TY - JOUR
AU - Armstrong, Lyle
AB - Abstract The function of the proteasome is essential for maintenance of cellular homeostasis, and in pluripotent stem cells, this has been extended to the removal of nascent proteins in a manner that restricts differentiation. In this study, we show enhanced expression of genes encoding subunits of the 20S proteasome in human embryonic stem cells (hESCs) coupled to their downregulation as the cells progress into differentiation. The decrease in expression is particularly marked for the alternative catalytic subunits of the 20S proteasome variant known as the immunoproteasome indicating the possibility of a hitherto unknown function for this proteasome variant in pluripotent cells. The immunoproteasome is normally associated with antigen-presenting cells where it provides peptides of an appropriate length for antibody generation; however, our data suggest that it may be involved in maintaining the pluripotency in hESCs. Selective inhibition of two immunoproteasome subunits (PSMB9 and PSMB8) results in downregulation of cell surface and transcriptional markers that characterize the pluripotent state, subtle cell accumulation in G1 at the expense of S-phase, and upregulation of various markers characterizing the differentiated primitive and definitive lineages arising from hESC. Our data also support a different function for each of these two subunits in hESC that may be linked to their selectivity in driving proteasome-mediated degradation of cell cycle regulatory components and/or differentiation inducing factors. Immunoproteasome, Proteasome, Human embryonic stem cells, Maintenance of pluripotency Introduction Embryonic stem cells (ESCs) have the potential to differentiate into almost any of the cell types in the adult organism (reviewed in [1]), and our previously published data support the concept of superior maintenance and repair systems in these cells to ensure genomic stability [2–4]. An essential feature of ESC biology is their ability to preserve their genomic integrity [5], and there are two principal mechanisms by which this may be achieved. First, spontaneous mutation frequencies in ESC must be suppressed by low levels of stress generation, high activities of stress defense, and high activity and fidelity of repair mechanisms. Second, ESC that accumulate mutations or DNA damage must be eliminated from the stem cell population by activation of checkpoint signaling that may lead to induction of apoptosis, cell cycle arrest, and/or stem cell differentiation [6]. Multiple analyses have shown that ESC express higher levels of antioxidant enzymes than their differentiated progeny and have more effective DNA repair systems [2–6] endowing ESCs with a considerable survival advantage by ensuring that the transcriptome remains within closely defined limits required by the range of permitted ESC functions. A well-maintained transcriptome is only the first step in cellular homeostasis since cell death or dysfunction can result from unrepaired damage to macromolecules such as proteins and cell membranes just as effectively as from DNA mutations. In view of this, eukaryotic cells have evolved defense mechanisms that complement the activity of the antioxidant enzymes, and these are aimed at redox-dependent repair of damaged proteins or elimination of these if the damage is too great to repair. Unrestricted accumulation of damaged proteins and aggregates that form subsequently disrupts cellular processes, therefore their rapid removal is a critical for the maintenance of cell viability. The proteasome is the major system responsible for the removal of oxidized and misfolded proteins [7, 8] by proteolytic cleavage. Several complexes now have been assigned the general title of proteasome, but they all contain the barrel-shaped 20S proteasome core particle in which the proteolytic activities reside, capped by a 19S regulatory structure at one or both ends of the 20S core to give a structure generally known as the 26S proteasome [9]. ESCs express the genes encoding most of the 26S proteasome subunits at high levels [10] with a surprising degree of similarity to the gene expression profile of the mammalian oocyte [11], suggesting a conserved and therefore important role in cell types that are either pluripotent or are capable of giving rise to pluripotent cells. The precise role of proteasome-based degradation of damaged or redundant proteins in ESCs still requires clarification, but some insights have been gained from studies showing dynamic turnover of transcription factors at the promoters of differentiation-associated genes that are not expressed at the pluripotent cell stage [12]. The highly permissive chromatin environment of pluripotent cells can potentially allow “leaky” expression, and it is important to remove transcription factors from promoter sequences. The proteasome also targets cryptic promoter elements to prevent nonspecific assembly of the preinitiation complex and spreading of the modified chromatin domain [12]. The 20S core comprises seven alpha and seven beta subunits in stacked rings with subunits occupying alternate (αββα) positions. Only three of the subunits possess catalytic activity, β1 (PSMB6), β2 (PSMB7), and β5 (PSMB5) [13]. The alpha subunits have no catalytic activity but are nonetheless essential for controlling access to the 20S core and for binding to regulatory elements such as the 19S “lid” or alternative regulators (PA28αβ [PSME1/2], PA28γ [PSME3], and PA200 [PSME4]). These are needed to activate the 20S proteasome by altering the structures of the alpha subunits to allow the entry of protein and peptides targeted for degradation. A specialized form of the 20S proteasome is generated by replacement of the catalytic beta subunits β1, β2, and β5 by β1i (LMP2, PSMB9), β2i (MECL-1, PSMB10), and β5i (LMP7, PSMB8) subunits, respectively, to form a variant known as the immunoproteasome [14, 15] so named because of its prevalence in cells capable of generating peptides for major histocompatibility complex 1 (MHC1) antigen processing. A further control complex exists, namely the 11S or PA28 (PSME) regulatory particle that can also bind to the ends of the 20S proteasome core, dramatically increasing its ability to degrade small peptides. Two components of the PA28 regulator, PA28α and PA28β, are also upregulated in response to γ-interferon, but the PA28 complex is unable to recognize ubiquitin-tagged proteins; therefore, it is unlikely to be able to unfold large proteins and drive them into the 20S proteasome for degradation. A further subunit of the PA28 regulator, PA28γ, exists and this appears to have several possibly unique functions concerning the degradation of the cell cycle proteins p1, p16INK4, and p19 [16]. The immunoproteasome has been linked to the PA28αβ (11s) regulator, but several key studies have indicated that both the 20S core and immunoproteasome structures are more likely to be responsible for the ATP-independent degradation of oxidized proteins than the 26S proteasome that actually appears to be largely unreactive with proteins that have been damaged in this manner [17–22]. Moreover, intact 20S particles isolated from somatic cells are able to degrade oxidized proteins [23], but importantly, the expression of immunoproteasome and 20S core subunits increases when these cells are subjected to oxidative stress such as the addition of hydrogen peroxide or nitric oxide to the culture medium [24]. This results in disassembly of the 26S proteasome that has been suggested to be a method to increase the numbers of free 20S proteasome complexes [25] and a corresponding increase in the proteolytic activities of the 20S and immunoproteasome. It is not absolutely clear how oxidized proteins are recruited to the 20S core, although one suggestion is that it may be able to recognize and interact with abnormally exposed hydrophobic regions present on the outer surfaces of the damaged proteins. Our previous investigations have indicated that the expression levels of several proteasome subunits genes undergo a marked downregulation upon differentiation of pluripotent cells; therefore, the basis of this work was a characterization of expression and function of proteasome subunit gene expression during differentiation of human ESCs (hESCs). This was extended to include analyses of proteasome activity during hESC differentiation and the response of these parameters to inhibition of catalytic subunits specific to the immunoproteasome variant of the normal 20S proteasome. Our data presented herein indicate an important role for the immunoproteasome in the maintenance of hESC homeostasis, cell cycle regulation, and pluripotency. Materials and Methods Culture and Differentiation of hESC The hESC line H9 (WiCell, Madison, WI) and two human induced pluripotent stem cell (hiPSC) lines (clone 1 and clone 4 [4]) were routinely passaged and maintained in hESC media on mitotically inactivated mouse embryonic fibroblast feeder layers [26]. One to two passages prior to experiments, hESCs were transferred to Matrigel-coated plates with feeder-conditioned media. Embryoid bodies (EBs) were formed by collagenasing hESC colonies from feeders and then incubating in differentiation medium (10% fetal bovine serum (FBS)) in low-attachment plates. Monolayer differentiation was performed by transferring fragments of hESC colonies to gelatin-coated T75 flasks and incubating with differentiation medium. RT-PCR Total RNA was extracted using TRIzol reagent (Invitrogen, Paisley, U.K., http://www.invitrogen.com) according to manufacturer's instructions. Following DNaseI treatment using RQ1 DNaseI (Promega, Mannheim, Germany, http://www.promega.com), cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen). Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed using SYBR GreenER qPCR SuperMix Universal mix (Invitrogen). All samples were analyzed using a Roche LC480 and were normalized to multiple housekeeping genes (G6PD, GAPDH, RPL13a, SDHA, and TBP). All primers were designed using the NCBI Primer-Blast Software and were optimized for use in quantitative PCR (qPCR) reactions and are displayed in Supporting Information Table S1. Cell Cycle Analysis hESCs were collected using Accutase (Chemicon, Temecula, CA, http://www.millipore.com/offices/cp3/temecula). Cell cycle analysis was performed using the CycleTest Plus DNA reagent kit (Becton Dickinson, Oxford, UK, http://www.bd.com/uk) with FACS Calibur (BD Biosciences, CA). The data were analyzed using ModFit software to generate percentages of cells in G1, S, and G2/M phases. Cell Surface Marker Staining Detection of human stem cell surface markers was performed by multicolor flow cytometry using the StemFlow Human Pluripotent Stem Cell Sorting and Analysis Kit (BD Biosciences). The kit uses fluorescent-conjugated antibodies against two human stem cell-specific, stage-specific embryonic antigen-3 (SSEA-3) and TRA-1-81, and a differentiation-specific, SSEA-1, cell surface markers. The manufacturer's protocol was followed, briefly; following incubation of hESCs with UK101 inhibitor for 96 hours, cells were treated with Accutase to obtain a single-cell suspension and counted. 2.5 × 105 cells per sample were fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature. Cells were washed and resuspended in 25 μl phosphate buffered saline (PBS). The cell suspension was incubated with 5 μl of the three antibodies at room temperature in the dark for 30 minutes. Cells were washed and resuspended in PBS. Stained cells were then analyzed on a FACS Canto II using FACSDiva software. Isotype controls conjugated to the same fluorochrome as the corresponding antibody were run alongside the antibody stained cells. BD CompBead Plus positive and negative beads were used to calculate fluorescence compensation. Western Immunoblotting Cell samples were washed with ice-cold PBS and lysed in RIPA buffer (50 mm Tris-HCl pH 8.0, 150 mm NaCl, 1% IGEPAL CA 630, 0.5% Na-DOC, and 0.1% SDS). Phenylmethylsulfonyl fluoride (PMSF) (1 mM) and Roche protease inhibitors were added before treatment of cells. After 30 minutes on ice, the lysates were homogenized and centrifuged at 13,000 rpm for 15 minutes. The total protein concentration was determined by Bradford Kit (Bio-Rad Laboratories Ltd., Hemel Hempstead, U.K., http://www.bio-rad.com) using the manufacturer's instructions. Lysates (10–20 μg total protein) were electrophoresed on a 10%–12% SDS–PAGE gel and electrophoretically transferred to a polyvinylidene difluoride membrane (Hybond-P [hydrophobic polyvinylidene difluoride membrane, cat no. RPN303F]; Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). Membranes were blocked in Tris-buffered saline with 5% milk and 0.1% Tween. The blots were probed with antibodies against PSMB5 (PW8895), PSMB6 (PW8140), PSMB7 (PW8145), PSMB8 (PW8845), PSMB9 (PW8205), PSMB10 (PW8350) (All; Biomol International, Exter, UK, http://www.enzolifesciences.com), CDK6 (sc-177), CDK2 (sc-163), CYCLIN E (sc-25303), p57 (sc-8298), p27 (sc-1641), p53 (sc-126), p21 (sc-6246), PCNA (sc-7907), CYCLIN A (sc-596), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Abcam) overnight and revealed with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibodies (Amersham Biosciences). Antibody/antigen complexes were detected using ECL (Amersham Biosciences), and images were acquired using a luminescent image analyzer FUJIFILM and LAS-3000 software (FUJI, Abingdon, U.K., www.rndsystems.com). Immunoproteasome Assays Undifferentiated hESCs and samples from EB differentiation were taken every 2 days through 16 days. Each sample was lysed in 500 μl of proteasome buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 mM ATP, 20% glycerol, 4 mM DTT, and then kept on ice for 30 minutes, vortexing every 10 minutes followed by brief sonication (2 × 20 seconds at 2.0 W on ice). Following centrifugation (13,000 rpm for 10 minutes at 4°C), supernatants were moved to fresh tubes and protein concentration adjusted to 1 mg/ml. Two micrograms of protein was added to 197 μl assay buffer (0.05 M Tris-HCl, 0.5 mM EDTA) for each assay using 40 μM of Bz-VGR-AMC (trypsin-like activity) (BW9375; Biomol International), Z-LLE-AMC (caspase-like activity) (ZW9345; Biomol International), and Suc-LLVY-AMC (chymotrypsin-like activity) (P802; Biomol International) in dimethylsulfoxide (DMSO) and 0.1 mM AMC (A9891; Sigma-Aldrich, Dorset, UK, http://www.sigmaaldrich.com/united-kingdom.html). Protein assays were incubated in the dark for 60 minutes at 37°C and then assayed in a luminometer for free-AMC detection (380 nm excitation and 440 nm emission). Samples were also tested again at 2 and 3 hours, and readings were adjusted to the free AMC control. RNA Interference hESCs were cultured under feeder-free conditions with feeder-conditioned media free of antibiotics for at least 4 days prior to transfections. PSMB8 and PSMB9 small interfering RNAs (siRNAs) were purchased from Invitrogen. The sequences are shown in Supporting Information Table S2. Transfection with a scrambled control siRNA was used as a negative control. Transfection of siRNAs into hESC was performed using the high efficiency nucleofection kit L from Amaxa (Amaxa Biosystems, Cologne, Germany, http:/amaxa.com) and 80 pmol siRNA (in 2 ml medium), as outlined in manufacturer's instructions (program A-023). PSMB8 and PSMB9 Inhibition PSMB9 was specifically inhibited using the UK101 inhibitor at concentration of 2 μM, while PSMB8 was specifically inhibited with PR-957 at a concentration of 2 μM. Cells were treated over a 6-day time course on Matrigel with conditioned medium with drug and medium changed every 24 hours. Control cells were treated with a similar amount of DMSO as drug. Cells were collected after 2, 4, and 6 days to be used for further analysis. Statistical Analysis Two-tailed pairwise Student's t test was used to analyze results obtained from two samples with one time point. The results were considered significant if p < .05. Results Expression of Immunoproteasome Subunit Encoding Genes Undergoes Downregulation During hESC Differentiation Analysis of the gene expression levels for proteasome subunit genes, known regulators, and chaperones presented in Supporting Information Figures S1–S8 showed different expression patterns during hESC differentiation. For example, analysis of PSMA gene expression (1-7, PSMA8 was undetectable) showed a 20%–30% reduction in expression during 16 days of EB-mediated differentiation, with only PSMA7 maintaining a steady state of mRNA expression during the differentiation process (Supporting Information Fig. S1). Expression of the majority of PSMB genes (PSMB1-10) showed similar reductions, but strikingly the expression of the immunoproteasome subunits PSMB8-TV1 and PSMB8-TV2 and PSMB9 (Supporting Information Fig. S2 and Fig. 1) was reduced dramatically during differentiation, similar to the kinetics seen for the reduction in mRNA levels of pluripotency markers such as NANOG and OCT4. PSMC gene [1–6] expression was stable (PSMC1-3), but some subunits showed up to 50% reduction in expression (PSMC4-6; Supporting Information Fig. S3). PSMD gene expression (PSMD1, PSMD2, PSMD11, PSMD12, and PSMD14) also showed some changes, with again a 40%–60% reduction in mRNA levels (Supporting Information Fig. S4). Among the PSME (Supporting Information Fig. S5), PSMF (Supporting Information Fig. S6), and PSMG (Supporting Information Fig. S7) clusters, the most interesting expression pattern was observed for PSME3 (an immunoproteasome subunit) and PSMG3 showing a 50%–60% reduction during hESC differentiation. Proteasome chaperones that mediate the construction of the proteasomes also showed similar changes to the other proteasome subunits. Proteasome maturation protein (POMP) is understood to play a major role in the formation of the 20S proteasome and seems to be overexpressed in ESC, where after its expression reduces akin to the other subunits (Supporting Information Fig. S8). As outlined above, the most striking observations are the rapid and extensive decrease in PSMB8 (transcript variants 1 and 2) and PSMB9 coupled to a rapid but transient increase in the expression of PSMB10 (Fig. 1). PSMB8 and 9 are of particular interest, since they encode the immunoproteasome subunits β5i (LMP7) and β1i (LMP2), whereas β2i (MECL-1) is encoded by PSMB10. These changes imply a significant change in 20S core proteasome composition and may suggest that the immunoproteasome variant has a role to play in pluripotent cells. 1 Open in new tabDownload slide Downregulation of immunoproteasome subunits PSMB8 and PSMB9 expression during human embryonic stem cell (hESC) differentiation. Expression analysis of PSMB5-10 subunits during hESC differentiation (embryoid body method) by quantitative real time RT-PCR. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each differentiation time point was compared to hESC (D denotes day during differentiation window). 1 Open in new tabDownload slide Downregulation of immunoproteasome subunits PSMB8 and PSMB9 expression during human embryonic stem cell (hESC) differentiation. Expression analysis of PSMB5-10 subunits during hESC differentiation (embryoid body method) by quantitative real time RT-PCR. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each differentiation time point was compared to hESC (D denotes day during differentiation window). To understand if PSMB8 and PSMB9 downregulation was a common phenomenon associated with hESC differentiation, we undertook PSMB5-10 expression analysis, using a 4-week monolayer differentiation. Monolayer differentiation of hESCs led to a 50% decrease in PSMB5, PSMB6, and PSMB7 over 4 weeks (Supporting Information Fig. S9A). In line with EB differentiation method, PSMB8 expression was reduced to 25%, although TV1 expression did recover by week 4, PSMB9 expression was reduced to 10% by week 2 while PSMB10 expression varied across the 4-week differentiation time course (Supporting Information Fig. S9B). A similar expression analysis in hiPSC (Supporting Information Fig. S10) corroborated the data generated in hESC and indicated that the decrease in PSMB8 and PSMB9 expressions at the mRNA level is a common phenomenon for PSC differentiation. Western analysis of the immunoproteasome subunits corroborates some of the changes seen at the mRNA level (Fig. 2). While PSMB5 protein remained unchanged through the 16 days differentiation, both PSMB6 and 7 seemed to have increased expression after day 4. PSMB8 expression decreased after day 6 of differentiation mimicking the downregulation observed at transcriptional level; however, low expression of this protein was observed toward the end of differentiation period (Fig. 2). The expression of mature form of PSMB9 underwent a slight upregulation at day 4 of differentiation, followed by downregulation from day 10 onward (Fig. 2). The PSMB10 gene product was not detectable by immunoblotting, albeit detectable at mRNA level (Fig. 1). It is interesting to note that although the PSMB8 and PSMB9 are both downregulated at the mRNA level, there is a different pattern in the way both of these proteins change their expression during hESC differentiation, suggesting perhaps a different requirement for these two immunoproteasome subunits in hESC. 2 Open in new tabDownload slide Expression analysis of PSMB5-10 during human embryonic stem cell (hESC) differentiation by Western immunoblotting. The images are representative of at least three independent experiments. GAPDH served as a loading control for all Western blots. Embryoid body method was used to induce hESC differentiation. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 2 Open in new tabDownload slide Expression analysis of PSMB5-10 during human embryonic stem cell (hESC) differentiation by Western immunoblotting. The images are representative of at least three independent experiments. GAPDH served as a loading control for all Western blots. Embryoid body method was used to induce hESC differentiation. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Proteolytic Activity of the 20S Proteasome Decreases During hESC Differentiation Three proteolytic activities (caspase-, trypsin-, and chymotrypsin-like activity) were analyzed by selective cleavage of fluorescent substrates specific to each proteolytic class (Fig. 3). Caspase-like activity showed a spike more than day 2 of differentiation while chymotrypsin-like activity was reduced over the first 6 days of differentiation and trypsin-like activity remained at the same level. This time-line corroborates results obtained by qPCR and immunoblotting, which suggest alteration in the catalytic subunits of the proteasome (Figs. 1, 2). It is possible that the observed reduction in these activities is simply due to the overall downregulation of 20S proteasome component gene expression; however, since the catalytically active constitutive 20S proteasome subunits (PSMB5, 6, and 7) seem to maintain constant (PSMB5) or increasing concentrations (PSMB6 and 7) while those of PSMB9 decrease, a partial or complete replacement of subunits, such as PSMB9, may explain the changes in proteolytic activity. The chymotrypsin-like site cleaves peptide bonds after hydrophobic residues, and the trypsin-like site cuts after basic residues, whereas the caspase-like site cuts preferentially after acidic residues [27]. It is not clear why a different profile of enzymatic activities is needed in the differentiating cells, but this may be related to a change in the type of substrates requiring proteasome-mediated degradation during hESC differentiation. 3 Open in new tabDownload slide Assessment of proteolytic activities (caspase-, trypsin-, and chymotrypsin-like activity) during human embryonic stem cell (hESC) differentiation. Embryoid body method was used to induce hESC differentiation. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each differentiation time point was compared to hESC (D denotes day during differentiation window). 3 Open in new tabDownload slide Assessment of proteolytic activities (caspase-, trypsin-, and chymotrypsin-like activity) during human embryonic stem cell (hESC) differentiation. Embryoid body method was used to induce hESC differentiation. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each differentiation time point was compared to hESC (D denotes day during differentiation window). Cell Cycle-Specific Expression of Immunoproteasome Subunits Proteasomal degradation of cell cycle regulators is an important level of regulation [28]. Our work and others have shown that hESCs display a short G1 and an extended S-phase, regulation of which is tightly linked to maintenance of pluripotency [29, 30]. For this reason, we sought to understand if PSMB8 and PSMB9 expression was linked to cell cycle regulation. hESCs were synchronized in different stages of cell cycle using mitotic inhibitors as described in one of our previous publications [31]. Expression analysis showed that while PSMB5, PSMB6, and PSMB7 mRNA expression remained similar throughout the cell cycle (Fig. 4A), PSMB8, PSMB9, and PSMB10 expression seemed to be specifically expressed at a higher level during G1 phase of the cell cycle (Fig. 4B). Western immunoblotting indicated a higher expression of PSMB8 in S and G2/M phase of the cell cycle (Fig. 4C). Higher expression of the mature form of PSMB9 was observed in S-phase of the cell cycle (Fig. 4C). This is not surprising given that G1 duration is short in hESC (2.5–4 hours; [31]) and a few hours may be needed to proceed from mRNA to protein synthesis. Cell cycle stage-specific expression of PSMB8 and PSMB9 may suggest a role for the immunoproteasome system in rapid processing of key cell cycle components needed for regulation of cell cycle in hESC. 4 Open in new tabDownload slide Cell cycle-specific expression of immunoproteasome subunits. (A, B): Expression analysis of PSMB5-10 subunits in unsynchronized and synchronized human embryonic stem cell (hESC) by quantitative real time RT-PCR. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test. (C): Expression of PSMB8 and 9 at each stage of cell cycle by Western blotting. Represented images are shown (n = 3). Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 4 Open in new tabDownload slide Cell cycle-specific expression of immunoproteasome subunits. (A, B): Expression analysis of PSMB5-10 subunits in unsynchronized and synchronized human embryonic stem cell (hESC) by quantitative real time RT-PCR. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test. (C): Expression of PSMB8 and 9 at each stage of cell cycle by Western blotting. Represented images are shown (n = 3). Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To further analyze the link between PSMB8 and PSMB9 and cell cycle regulation in hESC, we performed expression analysis in NANOG overexpressing cells (Fig. 5), which we have reported to undergo a faster G1/S and display an enhanced proliferation [30]. While PSMB5, PSMB6, and PSMB7 expression remained at a similar level in H9, and the NANOG overexpressing cells (Fig. 5A), PSMB8-TV1/TV2, PSMB9, and PSMB10, expression showed a significant increase in NANOG overexpressing hESC (Fig. 5B). At the mRNA level, PSMB8 and PSMB9 are upregulated in G1. In view of this, it is unlikely that the increased expression of PSMB8 and PSMB9 in the NANOG overexpressing hESC reflects the change in hESC subpopulation (containing more cells in S-phase). It is more likely that higher expression of PSMB8 and 9 in NANOG overexpressing cells reflects a role for PSMB8 and PSMB9 in regulating S-phase in hESC. 5 Open in new tabDownload slide Enhanced expression of PSMB8, PSMB9, and PSMB10 subunits in NANOG overexpressing human embryonic stem cell (hESC) by quantitative real time RT-PCR. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each differentiation time point was compared to control hESC line. Abbreviation: OE, overexpressing lines. 5 Open in new tabDownload slide Enhanced expression of PSMB8, PSMB9, and PSMB10 subunits in NANOG overexpressing human embryonic stem cell (hESC) by quantitative real time RT-PCR. The value for the hESC was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each differentiation time point was compared to control hESC line. Abbreviation: OE, overexpressing lines. Inhibition of PSMB8 and PSMB9 Results in Subtle Loss of Pluripotency Markers and Changes in Cell Cycle Regulation in hESC We reasoned that if immunoproteasome was to play a significant role in maintenance of pluripotency and regulation of cell cycle, loss of the PSMB8 and PSMB9 subunits in a similar manner to that observed early in hESC differentiation might be sufficient to induce differentiation. Single (data not shown) and dual PSMB8 (transcript variants 1 and 2) and PSMB9 RNAi-mediated knockdown (Supporting Information Fig. S11A) resulted in substantial decrease of mRNA expression (50%, 95%, and 90% loss of mRNA at 24 hours and 50%, 60%, and 90%, respectively, at 48 hours post-RNAi transfection). However, wide ranging mRNA analysis of pluripotency and differentiation markers showed either very little change (Supporting Information Fig. S11B) or no change at all. This may be due to the transient nature of RNAi-mediated knockdown, functional redundancy between proteasome subunits, or rather stable expression of PSMB8 and PSMB9 proteins. The later possibility is corroborated by Western immunoblotting data of Figure 2 showing a longer expression in time for both PSMB8 and 9 when compared with their relative transcript expression in Figure 1. To work around this possibility, we pursued a different inhibition study using two small-molecule inhibitors: UK101 [32] that specifically inhibits PSMB9 activity and PR-957 that specifically inhibits PSMB8 activity [33]. hESCs were treated more than 6 days with UK101 and PSMB8 inhibitor with medium and drug added fresh every day. When UK101 was applied, cell morphology remained at a similar level, but significant changes in the expression of pluripotency-associated genes, differentiation-associated genes, and proteasome genes were observed. NANOG and OCT4-TV1 expression was consistently decreased by 20%–25% at days 2, 4, and 6 (Fig. 6A, 6B). We also observed a significant increase in expression of differentiation associated genes that mark development of embryonic and extraembryonic lineages, such as BRACHYURY, CDH3, FGF5, NESTIN, PAX6, and GATA4 (Fig. 6C). Analysis of cell cycle regulation indicated a small but significant and reproducible accumulation of cells in G1 at the expense of reduction in S-phase of the cell cycle (Fig. 7A) as well as a reduction in numbers of cells expressing the typical pluripotency cell surface markers TRA-1-81+/SSEA-1− (84.3% in the control vs. 68.63% in the UK101-treated group, n = 3) and TRA-1-81+/SSEA3+ (83.7% in the control vs. 70.3% in the UK101-treated group, n = 3). Alkaline phosphatase staining of colonies at day 4 after treatment with UK101 inhibitor showed a significant decline in the percentage of alkaline phosphatase-positive colonies (82% in the control vs. 52.6% in the UK101-treated group, n = 4; p < .005). 6 Open in new tabDownload slide Inhibition of PSMB9 by small-molecule inhibitor, UK101, results in downregulation of OCT4 and NANOG expression and upregulation of differentiation markers. Expression analysis of NANOG (A), OCT4 (B), and lineage markers (C) by quantitative real time RT-PCR. The value for the control group (DMSO) was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each treatment time point was compared to control group. D denotes day during post-treatment with inhibitor. 6 Open in new tabDownload slide Inhibition of PSMB9 by small-molecule inhibitor, UK101, results in downregulation of OCT4 and NANOG expression and upregulation of differentiation markers. Expression analysis of NANOG (A), OCT4 (B), and lineage markers (C) by quantitative real time RT-PCR. The value for the control group (DMSO) was set to 1 and all other values were calculated with respect to that. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each treatment time point was compared to control group. D denotes day during post-treatment with inhibitor. 7 Open in new tabDownload slide Inhibition of PSMB9 by small-molecule inhibitor, UK101, results in subtle changes in cell cycle profile and loss of pluripotency markers. (A): Cell cycle changes analyzed by flow cytometry in control-and UK101-treated group at day 4 after application of UK101. Data are presented as mean ± SEM (n = 4). Statistical analysis was performed using Student's t test where each differentiation time point was compared to control group; (B): expression analysis of PSMB5-10 expression by quantitative real time RT-PCR in UK101- and control-treated group. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each treatment time point was compared to control group. 7 Open in new tabDownload slide Inhibition of PSMB9 by small-molecule inhibitor, UK101, results in subtle changes in cell cycle profile and loss of pluripotency markers. (A): Cell cycle changes analyzed by flow cytometry in control-and UK101-treated group at day 4 after application of UK101. Data are presented as mean ± SEM (n = 4). Statistical analysis was performed using Student's t test where each differentiation time point was compared to control group; (B): expression analysis of PSMB5-10 expression by quantitative real time RT-PCR in UK101- and control-treated group. Data are presented as mean ± SEM (n = 3). Statistical analysis was performed using Student's t test where each treatment time point was compared to control group. Application of PSMB8 inhibitor also resulted in reduction in NANOG and OCT4-TV1 expression (Supporting Information Fig. S12A). In contrast to application of PSMB9 inhibitor, we observed a decrease in expression of BRACHUYRY, CDH3, and NESTIN suggesting an inhibition of spontaneous differentiation toward primitive mesodermal, trophoectodermal, and ectodermal lineages. A significant increase in expression of FGF5 and GATA4 was noticed, suggesting that PSMB8 inhibition encourages differentiation to primitive ectodermal and endodermal lineages (Supporting Information Fig. S12A). Analysis of cell cycle regulation indicated a small but significant and reproducible accumulation of cells in G1 at the expense of reduction in S-phase of the cell cycle (Supporting Information Fig. S12B). Application of PSMB9 inhibitor also resulted in a reduction in numbers of cells expressing the typical pluripotency cell surface markers TRA-1-81+/SSEA-1− (84.3% in the control vs. 75.53% in the PSMB9 inhibitor-treated group, n = 3) and TRA-1-81+/SSEA3+ (83.7% in the control vs. 72.8% in the PSMB9 inhibitor-treated group, n = 3). Alkaline phosphatase staining of colonies at day 4 after treatment with PSMB9 inhibitor showed a significant decline in the percentage of alkaline phosphatase-positive colonies (82% in the control vs. 49.1% in the PSMB9 inhibitor-treated group, n = 4; p < .005). Taken together, these data suggest that PSMB8 and PSMB9 inhibition affect maintenance of hESC pluripotency and encourage hESC differentiation, albeit this may be achieved via different mechanisms. To understand further how PSMB8 and PSMB9 inhibition may affect cell cycle regulation and in particular the accumulation of cells in G1, we performed Western immunoblotting for expression of key cell cycle regulators and inhibitors (Supporting Information Fig. S12C). This indicated that application of PSMB9 inhibitor resulted in an increase in CYCLIN E expression, suggesting that this could be one of the cell cycle component targeted by the proteasome degradation system. Application of PSMB8 inhibitor instead resulted in the increase in expression not only of CYCLIN E but also of p53 and its transcriptional target, cell cycle inhibitor p21 (Supporting Information Fig. S12C). With this data, we cannot establish whether cell accumulation in G1 occurs prior to hESC differentiation and is thus the main causes for the differentiation to occur. Work performed in our group has shown that activation of p53 and subsequently p21 in hESC results in cell accumulation in G1 and rapid differentiation to trophoectodermal lineages [34]. Increase in p53 expression upon PMSB8 inhibition together with similarities in cell cycle regulation and differentiation phenotype suggests that p53 may indeed be a target of PSMB8 driven degradation in hESC, although this has to be proven experimentally. It must be noted that most of the changes observed in both gene expression and cell cycle regulation after PSMB9 inhibition, although significant, are not drastic (at the level of 20%–25%). This can be perhaps explained by functional redundancy within the immunoproteasome or presence of a feedback loop that results in increases in activity of other 20S proteasome subunits upon PSMB9 inhibition. The latter was confirmed by our expression analysis, PSMB5-10 gene expression analysis, which indicated a significant upregulation of all components (Fig. 7B). Such an increase in expression and potential activity of other proteasome subunits can dampen the effects of PSMB9 inhibition by UK101 and can be the reason behind the significant but less drastic effects observed in lack of pluripotent marker expression and cell cycle regulation reported above. Discussion A number of publications have highlighted the importance of the 26S proteasome for the maintenance of the pluripotent phenotype of ESCs [12, 35, 36], although the mechanism by which proteolytic cleavage helps to prevent ESC differentiation is not clear. It has been suggested that targeted assembly of the 26S proteasome subunits at tissue-specific genomic loci promotes a dynamic turnover of transcription factor and RNA polymerase II binding that in turn prevents leaky expression from these loci. The genes subjected to this mechanism tend to be located in regions of open chromatin so the transcriptional machinery of the cell enjoys easier access to the genes information content than in some other genomic loci so the mechanism involving the proteasome was invoked to explain how some genes could remain potentiated for rapid upregulation at the appropriate stage of differentiation while avoiding inappropriate expression at the undifferentiated stage. Our previous studies have shown that human and murine ESC possess enhanced stress defense mechanisms [3, 4]. Such mechanisms focus mostly on restricting the damage caused by reactive oxygen species; however, the early stages of murine ESC differentiation are accompanied by a marked decrease in the levels of proteins carrying oxidative carbonylation damage [37]. We have made a similar observation using hESC (data not shown) perhaps suggesting that the removal of damaged proteins is required for differentiation. This phenomenon is also observed during mouse development (blastocyst to trophoectoderm). Removal of damaged proteins is an important process as high levels of accumulated protein damage are indicative of cellular dysfunction, disease, and senescence. Therefore, hESC or even the cells of the inner cell mass of the blastocysts may require the removal of protein damage for proper function of the daughter cells. Since the 20S proteasome is the primary mechanism through which damaged or dysfunctional proteins are degraded, then the observed elimination of carbonylated proteins during mouse ESC/hESC differentiation may reflect changes in the processing capacity or substrate specificity of the proteasome. Immunoproteasome subunits are overexpressed in hESC compared to somatic cells derived from hESC differentiation. Our data do indicate that both the trypsin-like and chymotrypsin-like activities undergo similar decreases during ESC differentiation. The immunoproteasome, the 20S proteasome, and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes [38], and increased expression of PSMB8 and PSMB9 is observed in retina in response to noninflammatory injury [39]; therefore, the putative higher immunoproteasome activity of hESC may be linked to their enhanced oxidative stress defenses. In addition, there is the possibility that immunoproteasome activity has some hitherto unknown role in the maintenance of pluripotency. Indeed data presented in this manuscript indicate that inhibition of PSMB9 and PSMB8 does seem to alter the cell cycle profile and expression of pluripotency-associated genes in addition to increasing the expression of certain genes involved in the early differentiation of hESC. Viable and fertile mice lacking one or more of the inducible immunoproteasome subunits have been generated [40, 41]. If knockout models of immunoproteasome function in mouse are not embryonic lethal in all cases, this could imply that the proposed pluripotency maintenance function in ESC is not involved in embryonic development or its loss can be compensated by redundant systems. There is little data describing the efficiency of generating PSMB8/PSMB9 knockout mice so we cannot determine if the lack of an immunoproteasome influences embryonic survival, and the relative amounts of damaged proteins accumulating in the inner cell mass of PSMB8/PSMB9 knockout blastocysts have not been determined. It may be envisaged that the immunoproteasome functions in the rapid turnover of factors involved in pluripotent cell cycle progression and that inhibition of its proteolytic activity may lengthen G1 to the point that the hESCs are forced to begin differentiation [42]. This is corroborated by data presented in this manuscript and showing that inhibition of PSMB8 results in an increase in expression of CYCLIN E, p53, and p21, suggesting that probably both p53 and CYCLIN E are targets of immunoproteasome degradation in hESC. Although activation of p53 and subsequently p21 is expected to cause hESC accumulation in G1 as shown in one of our recent manuscripts [34], the increase in CYCLIN E expression is more likely to result in acceleration of G1 to S entry [6]. The contradictory action between stalling (p53/p21) and promoting G1/S entry (CYCLIN E) can be the main underlying factor behind the subtle changes observed in cell cycle regulation of hESC upon inhibition of PSMB8. We cannot determine at this stage whether hESC stalling in G1 is the main factor causing hESC differentiation; however, the similarity in cell differentiation phenotype between hESC with activated p53 [34] and the ones described herein upon inhibition of PSMB8 suggests that is likely to be the case. Current work in our group is investigating this further using double inhibition of p53 and PSMB8. Our current data also suggest that the numbers of cells in G1 phase increase upon PMSB9 inhibition although the ensuing cell differentiation events are more widespread across the lineages and different to the ones we observed upon PSMB8 inhibition, suggesting different mechanisms of action in hESC, which is also corroborated by their different pattern of protein expression during hESC differentiation. Unlike inhibition of PSMB8, PMSB9 inhibition only results in CYCLIN E expression that is expected to promote G1/S entry; instead, we observe a subtle hESC accumulation in G1, suggesting that in this case, hESC differentiation and not G1 stalling is perhaps the first event and may be linked to role of PSMB9 in driving proteasome-dependent degradation of differentiation promoting factors. Indeed, a recent report has shown the existence of phosphorylated form of Klf4 (Ser 123) that induces ESC differentiation. This phosphorylation event though causes recruitment of components of ubiquitin E3 ligase resulting in degradation of this specific Klf4-differentiation inducing in ESC [43]. It could be envisioned that PSMB9 is involved in similar proteasome-mediated degradation events and therefore its inhibition can result in accumulation of differentiation promoting factors. In support of this hypothesis, it is also interesting to note that the promoter regions of PSMB8, PSMB9, and PSMB10 show binding sites for NANOG, OCT4, and SOX2 (11 sites), whereas PSMB5, 6, and 7 have relatively few which may imply a greater degree of control over immunoproteasome subunits in pluripotent cells. This is also supported by recent data generated in our group and showing that application of PSMB8 and PSMB9 inhibitors during iPSC-mediated reprogramming using a polycystronic OSKM vector [44] abolishes formation of pluripotent colonies (control group = 0.7%, UK101 and PR-957 = 0%). Conclusions In conclusion, we have provided data supporting a role for the immunoproteasome in the maintenance of hESC pluripotency and cell cycle regulation albeit via different action mechanism for different components of the immunoproteasome. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: S.P.A. and J.C.: collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; I.N.: provided necessary reagents for this work, data analysis and interpretation, and final approval of manuscript; G.A.: performed some of the work and final approval of the manuscript; K.B.K.: provided necessary reagents for this work and final approval of manuscript; M.L. and L.A.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript. S.P.A. and J.C. contributed equally to this article. Disclosure of potential conflicts of interest is found at the end of this article. First published online in STEM CELLSEXPRESS April 24, 2012. Telephone: 44-191-241-8695; Fax: 44-191-241-8666; Copyright © 2012 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - A Putative Role for the Immunoproteasome in the Maintenance of Pluripotency in Human Embryonic Stem Cells
JF - Stem Cells
DO - 10.1002/stem.1113
DA - 2012-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-putative-role-for-the-immunoproteasome-in-the-maintenance-of-N7giJ4NGuO
SP - 1373
EP - 1384
VL - 30
IS - 7
DP - DeepDyve
ER -