues. Autotaxin (ATX) is a key enzyme that regulates lysophosphatidic acid (LPA) levels in biological fluids, which exerts a wide range of cellular functions. However, the biological role of ATX in human umbilical cord blood-derived mesenchymal stem cells (hMSCs) migration remains to be fully elucidated. In this study, we observed that hMSCs, which were stimulated with LPA, accelerated wound healing, and LPA increased the migration of hMSCs into a wound site in a mouse skin wound healing model. In an experiment to investigate the effect of LPA on hMSC migration, ATX and LPA increased hMSC migration in a dose-dependent manner, and LPA receptor 1/3 siRNA transfections inhibited the ATX-induced cell migration. Furthermore, LPA increased Ca2+ influx and PKC phosphorylation, which were blocked by Gαi and Gαq knockdown as well as by Ptx pretreatment. LPA increased GSK3β phosphorylation and β-catenin activation. LPA induced the cytosol to nuclear translocation of β-catenin, which was inhibited by PKC inhibitors. LPA stimulated the binding of β-catenin on the E-box located in the promoter of the CDH-1 gene and decreased CDH-1 promoter activity. In addition, the ATX and LPA-induced increase in hMSC migration was blocked by β-catenin siRNA transfection. LPA-induced PKC phosphorylation is also involved in Rac1 and CDC42 activation, and Rac1 and CDC42 knockdown abolished LPA-induced F-actin reorganization. In conclusion, ATX/LPA stimulates the migration of hMSCs through LPAR1/3-dependent E-cadherin reduction and cytoskeletal rearrangement via PKC/GSK3β/β-catenin and PKC/Rho GTPase pathways. Stem Cells 2015;33:819–832 Autotaxin, Lysophosphatidic acid, Adherent junction, Cytoskeleton rearrangement, Migration, Wound healing Introduction Bioactive molecules and stem cell-based regenerative engineering is emerging a promising approach for regenerating tissues. A growing number of reports clearly demonstrate that lipid metabolisms determine the fate of stem cells [1-3]. Among lipid metabolites, lysophosphatidic acid (LPA), a potent lysophospholipid, has attracted a great amount of attention because it is a critical regulator of proliferation, migration, and differentiation of adult stem cells as well as embryonic stem cells [4-9]. Autotaxin (ATX) is a mitogenic-like phosphodiesterase originally identified as an “autocrine motility factor,” which was shown to have lysophospholipase D (lysoPLD) activity, converting lysophosphatidylcholine to LPA [10-12]. The temporal and spatial expression pattern of ATX in the developing mouse embryo are highly dynamic, and the importance of ATX in embryogenesis has been demonstrated by the observation that ATX-deficiency mice die at embryonic day 9.5 (E9.5) with profound vascular and neural tube defects [13-16]. LPA has been shown to have diverse roles in many biological processes and can bind to seven G protein-coupled receptors (GPCRs; LPAR1–7) triggering both overlapping and distinct signaling pathways by coupling to multiple G proteins that clearly illustrate the complexity of lysophospholipid signaling [17, 18]. It is apparent that stem cells express different lysophospholipid receptors not only depending on their source of origin but also on the conditions used for their isolation and/or culture. Recent evidence is emerging that LPA is involved in a variety of physiological and pathophysiological responses including wound healing, production of angiogenic factors, chemotaxis, and cell cycle progression [4, 19-21]. Furthermore, the previous report that LPA decreased cell adhesion in the myeloid progenitor cell line through a Rho-dependent pathway [22] provides an emerging picture for the importance of LPA in regulating the mobilization and homing of stem cells. A key event in human umbilical cord blood-derived mesenchymal stem cells (hMSCs) migration is disruption of cell-cell contacts via modulation of intracellular junctional components including cadherins and cytoskeletal reorganization [23]. In addition, Rho family GTPase have been described as important signaling molecules to the cytoskeleton, regulating the coordinated assembly and activation of actin with actin-binding proteins which modulates cellular migration [24]. LPA largely antagonizes the apoptosis process and regulates cytoskeleton dynamics in vitro [21, 25, 26], which prompted us to examine the beneficial effects of LPA on hMSCs. In addition, the future challenge remains to understand how the multifaceted interplay between signaling pathways mediated by lysophospholipids is integrated in the functional regulation of stem cells. hMSCs are considered a suitable source for cell therapy due to their ready availability, nonimmunogenicity, and ability to differentiate into multiple tissue types [27]. In particular, hMSCs can play a critical role in tissue repair by serving as support cells that secrete trophic factors to accelerate angiogenesis and wound healing [28]. The ATX/LPA signaling is upregulated in a variety of inflammatory conditions [29, 30], which suggest the possibility that ATX-LPA signaling promotes the migration of hMSCs to wound areas. The mechanism underlying the migration of hMSCs has garnered significant attention secondary to the potential of using exogenously administered or endogenously recruited MSCs for tissue repair [31, 32]. However, the responses to LPA on stem cells are various depending on the cell types and origins. Thus, elucidation of these lysophospholipid-mediated signaling pathways, either through targeting the lysophospholipid GPCRs or enzymes involved in lysophospholipid metabolisms, could provide opportunities for future cell therapy. Therapeutic interventions to elevate LPA levels and/or administration of pharmacological LPA mimetics could be novel and effective cell-based therapeutic strategies. Therefore, in this study, we investigated the effect of LPA on wound healing in a mouse skin wound model and the molecular mechanisms underlying the ATX/LPA-mediated regulations of hMSC migration. Materials and Methods Materials hMSCs were obtained from the Medipost, Co. (Seoul, Korea). Fetal bovine serum (FBS) was purchased from BioWhittaker, Inc. (Walkersville, MO). Recombinant human ENPP-2/ATX was obtained from the R&D System (Minneapolis, MN). Oleoyl-l-α-lysophosphatidic acid sodium salt (LPA), staurosporine, bisindolylmaleimide I, methyl-β-cyclodextrin (MβCD), and pertussis toxin (Ptx) obtained from Sigma Chemical Company (St. Louis, MO). The vehicles of inhibitor did not affect the results of each experiment, respectively (Supporting Information Fig. S1). Alexa Fluor 488 Phalloidin and Fluo-3AM were purchased from Life Technologies, Corp. (Carlsbad, CA). LPAR1, LPAR2, LPAR3, caveolin-1, β-actin, PKCα, PKCδ, PKCθ, PKCε, PKCζ, pan-cadherin, phospho-GSK3β, β-catenin, snail, slug, TCF, LEF, lamin A/C, Rac1, RhoA, CDC42, phospho-Cofilin, Profilin, normal IgG, Gαq, and E-cadherin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Gαi, Gα12, and phospho-pan PKC antibodies were purchased from Cell Signaling (Beverly, MA). F-actin and active-β-catenin antibodies were obtained from Abcam plc. (Cambridge, U.K.). All other reagents were of the highest purity commercially available and were used as received. Culture of Human UCB-MSCs hMSCs were cultured without a feeder layer in the α-minimum essential medium and (α-MEM; Thermo, MA) supplemented with 1% penicillin and streptomycin, and 10% FBS. Cells were grown in 100-mm diameter culture dishes in an incubator maintained at 37°C with 5% CO2 for each experiment. The medium was replaced with serum-free α-MEM at 24 hours before experiments. Cells were washed twice with phosphate-buffered saline (PBS) following incubation and were maintained in a serum-free α-MEM including all supplements and indicated agents. Mouse Skin Wound Healing Model Eight-week-old age male ICR mice were used. Experiments with animals were carried out under approval by the Institutional Animal Care and Use Committee of Seoul National University (SNU-140123-6) and in accordance of the NIH Guide for the Care and Use of Laboratory Animals. Mice were anesthetized using a 2:1 mixture of Zoletil 50 (20 mg/kg, Virbac Laboratories, Carros, France) and Xylazine HCl (10 mg/kg, Rompun, Bayer, Germany) via intraperitoneal injection. Mouse dorsal skin wounding and cell implantation were performed as described previously [33, 34]. For ex vivo activation of hMSCs, the cells were cultured with/without LPA for 24 hours and thoroughly washed with PBS three times. Vehicle, hMSCs, LPA, or ex vivo LPA-stimulated hMSCs were injected into the dermis around the wound. After that, we attached silicone splint to peri-wound site using dermal glue and 6-0 nylon sutures. Images of wounds were made with a digital camera system (D50, Nikon, Tokyo, Japan) at the same camera/subject distance (30 cm). The sizes of wound closure were determined using Image J program and represent as percentage of original wound area. At day 12, the mice were sacrificed and the dorsal skin including wound was excised and the image of inner side of skin was acquired to evaluate vascularity. The wound tissues were embed in O.C.T. compound (Sakura Finetek, CA), cut the samples to 6-µm-thick frozen sections for hematoxylin and eosin staining. To examine the migration of transplanted hMSCs into wound site, the hMSCs were prestained with DAPI (2 µg/ml) for 2 hours. The experimental animals were divided into two groups; DAPI-stained hMSCs (n = 3) and DAPI-stained ex vivo LPA-stimulated hMSCs (n = 3). Each group of cells was suspended in 1:1 mixture of α-MEM and growth factor-reduced Matrigel and injected into dermis around wound site. At day 12, the mice were euthanized and the samples were prepared as mentioned above. The DAPI-positive hMSCs were observed using fluorescence microscope. Individual sections were digitized with a Cascade 512B camera (Roper Scientific, Tucson, AZ) and analyzed with MetaMorph v.7.01 software (Molecular Devices Corporation, PA). We maintained a constant threshold of each image and compensated subtle variability of the background intensity. The cells that were brighter than the average gray level of each image at least 70% were counted. The average number of DAPI-positive cells per section from wound of each group was calculated. Oirs Cell Migration Assay hMSCs were seeded at 3 × 102 cells/100 µl in Oirs well (Platypus Technologies, WI) and incubated for 24 hours to permit cell adhesion. Inserts were carefully removed when the cell reached around 70% confluence, and the wells were washed with culture medium. Cells were stained with 5 µM calcein AM for 30 minutes and migrated cells were quantified through measurement of fluorescence signals using a Victor3 luminometer (PerkinElmer, Inc., Waltham, MA) at excitation and emission wavelengths of 485 and 515 nm, respectively. The measurements were shown as relative fluorescence unit. Reverse Transcription-Polymerase Chain Reaction and Real-Time PCR The hMSCs were maintained in serum-free α-MEM for 24 hours. After refreshing the culture media with serum-free α-MEM, the cells were incubated for 24 hours with/without LPA and the total RNA was extracted from the cells using RNeasy mini kit (QIAGEN; Valencia, CA). Real-time quantification of target RNA was performed in a Rotor-Gene 6000 (Corbett Research; NSW, Australia) using QuantiTect SYBR Green PCR Kits (QIAGEN). The primers used are described in the Supporting Information Table S1. The reaction mixture (20 µl) contained 200 ng of RT products, 0.05 µM of each primer, and appropriate amounts of enzymes and fluorescent dyes as recommended by the supplier. The PCR and real-time PCR were performed as follows: 15 minutes at 95°C for DNA polymerase activation; 15 seconds at 95°C for denaturing; and 30 cycles of 15 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at 72°C. The fluorescent intensity was acquired during the extension step (30 seconds at 72°C) and analysis was performed using the software provided by the manufacturer. Following real-time PCR, melting curve analysis was performed to verify the specificity and identity of the PCR products. Western Blot Analysis Cells were harvested, washed twice with PBS, and lysed in lysis buffer (20 mM Tris [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride, and 0.5 mM sodium orthovanadate) for 30 minutes on ice. The lysates were centrifuged (30 minutes at 15,000 rpm, 4°C), and the protein concentration was determined using the Bradford method [35]. Equal amounts of protein (20 µg) were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, and 0.01% Tween-20) containing 5% skim milk for 1 hour, and incubated with the appropriate primary antibodies at the dilutions recommended by the suppliers. The membranes were then washed and the primary antibodies were recognized with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. The membrane was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech; Buckinghamshire, U.K.). The results were measured optical density using Image J program and presented as means ± SEM. Measurement of [Ca2+]i Changes in [Ca2+]i were measured using Fluo-3AM dissolved in dimethylsulfoxide. The hMSCs in confocal 35 mm coverglass bottom dishes were rinsed twice with a Bath Solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, 5.5 mM HEPES, pH 7.4), incubated in a Bath Solution containing 3 mM Fluo-3AM with 5% CO2−95% O2 at 37°C for 60 minutes, rinsed twice with the Bath Solution, and scanned every second with confocal microscope (Fluoview 300, Olympus, Hamburg, Germany) at excitation and emission wavelengths of 488 and 515 nm, respectively. A 23187 (Ca2+ ionophore) was treated to the cells as a positive control in order to verify the assay. All [Ca2+]i analyses were processed at a single-cell level and were expressed as the relative fluorescence intensity. Live Cell Imaging Microscopy Cells cultured in ibid insert dish were stained with DAPI (2 µg/ml) for 1 hour and washed with PBS. After refresh with serum-free α-MEM, placed in temperature/CO2 control chambers (Tokai, Tokyo, Japan) attached to an Olympus IX81-ZDC zero-drift microscope. DIC and DAPI images continued to be collected for 0–24 hours at 5-minute intervals, using a Cascade 512B camera (Roper Scientific) operated by the multidimensional acquisition package of MetaMorph v. 7.01 software (Molecular Devices, Sunnyvale, CA). To measure the migrated cell number and migration distance, we maintained a constant threshold for each image, compensated for subtle variability of the background intensity, and analyzed cells that were at least 70% brighter than the average gray level of each image after background subtraction and shading correction. Immunoprecipitation The binding between LPAR1–3 with Gαi, Gαq, and Gα12 was analyzed by immunoprecipitation. Cells were lysed with the coimmunoprecipitation buffer (1% Triton X-100 in 50 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 2.5 mM Na4PO7, 100 mM NaF, 200 nM microcystin lysine-arginine, and protease inhibitors). Cell lysates (300 µg) were mixed with anti-β-LPAR1–3 antibodies, and then incubated for 4 hours, prior to addition of Protein A/G PLUS-agarose immunoprecipitation reagent (Pierce; Rockford, IL). The samples were incubated for an additional 12 hours. The beads were washed four times with the coimmunoprecipitation buffer, and the bound proteins were eluted by boiling in laemmli sample buffer for 5 minutes. Samples were analyzed by Western blotting with anti-Gαi, Gαq, and Gα12 antibodies. Small Interfering RNA Transfection Cells were grown until 75% confluence, and transfected for 24 hours with LPAR1–5, Gαi, Gαq, Gα12, RhoA, Rac1, CDC42, Cofilin, Profilin-specific siRNA (25 nM; Dharmacon, Lafayette, CO), or nontargeting siRNA (25 nM) as a negative control (Dharmacon) using DharmaFECT transfection reagent (Dharmacon). The siRNA used is described in Supporting Information Table S2. After 6 hours of incubation, the culture media were replaced with transfection mixture-free and serum-free media and the cells were maintained until 24 hours. The efficacies of siRNAs used in this study were confirmed in our experimental condition (Supporting Information Fig. S2). Affinity Precipitation Activation of Cdc42, Rac1, and RhoA activities was determined using an affinity precipitation assay kits (EMD Millipore, Billerica, MA) according to the manufacturer's instructions. Cells were lysed in ice-cold MLB lysis buffer, and 400 µg of lysates was incubated for 1 hour with agarose beads coupled with the Rho-binding domain of rhotekin (GST-Rhotekin-RBD) or with Cdc42/Rac binding domain (GST-PAK-PBD), respectively. And then the bound Cdc42, Rac1, and RhoA proteins were eluted with 2× Laemmli sample buffer and subjected to Western blot using anti-Cdc42, anti-Rac1, and anti-RhoA antibodies, respectively. Chromatin Immunoprecipitation Chromatin immunoprecipitation (ChIP) was carried out using an EZ-ChIP-Chromatin Immunoprecipitation Kit (EMD Millipore) according to the manufacturer's instructions. Chromatin-protein complexes were immunoprecipitated using anti-β-catenin, TCF, LEF, Snail, Slug, and PolII antibodies. The normal IgG was used as a negative control. After overnight incubation, immune complexes were eluted with 200 µl (two times of 100 µl each) elution buffer (1% SDS, 50 mM Tris-HCl, pH 7.5, and 10 mM EDTA), and then were incubated with RNase for 1 and 4 hours with proteinase K at 65°C. DNA was extracted and amplified by PCR using the E-box primer (Supporting Information Table S1), which is a 93-bp fragment encompassing the human E-box elements 1 and 3 in the E-cadherin promoter [36]. As inputs, we used products that corresponded to PCR reactions containing 1% of the total chromatin extract used in the immunoprecipitation reactions. Subcellular Fractionation hMSCs were lysed in hypotonic buffer (20 mM HEPES [pH 7], 10 mM KCl, 2 mM MgCl2, 0.5% Nonidet P40, 1 mM sodium orthovanadate) containing a cocktail of protease inhibitors. Lysates were homogenized gently (30 strikes in a syringe with a 19-gauge needle, and 30 strikes with a 25-gauge needle) and centrifuged at 8,000 rpm for 5 minutes at 4°C. The pellets, containing the nuclear fraction, were washed three times in hypotonic buffer. After homogenation in hypertonic buffer (hypertonic buffer containing 0.5 M NaCl and a cocktail of protease inhibitors), they were centrifuged at 15,000 rpm for 30 minutes, and the supernatants (nuclear fraction) were conserved at −70°C. The supernatants, containing the cytosol and membrane fraction, were cleared by centrifugation at 15,000 rpm for 5 minutes and then the supernatants (cytosol fraction) were conserved at −70°C. The pellets (containing membrane fraction) were washed with hypotonic buffer twice, and were centrifuged at 12,000g for 1 hour after lysing with RIPA buffer. Cell Transfection and Luciferase Assay A total 1 × 104 cells were seeded in 96-well culture dish 24 hours before transfection. CDH-1 (200 ng/well) promoter construct (Genecopoeia, Rockville, MD), a dual reporter system with Gauusia luciferase (Gluc) and secreted alkaline phosphatase (SEAP), was transfected using Lipofectamine LTX reagent (Life Technologies, Corp., Carlsbad, CA) and then the cells were treated with LPA for 24 hours after 48 hours transfection. Luciferase activities of cell culture medium were measured using Secrete-Pair Dual Luminescence Assay Kit (Genecopoeia). Light emission was measured for more than 3 seconds using a Victor3 luminometer (PerkinElmer, Inc., Waltham, MA). Efficiency of transfection was normalized using SEAP activity. The changes in ratio of Gluc and SEAP activities were expressed as relative CDH-1 promoter activity. Statistical Analysis Results are expressed as mean value ± SEM. All experiments were analyzed by ANOVA, and some experiments that needed to compare with more than or equal to three groups were examined using a Bonferroni-Dunn test. A p value of <.05 was considered statistically significant. Results hMSCs Stimulated with LPA Enhanced Skin Wound Healing in a Mouse Model To investigate the wound healing effect of hMSCs which pretreated with LPA ex vivo, wound area reduction and neovascularization were examined, and histological examinations were performed in a mouse skin wound healing model. The wound area was significantly decreased by hMSC transplantation or LPA treatment, and the LPA treated group accelerated the wound healing compared to the hMSC transplantation group. It was noted that transplantation of ex vivo LPA-stimulated hMSC significantly enhanced the wound healing from day 9 compared to the control. At day 12, a significant reduction in wound size was observed in order of the ex vivo LPA-stimulated hMSC, LPA alone, hMSC alone, and control group (Fig. 1A, 1B). In the wounds of the control and hMSC transplanted groups, thin blood vessels were seen and had a few branches in the skin surrounding the wounds, which were not completely closed. In contrast, in the wounds of the LPA treatment and ex vivo LPA-stimulated hMSC transplantation groups, thick vessels and their fine branches extended into the wounds, forming complex networks (Fig. 1C). Histological examination at day 12 showed that wound site was covered by epithelium, but not completely filled with granulation tissue in the control or hMSC alone transplanted mice. In LPA alone or ex vivo LPA-stimulated hMSCs group, the epidermal hyperplasia was observed, and the wound sites were completely filled with granulation tissue (Fig. 1D). Open in new tabDownload slide Effect of LPA and hMSC on skin wound healing in mice model. Mouse skin wounds were made by 6-mm-diameter biopsy punch. Experimental animals were divided into four groups; control group (n = 8) which received 50 µl of 1:1 mixture of α-MEM and growth factor-reduced Matrigel (BD Biosciences, NJ) as a vehicle, LPA group (n = 8), hMSCs (1 × 106 cells) group (n = 8), and ex vivo LPA-stimulated hMSCs (1 × 106 cells) group (n = 8). (A): Representative gross images on skin wound healing at days 0, 5, 7, 9, and 12 are shown. (B): Quantifications of wound sizes relative to original wound size are shown. Error bars represent the mean ± SEM. n = 8. *, p < .05 versus control, #, p < .05 versus hMSCs alone, †, p < .05 versus LPA alone. (C): Representative images of vascularity wound area at day 12 in mice treated with vehicle medium (control), hMSCs, LPA, or ex vivo LPA-stimulated hMSCs are shown. (D): Wound tissues were stained with hematoxylin and eosin at day 12 after wounded, and the representative wound histological images are shown. Star markers indicate wound sites. Abbreviations: CL, cornified layer; D, dermis; Ep, epidermis; GT, granulation tissue; hMSC, human umbilical cord blood-derived mesenchymal stem cell; LPA, lysophosphatidic acid; SC, subcutaneous tissue. Open in new tabDownload slide Effect of LPA and hMSC on skin wound healing in mice model. Mouse skin wounds were made by 6-mm-diameter biopsy punch. Experimental animals were divided into four groups; control group (n = 8) which received 50 µl of 1:1 mixture of α-MEM and growth factor-reduced Matrigel (BD Biosciences, NJ) as a vehicle, LPA group (n = 8), hMSCs (1 × 106 cells) group (n = 8), and ex vivo LPA-stimulated hMSCs (1 × 106 cells) group (n = 8). (A): Representative gross images on skin wound healing at days 0, 5, 7, 9, and 12 are shown. (B): Quantifications of wound sizes relative to original wound size are shown. Error bars represent the mean ± SEM. n = 8. *, p < .05 versus control, #, p < .05 versus hMSCs alone, †, p < .05 versus LPA alone. (C): Representative images of vascularity wound area at day 12 in mice treated with vehicle medium (control), hMSCs, LPA, or ex vivo LPA-stimulated hMSCs are shown. (D): Wound tissues were stained with hematoxylin and eosin at day 12 after wounded, and the representative wound histological images are shown. Star markers indicate wound sites. Abbreviations: CL, cornified layer; D, dermis; Ep, epidermis; GT, granulation tissue; hMSC, human umbilical cord blood-derived mesenchymal stem cell; LPA, lysophosphatidic acid; SC, subcutaneous tissue. ATX Stimulated hMSC Migration in a LPAR1/3-Dependent Manner To examine whether the ex vivo LPA stimulation of hMSCs on wound healing is involved with hMSC motility enhancement, the hMSCs were stained with DAPI and then transplanted into the dermis around the wound in the mouse skin wound model. In the histological sections of the wound site, the DAPI-positive cells located in the granulation tissue were significantly increased in the ex vivo LPA-stimulated hMSCs group compared to the hMSCs alone group (Fig. 2A) suggest that LPA enhanced the migration of hMSCs to the wound site and accelerated wound healing. Thus, we investigated the effect of ATX and LPA on hMSC migration regulation. As shown in Figure 2B, an increasing concentration of ATX from 0.1 to 1 nM induced significantly increased hMSC migration. To confirm the existence of LPA receptors in hMSCs, the total mRNA was extracted, and the mRNA expression of the LPA receptors was detected with reverse transcription-polymerase chain reaction (Fig. 2C). When the cells were treated with ATX or LPA, the mRNAs of the LPA receptors were significantly increased, and this result was correlated with the protein expression level (Fig. 2D, 2E). In addition, as shown in Figure 1F, LPAR1–3 were located in the lipid rafts (Fig. 2F). In next, we examined the ATX-induced hMSC migration was mediated by LPA production and the subsequent LPA receptor activation. LPA significantly increased hMSC migration in a dose-dependent manner (0.1–5 µM) and lamellipodia formation (Fig. 2G, 2H). In addition, ATX promoted a substantial cell migration into the denuded area and increased the lamellipodia formation of the cells present in the denuded area which were inhibited by LPAR1 and LPAR3 siRNA, but not by LPAR2, LPAR4, and LPAR5 siRNA transfection (Fig. 2I). Open in new tabDownload slide Effect of autotaxin in MSC migration and involvement of LPA receptors. (A): The DAPI-stained hMSCs with/without ex vivo LPA stimulation were injected into dermis around the wound in mouse skin wound model. After 12 days, histological sections of wound site were observed and the DAPI-positive cells were counted. All images were acquired digitally (×200) and the inset shows magnified image. The below panel depicts the mean ± SEM of DAPI-positive cell number. *, p < .05 versus control. Scale bar represents 200 µm. (B): Cells were treated with ATX (0–1 nM) for 24 hours and cell migration was detected with Oris cell migration assay. The changes in fluorescence were expressed as relative fluorescence units (RFU). (C): The transcriptional levels of the LPA receptors 1–5 (LPAR1–5) were detected with reverse transcription-polymerase chain reaction. (D, E): hMSCs were treated with LPA or ATX and LPAR1–5 mRNA and LPAR1–3 protein expression levels were detected using real-time PCR and Western blot analysis, respectively. The right panel of Figure 1D depicted by bars denote mean ± SEM of six experiments for each condition calculated by fold of control. And the right panel of Figure 1E depicted by bars denotes mean ± SEM of six experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control. N.S.: not significant. (F): The total cell lysates were subjected to discontinuous sucrose density gradient fractionation, and then LPAR1–3 and cav-1 were detected. The changes in fluorescence were expressed as RFU. (G): hMSCs were treated with various concentrations of LPA (0–5 µM) for 24 hours and cell migration was detected with Oris cell migration assay. Data of (B) and (F) represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control. (H): The cells were treated with various concentration of LPA. Scale bar represents 50 µm. (I): hMSCs cultured in ibid insert dish were transfected with LPAR1–5-specific siRNA prior to ATX treatment. The cells of (H) and (I) were stained with Phalloidin and propidium iodide. Scale bar represents 100 µm. Abbreviations: ATX, autotaxin; hMSC, human umbilical cord blood-derived mesenchymal stem cell; LPA, lysophosphatidic acid. Open in new tabDownload slide Effect of autotaxin in MSC migration and involvement of LPA receptors. (A): The DAPI-stained hMSCs with/without ex vivo LPA stimulation were injected into dermis around the wound in mouse skin wound model. After 12 days, histological sections of wound site were observed and the DAPI-positive cells were counted. All images were acquired digitally (×200) and the inset shows magnified image. The below panel depicts the mean ± SEM of DAPI-positive cell number. *, p < .05 versus control. Scale bar represents 200 µm. (B): Cells were treated with ATX (0–1 nM) for 24 hours and cell migration was detected with Oris cell migration assay. The changes in fluorescence were expressed as relative fluorescence units (RFU). (C): The transcriptional levels of the LPA receptors 1–5 (LPAR1–5) were detected with reverse transcription-polymerase chain reaction. (D, E): hMSCs were treated with LPA or ATX and LPAR1–5 mRNA and LPAR1–3 protein expression levels were detected using real-time PCR and Western blot analysis, respectively. The right panel of Figure 1D depicted by bars denote mean ± SEM of six experiments for each condition calculated by fold of control. And the right panel of Figure 1E depicted by bars denotes mean ± SEM of six experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control. N.S.: not significant. (F): The total cell lysates were subjected to discontinuous sucrose density gradient fractionation, and then LPAR1–3 and cav-1 were detected. The changes in fluorescence were expressed as RFU. (G): hMSCs were treated with various concentrations of LPA (0–5 µM) for 24 hours and cell migration was detected with Oris cell migration assay. Data of (B) and (F) represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control. (H): The cells were treated with various concentration of LPA. Scale bar represents 50 µm. (I): hMSCs cultured in ibid insert dish were transfected with LPAR1–5-specific siRNA prior to ATX treatment. The cells of (H) and (I) were stained with Phalloidin and propidium iodide. Scale bar represents 100 µm. Abbreviations: ATX, autotaxin; hMSC, human umbilical cord blood-derived mesenchymal stem cell; LPA, lysophosphatidic acid. Gαi and Gαq Activation Are Involved in the LPA-Induced hMSC Migration LPA stimulated the detachment of the Gαi, Gαq, and Gα12 subunits from LPAR, but we could not distinguish the specific isotype of the LPAR and Gα subunits activated by LPA (Fig. 3A). However, lipid raft disruption using MβCD and LPAR1/3 inhibition (Ki16425) attenuated the LPA-induced hMSC migration. In addition, Gαi inhibition by Ptx pretreatment also decreased hMSC migration (Fig. 3B, 3C). Also, LPAR1 and 3 siRNA and Gαi and Gαq siRNA transfections inhibited LPA-induced F-actin reorganization (Fig. 3D, 3E). Consistent with these results, Gαi and Gαq siRNA transfections abolished the LPA-induced hMSC migration and lamellipodia formation but Gα12 siRNA transfection had no affect by itself (Fig. 3F, 3G). Open in new tabDownload slide Involvement of Gα subunits in LPA-induced mesenchymal stem cell (MSC) migration. (A): Human umbilical cord blood-derived MSCs (hMSCs) were treated with LPA for 30 minutes. The total cell lysates and immunoprecipitation of anti-LPAR1–3 were analyzed by Western blotting with antibodies that recognize Gαi, Gαq, or Gα12. Cells were pretreated with MβCD, Ki16425, or Ptx for 1 hour prior incubation with LPA for 24 hours. (B): The cells were stained with phalloidin and propidium iodide (PI) and observed with confocal microscope or (C) cell migration was detected with Oris cell migration assay. Data represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control, **, p < .05 versus LPA alone. Scale bar represents 100 µm. (D, E): The cells were transfected with LPAR1–3, Gαi, Gαq, or Gα12 specific siRNA prior to treatment of LPA for 24 hours, and the total cell lysates were subjected to SDS-PAGE for detection of F-actin expression level. The right part depicted by bars denotes mean ± SEM of three experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control, **, p < .05 versus LPA alone. hMSCs were transfected with Gαi, Gαq, or Gα12 specific siRNA for 24 hours prior to treatment of LPA for 24 hours. (F): The cells were stained with phalloidin and PI and observed with confocal microscope or (G) cell migration was detected with Oris cell migration assay. Data represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control, **, p < .05 versus LPA alone. Scale bar represents 100 µm. Abbreviations: LPA, lysophosphatidic acid; RFU, relative fluorescence units. Open in new tabDownload slide Involvement of Gα subunits in LPA-induced mesenchymal stem cell (MSC) migration. (A): Human umbilical cord blood-derived MSCs (hMSCs) were treated with LPA for 30 minutes. The total cell lysates and immunoprecipitation of anti-LPAR1–3 were analyzed by Western blotting with antibodies that recognize Gαi, Gαq, or Gα12. Cells were pretreated with MβCD, Ki16425, or Ptx for 1 hour prior incubation with LPA for 24 hours. (B): The cells were stained with phalloidin and propidium iodide (PI) and observed with confocal microscope or (C) cell migration was detected with Oris cell migration assay. Data represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control, **, p < .05 versus LPA alone. Scale bar represents 100 µm. (D, E): The cells were transfected with LPAR1–3, Gαi, Gαq, or Gα12 specific siRNA prior to treatment of LPA for 24 hours, and the total cell lysates were subjected to SDS-PAGE for detection of F-actin expression level. The right part depicted by bars denotes mean ± SEM of three experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control, **, p < .05 versus LPA alone. hMSCs were transfected with Gαi, Gαq, or Gα12 specific siRNA for 24 hours prior to treatment of LPA for 24 hours. (F): The cells were stained with phalloidin and PI and observed with confocal microscope or (G) cell migration was detected with Oris cell migration assay. Data represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control, **, p < .05 versus LPA alone. Scale bar represents 100 µm. Abbreviations: LPA, lysophosphatidic acid; RFU, relative fluorescence units. LPA Induced LPAR1/3-Dependent Ca2+ Influx and PKC Activation LPA increased the Ca2+ influx, which was blocked by pretreatment with EGTA/BAPTA-AM, MβCD, Ki16425, and Ptx (Fig. 4A). In addition, LPA increased the phosphorylation of PKC in a time-dependent manner (the maximum activation was observed at 15–90 minutes after LPA treatment) (Fig. 4B). In an experiment to identify the activated specific PKC isoforms activated by LPA, LPA stimulated the translocation of PKC α, δ, and ζ from cytosol to membrane, which has been considered the hallmark of PKC activation (Fig. 4C). Furthermore, Ki16425 pretreatment blocked the LPA-induced phosphorylation of PKC (Fig. 4D), and these results are supported by the results of the LPAR siRNA and Gα subunit siRNA transfection experiments in which the LPAR1/3 siRNA and Gαi/Gαq siRNA transfections abolished the LPA-induced PKC activation (Fig. 4E, 4F). The LPA-induced hMSC migration and lamellipodia formation were inhibited by PKC inhibition using staurosporine and bisindolylmaleimide I (Fig. 4G). Open in new tabDownload slide Involvement of Ca2+/PKC in LPA-induced mesenchymal stem cell (MSC) migration. (A): Human umbilical cord blood-derived MSCs (hMSCs) were pretreated with EGTA/BAPTA-AM, MβCD, Ki16425, or Ptx for 1 hour. And then the cells were loaded with Fluo-3AM in serum-free medium for 60 minutes and treated with LPA. A23187 (Ca2+ ionophore, 10−6 M) was used as a positive control. The changes in fluorescence were expressed as RFI. (B): Cells were treated with LPA for 0–180 minutes, analyzed by Western blotting with anti-p-pan PKC antibodies. (C): Cells were treated with LPA for 1 hour, and cytosolic and membrane fractions were prepared. The PKC α, δ, θ, ε, and ζ isoforms present in either cytosolic or membrane compartments were detected by Western blotting. (D): hMSCs were either transfected with LPAR1–3, Gαi, Gαq, or Gα12 specific siRNA for 24 hours or pretreated with Ki16425 for 1 hour, and then phosphorylation level of pan-PKC was detected. (E, F): The cells were transfected with LPAR1–3 siRNA or Gα subunits (i, q, 12) siRNA for 24 hours prior to incubation with LPA for 24 hours. The total lysates were subjected to SDS-PAGE for detection of pan-PKC phsophorylation level. The lower parts of (B), (D)–(F) depict the mean ± SEM of four experiments for each condition determined from densitometry relative to β-actin. *, p < .05 versus control, *, p < .05 versus LPA alone. (G): Cells cultured in ibid insert dish were treated with autotaxin for 24 hours, or pretreated with staurosporine and bisindolylmaleimide I for 1 hour prior to incubation with LPA for 24 hours. The cells were stained with Phalloidin and propidium iodide. Scale bars represent 100 µm. Abbreviations: LPA, lysophosphatidic acid; RFI, relative fluorescence intensities. Open in new tabDownload slide Involvement of Ca2+/PKC in LPA-induced mesenchymal stem cell (MSC) migration. (A): Human umbilical cord blood-derived MSCs (hMSCs) were pretreated with EGTA/BAPTA-AM, MβCD, Ki16425, or Ptx for 1 hour. And then the cells were loaded with Fluo-3AM in serum-free medium for 60 minutes and treated with LPA. A23187 (Ca2+ ionophore, 10−6 M) was used as a positive control. The changes in fluorescence were expressed as RFI. (B): Cells were treated with LPA for 0–180 minutes, analyzed by Western blotting with anti-p-pan PKC antibodies. (C): Cells were treated with LPA for 1 hour, and cytosolic and membrane fractions were prepared. The PKC α, δ, θ, ε, and ζ isoforms present in either cytosolic or membrane compartments were detected by Western blotting. (D): hMSCs were either transfected with LPAR1–3, Gαi, Gαq, or Gα12 specific siRNA for 24 hours or pretreated with Ki16425 for 1 hour, and then phosphorylation level of pan-PKC was detected. (E, F): The cells were transfected with LPAR1–3 siRNA or Gα subunits (i, q, 12) siRNA for 24 hours prior to incubation with LPA for 24 hours. The total lysates were subjected to SDS-PAGE for detection of pan-PKC phsophorylation level. The lower parts of (B), (D)–(F) depict the mean ± SEM of four experiments for each condition determined from densitometry relative to β-actin. *, p < .05 versus control, *, p < .05 versus LPA alone. (G): Cells cultured in ibid insert dish were treated with autotaxin for 24 hours, or pretreated with staurosporine and bisindolylmaleimide I for 1 hour prior to incubation with LPA for 24 hours. The cells were stained with Phalloidin and propidium iodide. Scale bars represent 100 µm. Abbreviations: LPA, lysophosphatidic acid; RFI, relative fluorescence intensities. LPA Attenuated E-Cadherin Expression Through the GSK3β/β-Catenin Pathway LPA stimulated GSK3β phosphorylation and active-β-catenin expression in a time-dependent manner (Fig. 5A). In addition, β-catenin, snail, and slug expression levels in both the cytosol and nuclear fraction were increased by LPA treatment (Fig. 5B, 5C). These results suggest that the inhibition of GSK3β increased cytosolic accumulation and nuclear translocation of β-catenin through disruption of β-catenin complex with GSK3β/APC/axin. Pretreatment with PKC inhibitors (staurosporine or bisindolylmaleimide I) attenuated the LPA-induced GSK3β phosphorylation and β-catenin activation as well as nuclear localization of β-catenin, snail, and slug (Fig. 5D). As shown in Figure 5E, chromatin immunoprecipitation with β-catenin, TCF-1, LEF-1, snail, and slug antibodies revealed that LPA treatment stimulates β-catenin, TCF-1, and LEF-1 binding on the E-box of the CDH-1 gene promoter region; however, snail and slug did not bind to the E-box (Supporting Information Fig. S3). To confirm the effect of LPA on CDH-1 promoter activity, the cells were transfected with CDH-1 promoter and the luciferase activity was detected. LPA and LiCl treatment decreased CDH-1 promoter activity which was blocked by β-catenin siRNA (Fig. 5F). In addition, ATX or LPA decreased E-cadherin expression in a time-dependent manner, but N-cadherin expression did not affected by LPA (Fig. 5G, 5H). Consistently, GSK3β inhibition using LiCl decreased E-cadherin expression and increased hMSC migration (Fig. 5I). In contrast, β-catenin siRNA transfection blocked the LPA-induced E-cadherin decrease and migration (Fig. 5J). Open in new tabDownload slide Effect of LPA on E-cadherin reduction via GSK3β/β-catenin pathway. (A): Human umbilical cord blood-derived mesenchymal stem cells (hMSCs) were treated with LPA for 0–180 minutes and the GSK-3β phsophorylation level and active-β-catenin expression level were detected. (B, C): Cells were treated with LPA for 2 hours and membrane, cytosol, and nuclear fractions were analyzed by Western blotting with antibodies that recognize β-catenin, snail, or slug. (D): hMSCs were pretreated with staurosporine or bisindolylmaleimide I prior to treatment of LPA. The GSK-3β phsophorylation level and active-β-catenin expression level were observed in total lysates and the β-catenin, snail, or slug expression levels were detected in nuclear fraction. (E): Cells were treated with LPA and the chromatin DNA was immunoprecipitated with antibodies against β-catenin, TCF, LEF, Pol II, or normal IgG. Pol II and IgG were used as positive and negative control, respectively. The resulting samples were amplified by PCR with the primers for E-box of CDH-1 gene promoter or GAPDH. The right panel depicted by bars denotes mean ± SEM of three experiments for each condition determined by real-time PCR. *, p < .05 versus control. (F): hMSCs were transfected with CDH-1 promoter construct vector (pEZX-LvPG04). And then the cells were pretreated with LiCl for 12 hours or cotransfected with β-catenin siRNA for 24 hours. After incubation with LPA for 24 hours, the Gaussia Luciferase (Gluc) and secreted alkaline phosphatase (SEAP) activities were detected in cell culture media. The changes in ratio of Gluc and SEAP activities were expressed as relative CDH1 promoter activity. *, p < .05 versus control, **, p < .05 versus LPA alone. (G): hMSCs were treated with LPA for 0–48 hours and the total cell lysates were subjected to SDS-PAGE for detection of E-cadherin and N-cadherin expression level. The right panel depicted by bars denotes mean ± SEM of three experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control. (H): hMSCs were treated with autotaxin or LPA for 24 hours and then stained with phalloidin and propidium iodide. Scale bar represents 50 µm. (I): Cells were treated with LiCl for 24 hours or transfected with β-catenin for 24 hours prior to LPA treatment. And the E-cadherin expression level was observed. The lower parts of (A)–(C), (G), and (I) depict the mean ± SEM of four experiments for each condition determined from densitometry relative to β-actin. *, p < .05 versus control, **, p < .05 versus LPA alone. (J): Cell migration was detected with Oris cell migration assay. Data represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control, **, p < .05 versus LPA alone. Abbreviation: LPA, lysophosphatidic acid. Open in new tabDownload slide Effect of LPA on E-cadherin reduction via GSK3β/β-catenin pathway. (A): Human umbilical cord blood-derived mesenchymal stem cells (hMSCs) were treated with LPA for 0–180 minutes and the GSK-3β phsophorylation level and active-β-catenin expression level were detected. (B, C): Cells were treated with LPA for 2 hours and membrane, cytosol, and nuclear fractions were analyzed by Western blotting with antibodies that recognize β-catenin, snail, or slug. (D): hMSCs were pretreated with staurosporine or bisindolylmaleimide I prior to treatment of LPA. The GSK-3β phsophorylation level and active-β-catenin expression level were observed in total lysates and the β-catenin, snail, or slug expression levels were detected in nuclear fraction. (E): Cells were treated with LPA and the chromatin DNA was immunoprecipitated with antibodies against β-catenin, TCF, LEF, Pol II, or normal IgG. Pol II and IgG were used as positive and negative control, respectively. The resulting samples were amplified by PCR with the primers for E-box of CDH-1 gene promoter or GAPDH. The right panel depicted by bars denotes mean ± SEM of three experiments for each condition determined by real-time PCR. *, p < .05 versus control. (F): hMSCs were transfected with CDH-1 promoter construct vector (pEZX-LvPG04). And then the cells were pretreated with LiCl for 12 hours or cotransfected with β-catenin siRNA for 24 hours. After incubation with LPA for 24 hours, the Gaussia Luciferase (Gluc) and secreted alkaline phosphatase (SEAP) activities were detected in cell culture media. The changes in ratio of Gluc and SEAP activities were expressed as relative CDH1 promoter activity. *, p < .05 versus control, **, p < .05 versus LPA alone. (G): hMSCs were treated with LPA for 0–48 hours and the total cell lysates were subjected to SDS-PAGE for detection of E-cadherin and N-cadherin expression level. The right panel depicted by bars denotes mean ± SEM of three experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control. (H): hMSCs were treated with autotaxin or LPA for 24 hours and then stained with phalloidin and propidium iodide. Scale bar represents 50 µm. (I): Cells were treated with LiCl for 24 hours or transfected with β-catenin for 24 hours prior to LPA treatment. And the E-cadherin expression level was observed. The lower parts of (A)–(C), (G), and (I) depict the mean ± SEM of four experiments for each condition determined from densitometry relative to β-actin. *, p < .05 versus control, **, p < .05 versus LPA alone. (J): Cell migration was detected with Oris cell migration assay. Data represent means ± SEM of five independent experiments with triplicate dishes. *, p < .05 versus control, **, p < .05 versus LPA alone. Abbreviation: LPA, lysophosphatidic acid. LPA-Induced Rho GTPase Family Activation Involved in F-Actin Reorganization In experiment to examine the involvement of Rho GTPase activation on the LPA-induced hMSC migration, LPA stimulated Rac1 and CDC42, but not RhoA, and they were blocked by pretreatment with staurosporine and bisindolylmaleimide I (Fig. 6A, 6B). In addition, Rac1 and CDC42 siRNA transfections attenuated the LPA-induced increase of Cofilin-1 phosphorylation and Profilin-1 expression levels (Fig. 6C, 6D). Cofilin-1 and Profilin-1 siRNA transfections attenuated the LPA-induced F-actin reorganization. These results in which LPA-induced hMSC migration is dependent on Rac1 and CDC42 activation is supported by the in vitro wound healing assay using RhoA, Rac1, and CDC42 siRNA transfections (Fig. 6E). Consistently, the time lapse-imaging of the hMSCs shows that ATX or LPA increased both the migration distance and number of migrated cells in Rac1 and CDC42-dependent manner (Fig. 6F). Open in new tabDownload slide Involvement of Rho GTPase activation on LPA-induced cytoskeletal rearrangement. (A): Human umbilical cord blood-derived mesenchymal stem cells (hMSCs) were treated with LPA for 0–60 minutes and the GTP-RhoA, GTP-Rac1, and GTP-CDC42 levels were detected with affinity precipitation. RhoA, Cdc42, and Rac1 expression levels were also detected in total lysates. (B): The cells were pretreated with staurosporine and bisindolylmaleimide I for 1 hour prior to incubation with LPA for 60 minutes and the GTP-Rac1 and GTP-CDC42 levels were detected with affinity precipitation. (C): After transfection with RhoA, Rac1, or CDC42 siRNA for 24 hours, the cells were treated with LPA for 24 hours, and then F-actin, phospho-Cofilin 1, or Profilin expression levels were detected. (D): hMSCs were transfected with Cofilin 1 or Profilin siRNA for 24 hours prior to treatment of LPA for 24 hours and the F-actin expression level was detected with Western blotting. The right parts of (A)–(D) depicted by bars denote mean ± SEM of three experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control, **, p < .05 versus LPA alone. (E): The cells cultured in ibid insert dish were transfected with RhoA, Rac1, or CDC42 siRNA prior to incubation with LPA for 24 hours, and then the cells were stained with phalloidin and propidium iodide. Scale bar represents 100 µm. (F): hMSCs were either treated with autotaxin (ATX) or LPA for 24 hours with/without RhoA, Rac1, or CDC 42 siRNA transfection. The cells were stained with DAPI before ATX or LPA treatment, and then time lapse image was acquired for incubation period. The right upper part of (F) depicting the line graph denotes the distance of cell migration and the right lower part denotes the number of migrated cell, which is presented as mean ± SEM of three experiments. *, p < .05 versus control, **, p < .05 versus LPA alone. Scale bar represents 100 µm. Abbreviation: LPA, lysophosphatidic acid. Open in new tabDownload slide Involvement of Rho GTPase activation on LPA-induced cytoskeletal rearrangement. (A): Human umbilical cord blood-derived mesenchymal stem cells (hMSCs) were treated with LPA for 0–60 minutes and the GTP-RhoA, GTP-Rac1, and GTP-CDC42 levels were detected with affinity precipitation. RhoA, Cdc42, and Rac1 expression levels were also detected in total lysates. (B): The cells were pretreated with staurosporine and bisindolylmaleimide I for 1 hour prior to incubation with LPA for 60 minutes and the GTP-Rac1 and GTP-CDC42 levels were detected with affinity precipitation. (C): After transfection with RhoA, Rac1, or CDC42 siRNA for 24 hours, the cells were treated with LPA for 24 hours, and then F-actin, phospho-Cofilin 1, or Profilin expression levels were detected. (D): hMSCs were transfected with Cofilin 1 or Profilin siRNA for 24 hours prior to treatment of LPA for 24 hours and the F-actin expression level was detected with Western blotting. The right parts of (A)–(D) depicted by bars denote mean ± SEM of three experiments for each condition determined by densitometry relative to β-actin. *, p < .05 versus control, **, p < .05 versus LPA alone. (E): The cells cultured in ibid insert dish were transfected with RhoA, Rac1, or CDC42 siRNA prior to incubation with LPA for 24 hours, and then the cells were stained with phalloidin and propidium iodide. Scale bar represents 100 µm. (F): hMSCs were either treated with autotaxin (ATX) or LPA for 24 hours with/without RhoA, Rac1, or CDC 42 siRNA transfection. The cells were stained with DAPI before ATX or LPA treatment, and then time lapse image was acquired for incubation period. The right upper part of (F) depicting the line graph denotes the distance of cell migration and the right lower part denotes the number of migrated cell, which is presented as mean ± SEM of three experiments. *, p < .05 versus control, **, p < .05 versus LPA alone. Scale bar represents 100 µm. Abbreviation: LPA, lysophosphatidic acid. Open in new tabDownload slide The proposed model for the signaling pathways involved in LPA-induced adherent junction disruption and hMSCs migration. LPA generated by autotaxin bound to LPA receptor 1/3 which stimulated PKC activation through Gβγi and Gαq. LPA-induced PKC activation inhibited GSK3β activation which resulted in β-catenin accumulation and translocation to nuclear. Subsequently, β-catenin inhibited E-cadherin transcription through binding to E-box of CDH-1 promoter. In addition, LPA-induced PKC activation also stimulated F-actin reorganization through Rac1/CDC42-cofilin-1/profiling-1 signaling pathway. Taken together, LPA-induced adherent junction disruption and lamellipodia formation stimulate hMSCs migration. Abbreviations: hMSCs, human umbilical cord blood-derived mesenchymal stem cells; LPA, lysophosphatidic acid; LPAR, LPA receptor; LPC, lysophosphatidylcholine; PKC, protein kinase C; PTX, pertussis toxin. Open in new tabDownload slide The proposed model for the signaling pathways involved in LPA-induced adherent junction disruption and hMSCs migration. LPA generated by autotaxin bound to LPA receptor 1/3 which stimulated PKC activation through Gβγi and Gαq. LPA-induced PKC activation inhibited GSK3β activation which resulted in β-catenin accumulation and translocation to nuclear. Subsequently, β-catenin inhibited E-cadherin transcription through binding to E-box of CDH-1 promoter. In addition, LPA-induced PKC activation also stimulated F-actin reorganization through Rac1/CDC42-cofilin-1/profiling-1 signaling pathway. Taken together, LPA-induced adherent junction disruption and lamellipodia formation stimulate hMSCs migration. Abbreviations: hMSCs, human umbilical cord blood-derived mesenchymal stem cells; LPA, lysophosphatidic acid; LPAR, LPA receptor; LPC, lysophosphatidylcholine; PKC, protein kinase C; PTX, pertussis toxin. Discussion In this study, we showed that ATX-induced LPAR1/3 activation decreased E-cadherin expression and increased lamellipodia formation through PKC-dependent β-catenin or Rho GTPase family activation, which sequentially stimulated the migration of hMSCs. In a mouse wound healing model, hMSCs or LPA application increased the degree of wound healing; furthermore, LPA more effectively healed the wound than that of hMSC transplantation. It has been reported that LPA stimulates the migration or proliferation of skin cells such as keratinocytes, fibroblasts and mesenchymal and epithelial cells, and hMSCs promote wound healing through paracrine signaling and differentiation [37, 38]. Furthermore, we acquired a higher capillary density in the LPA-treated hMSC group compared to the hMSC alone group. Our observations of enhanced vascularity in the LPA-treated skin wounds and subsequent accelerated wound healing suggest that both increased neovascularization and re-epithelization as a target for LPA effects. These observations may partly explain a mechanism by which LPA stimulates the clinical healing of skin wound. Indeed, previous reports showed that LPA enhances VEGF secretion by hMSCs [39, 40], suggesting that implanted hMSCs with LPA can stimulate angiogenesis by secreting multiple angiogenic cytokines in addition to LPA acting as a direct angiogenic factor in vivo. Interestingly, the ex vivo stimulation of hMSCs with LPA has a very potent effect on mouse skin wound healing, and ex vivo activation using LPA increased the migration of hMSCs to the wound site, suggesting the possibility that the LPA-induced hMSC migration enhances the wound healing progress and the valuable tools for modulating hMSC function using bioactive lipid metabolites in regenerative medicine. Our data show that LPA treatment significantly enhances hMSC migration, which provides a mechanistic explanation for the important role of LPA in tissue regeneration. To our knowledge, this is the first observation that LPA can lead to enhanced MSCs migration and angiogenesis in skin wound healing. Indeed, regenerative engineering technologies using bioactive molecules, stem cells, and advanced biomaterials have been proposed as promising approaches for tissue repair and regeneration. Therefore, dissecting the regulatory pathways and deciphering underlying molecular mechanisms of hMSCs migration would be necessary to advance stem cell therapy in the management of wound healing. Although ATX positively or negatively modulates cell motility depending on the cell type and LPA receptor subtypes [41], in this study, ATX stimulated hMSC migration and lamellipodia formation. hMSCs expressed LPAR1–5, but mouse MSCs expressed only three LPA receptors (LPAR1–3) [42-45]. Consistently, we observed expression of LPAR1–5 in hMSCs, but the ATX-induced hMSCs migration were occur in LPAR1 and 3-dependent manner, suggesting that the functions of LPA depend on its receptor subtypes. Similar to our previous report showing that S1P receptors 1–3 were located in lipid rafts [46], LPAR1–3 were also related with the lipid rafts, suggesting that the lipid rafts act as an important signaling platform for lysophospholipids in stem cells. Lipid rafts mediate various stem cell responses such as migration and proliferation [47, 48]. In addition, LPA receptor inhibition using siRNA transfection revealed that the ATX-induced hMSC migration is mediated by LPAR1 and LPAR3. Furthermore, LPA attenuated binding of Gαi, Gαq, and Gα12 to LPAR1–3, which suggests that LPA can elicit a response from LPAR1–3 and their coupled G proteins, and these results support the multiple actions of LPA in the functional regulation of stem cells. Although LPA treatment could stimulate LPAR1–3 and their downstream G proteins, Gαi and Gαq coupled with LPAR1/3 were closely involved in the LPA-induced hMSC migration. Interestingly, it has been reported that LPAR1–3 have different functions in cell motility regulation [49-52], suggesting that LPA exerts different responses in the distribution and expression levels of the LPAR receptors and their downstream signals depending on the specific cell type. Previous studies have shown that the activation of Gαq or Gβγi results in the stimulation of phospholipase C activity, leading to phosphatidylinositol-(4,5)-bisphosphate hydrolysis and Ca2+ mobilization from internal stores, and the subsequent activation of protein kinase C (PKC) [53, 54]. In addition, LPA stimulates Ca2+ signaling and PKC activation, which are involved in proliferation, migration, or gap junction regulation in both embryonic stem cells and MSCs [55-59]. In this study, LPA stimulates PKC α, δ, and ζ, which were abolished by Gαi and Gαq siRNA transfections as well as by Ptx pretreatment. Coincident with our results, LPA stimulated the translocation of the PKC isoforms α, β, ε, and ζ in a Ptx-sensitive manner [60], suggesting that the LPA-induced PKC activation could be elicited by Gαq-dependent PLD activation as well as in a Gβγi-dependent manner. In addition, it has been reported that PKC activation is involved in the migration process such as adherent junction disruption and cytoskeletal reorganization [61-63]. Adherent junction molecules, such as E-cadherin, have been shown to influence the proliferation of stem/progenitor cells [64]. Furthermore, the loss of cell-cell junctions elicited by E-cadherin fragmentation or downregulation by miRNA-10b resulting in an increase in motility of bone marrow-derived MSCs [65] suggests that E-cadherin is essential for embryogenesis as well as the regulation of stem cell motility. Interestingly, LPA did not affect on N-cadherin expression level, suggests that the possibility that the adherent junction disruption through E-cadherin reduction and N-cadherin recycling without alteration of N-cadherin expression level may be involved in LPA-induced hMSC migration. Several transcriptional repressors for E-cadherin have been recently identified, including Snail and Slug [66-69]. In this study, the data suggest that LPA-induced GSK3β phosphorylation elicits a release of β-catenin from the APC/Axin complex, and free β-catenin could be involved in snail and slug stabilization. Furthermore, β-catenin and the TCF/LEF family were bound to the E-box of the CDH-1 gene promoter, but snail and slug did not bind to the E-box, suggesting that the LPA-induced activation of β-catenin directly suppresses CDH-1 gene transcription; however, snail and slug may have other functions rather than cadherin suppression. In accordance with our results, LPA was previously reported to induce proliferation or migration of colon cancer cells and lung-residence MSCs via activation of the β-catenin pathway in a LPA receptor-dependent manner [70, 71]. Although it is still controversial whether LPA is mediated through its effects on structural organization and cadherin/β-catenin interactions or via its activation of nuclear transcription factors from the TCF/LEF family [72-74], our data suggest that the LPA-induced β-catenin activation directly downregulates E-cadherin expression through transcriptional regulation, which was supported by the results. The binding of β-catenin on the E-box of the CDH-1 gene promoter and the LPA-induced decrease of E-cadherin expression were observed later than β-catenin activation. In support of LPA-mediated hMSC migration, our results further elucidated the potential role of LPA in the Rho GTPase activity and its effect on the regulation of cytoskeletal reorganization-related proteins Profilin-1, Cofilin-1, and F-actin, which are critical requirements for stem cell migration [57, 75]. We found that LPA stimulated Rac1 and CDC42, but not Rho A, which are the best-characterized molecules among the Rho family GTPases; Rho controls the stress fibers and focal adhesion formation, and Rac1 and Cdc42 regulate membrane ruffling and filopodium formation [23]. It has been reported that Y27632-mediated inhibition of Rho kinase attenuated LPA induced human adipose tissue-derived MSC migration [39]. In contrast, the RhoA-Rho kinase pathway has an inhibitory effect on the LPA-induced migration of human bone marrow-derived MSCs [42]. These previous reports suggest that LPA-induced RhoA positively or negatively affects cell migration depending on gradient sensing, polarization, and orientation as well as specific cell types. Furthermore, it has been reported that the function of Rho was not dependent on Cdc42 or Rac [76]. In addition, several studies have shown that PKC isozymes translocate to the F-actin component of the cytoskeleton in intact cells [77-81], and provided evidence supporting a close association between PKC isozymes and members of the Rho family GTPases, and a convergence of corresponding signaling pathways [82-85]. The PKCα and ζ isoforms bind to the membrane-bound small Rho GTPase, Cdc42, and Rac1 [86], which mediate cell invasion and migration through lamellipodia, filopodia, podosome, or invadopodia formation [87]. These previous reports and our results suggest that the important role of PKC in signal transduction in hMSC motility and specific isoforms may be responsible for this motility. Thus, additional studies will be necessary to elucidate the relative contribution of each isoform in the regulation of hMSC migration. Moreover, in this study, LPA-induced Rac1 and CDC42 activation are involved in F-actin reorganization through Profilin-1 expression and Cofilin-1 phosphorylation. It is clearly shown that Profilin-1 and Cofilin-1 play key roles in enhancing actin assembly at the plasma membrane, thereby increasing F-actin expression that drives cell motility and other actin-linked processes [88, 89]. Conclusions In summary, our observations suggest the possible role of ATX/LPA signaling as a physiological regulator of migration of hMSCs, which will be important to further decipher the steps that lead to the migration activation of hMSCs. Our results could help to better engineer MSCs as improved cellular therapeutics in a cost-effective and readily applicable manner [42]. However, despite the encouraging outcomes regarding the use of MSCs in the management of tissue regeneration as reported by preclinical and clinical studies, many critical questions that remain should be addressed prior to their widespread clinical application. In conclusion, the ATX/LPA receptor axis stimulates hMSC migration through adherent junction disruption and cytoskeletal rearrangement via LPA receptor 1/3-dependent PKC/GSK-3β/β-catenin and PKC/Rho GTPase pathways. Acknowledgments This research was supported by National R&D Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2013M3A9B4076520). Author Contributions J.M.R.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; H.J.H.: administrative support, manuscript writing, final approval of manuscript, and financial support. 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TI - Autotaxin-LPA Axis Regulates hMSC Migration by Adherent Junction Disruption and Cytoskeletal Rearrangement Via LPAR1/3-Dependent PKC/GSK3β/β-Catenin and PKC/Rho GTPase Pathways
JO - Stem Cells
DO - 10.1002/stem.1882
DA - 2015-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/autotaxin-lpa-axis-regulates-hmsc-migration-by-adherent-junction-kKmA03yss8
SP - 819
EP - 832
VL - 33
IS - 3
DP - DeepDyve
ER -