The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function
2021; Springer Nature; Volume: 22; Issue: 10 Linguagem: Inglês
10.15252/embr.202052301
ISSN1469-3178
AutoresAnna Geueke, Giada Mantellato, Florian Kuester, Peter Schettina, Melanie Nelles, Jens M. Seeger, Hamid Kashkar, Catherin Niemann,
Tópico(s)Mesenchymal stem cell research
ResumoArticle2 August 2021Open Access Transparent process The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function Anna Geueke Anna Geueke Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Giada Mantellato Giada Mantellato Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Florian Kuester Florian Kuester Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Peter Schettina Peter Schettina Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Melanie Nelles Melanie Nelles Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Jens Michael Seeger Jens Michael Seeger Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), CECAD Research Center, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany Search for more papers by this author Hamid Kashkar Hamid Kashkar orcid.org/0000-0003-2796-1429 Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), CECAD Research Center, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany Search for more papers by this author Catherin Niemann Corresponding Author Catherin Niemann [email protected] orcid.org/0000-0002-7224-6216 Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Anna Geueke Anna Geueke Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Giada Mantellato Giada Mantellato Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Florian Kuester Florian Kuester Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Peter Schettina Peter Schettina Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Melanie Nelles Melanie Nelles Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Jens Michael Seeger Jens Michael Seeger Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), CECAD Research Center, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany Search for more papers by this author Hamid Kashkar Hamid Kashkar orcid.org/0000-0003-2796-1429 Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), CECAD Research Center, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany Search for more papers by this author Catherin Niemann Corresponding Author Catherin Niemann [email protected] orcid.org/0000-0002-7224-6216 Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany Search for more papers by this author Author Information Anna Geueke1,2, Giada Mantellato1,2, Florian Kuester1, Peter Schettina1, Melanie Nelles1, Jens Michael Seeger3, Hamid Kashkar1,3 and Catherin Niemann *,1,2 1Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 2Centre of Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany 3Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), CECAD Research Center, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany *Corresponding author. Tel: +49 221 47889511; E-mail: [email protected] EMBO Reports (2021)22:e52301https://doi.org/10.15252/embr.202052301 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti-apoptotic Bcl-2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl-2 antagonist ABT-199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro-apoptotic BH3-only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl-2 and the marked susceptibility to Bcl-2 antagonism. In line with these observations, conditional knockout of Bcl-2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl-2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl-2-induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche-instructive mechanism of Bcl-2-regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis. Synopsis Anti-apoptotic Bcl-2 protein function is required to maintain a hair follicle subpopulation of stem cells in murine skin. Blocking cell death by Bcl-2 overexpression affects the hair follicle stem cell niche and hair follicle regeneration. Systemic blocking of Bcl-2 specifically depletes supra-basal hair follicle stem cells. Loss of supra-basal stem cells interferes with the hair follicle regeneration cycle. Ectopic Bcl-2 expression results in the accumulation of quiescent stem cells and affects hair regeneration. Bcl-2 overexpression changes the hair follicle stem cell compartments and accelerates skin tumour formation. Introduction In most tissues, including the mammalian skin, SCs exist in heterogeneous states and localize to specific niches. On a functional level, SC quiescence, self-renewal and differentiation as well as the elimination of damaged SCs have to be tightly regulated to protect the tissue from degeneration or cancerous overgrowth. While it is well established that the niche instructs the activities of tissue-resident stem and progenitor cells (Morrison & Scadden, 2014; Yu et al, 2018), it is not sufficiently understood how SCs translate the input of their niche signals into an appropriate and robust molecular response. Epithelial SCs in hair follicles (HFs) are an excellent model to dissect the relevant niche factors and positional cues coordinating SC function. HFs undergo cycles of growth (anagen), regression (catagen) and rest (telogen) (Paus & Cotsarelis, 1999) (Fig EV1A). The cyclic transition from telogen to anagen relies on niche signals, such as Wnt, BMP, Shh and FGF (Kishimoto et al, 2000; Rendl et al, 2008; Greco et al, 2009; Woo et al, 2012), and is fuelled by SCs residing in the HF bulge, an anatomical niche found in the lower permanent part of HFs (Lavker et al, 2003). Click here to expand this figure. Figure EV1. Bcl-2 inhibition causes SC-specific cell death and loss of suprabasal BSCs Schematic of the hair cycle phases anagen (growth), catagen (regression) and telogen (rest). HF Schematic focusing on basal and suprabasal bulge SCs (bBSCs, sbBSCs), the hair germ (HG) and the dermal papilla (DP). IFE, interfollicular epidermis; SG, sebaceous glands. Timeline experimental ABT-199 treatment (P50) and analysis (P51) in telogen and Immunofluorescence staining of telogen tail whole mounts for Keratin 15 (Krt15) and active caspase-3 (aCas3) in control (Ctr) and Bcl-2 inhibitor (ABT-199)-treated mice. B, bulge; HF, hair follicle; SG, sebaceous gland; dashed lines, sebaceous glands. Scale bar, 100 µm. Quantification of tail HF with aCas3+ cells in bulge and hair germ of control (Ctr) and ABT-199-treated mice (n = 4 biological replicates; mean ± standard deviation (SD); ***P < 0.001, ns=not significant, two-tailed unpaired Student's t-test. Immunofluorescence staining of telogen tail whole mounts for tyrosinase-related protein-2 (Trp2) in wild-type (wt) animals (n = 3 mice). SG, sebaceous gland; B, bulge; HG: hair germ; dashed lines: sebaceous glands. Scale bar, 100 µm. Model for Bcl-2-mediated protection of suprabasal BSCs from apoptosis. Download figure Download PowerPoint It has been shown that SCs frequently comprise a heterogeneous cell population (Donati & Watt, 2015; Joost et al, 2016). How SC heterogeneity is orchestrated on the cellular and molecular levels and how it affects tissue function are currently not well understood. Previous reports showed that slow-cycling label-retaining cells (LRCs) are present in two prominent HFSC populations, basal and suprabasal bulge SCs (bBSCs, sbBSCs) (Cotsarelis et al, 1990; Blanpain et al, 2004) (Fig EV1B). CD34+/itga6low sbBSC and neighbouring CD34+/itga6high bBSCs are similar with both SC populations able to self-renew in vitro and to generate new HFs when grafted (Blanpain et al, 2004). Until now, the molecular mechanisms underlying a position-encoded bulge SC heterogeneity that lead to functional differences in tissue maintenance have not been reported. The anti-apoptotic Bcl-2 protein is a critical component of the intrinsic (mitochondrial) apoptosis and is highly expressed in HF BSCs (Sotiropoulou et al, 2010). The members of the Bcl-2 protein family control mitochondrial outer membrane permeabilization (MOMP) which results in cytochrome c release and triggers the activation of the initiator caspase-9 and executioner caspase-3 and caspase-7 (Taylor et al, 2008; Galluzzi et al, 2018). In the mammalian skin, Bcl-2 expression has been associated with increased resistance of BSCs to DNA damage-induced cell death (Sotiropoulou et al, 2010). In this study, we investigate the physiological function of Bcl-2 for BSCs, the relevance of BSC-specific Bcl-2 regulation and its role for SC-driven skin regeneration and tumour formation. Results Bcl-2 protects suprabasal bulge SCs To investigate the role of Bcl-2 in BSC homeostasis, we systemically treated mice with a highly selective Bcl-2 antagonist ABT-199/venetoclax which is currently implemented in cancer therapy (Souers et al, 2013; Thijssen & Roberts, 2019). Remarkably, a single i.p. injection of ABT-199 during the resting phase of the hair cycle (postnatal day, Pd50; Fig EV1A) resulted in apoptosis in HF BSCs. Active caspase-3 (aCas3) staining was detected exclusively in the lower part of HFs, including the HF bulge and hair germ (HG), but not in other SCs or tissue locations (Fig 1A and B). More detailed analysis showed that specifically sbBSCs, a subpopulation of BSCs between the new HF and the club hair, were aCas3 positive in ABT-199-treated skin (Figs 1A and EV1B). FACS analysis of BSCs revealed the specific loss of CD34+/Itga6low sbBSCs upon ABT-199 treatment (Fig 1C and D). Quantification showed a dramatic increase (˜ 60%) in HFs containing apoptotic BSCs following treatment with ABT-199 compared with controls (Fig 1B). ABT-199 treatment did not increase the number of HF with apoptotic cells in the HG (Fig 1B). Importantly, no aCas3+ cells were detected in the dermal papilla (DP), a cluster of specialized stromal fibroblasts that are crucial for the initiation of new HF growth (Fig 1B). Notably, apoptosis of BSCs was also increased in HFs of tail epidermis after ABT-199 treatment, where HFs differ in their architecture (Duverger & Morasso, 2009) (Fig EV1C and D), suggesting that tail BSCs also rely on a Bcl-2-mediated protection mechanism. Different body regions may have developed a general mechanism of safeguarding and maintaining HF SCs. Our findings showed that sbBSCs were distinct from bBSCs in their susceptibility to ABT-199 revealing SC specificity in dependency on anti-apoptotic factors. Next, we tested whether Bcl-2 is differentially expressed in the HF SC populations, which showed that the highest expression of Bcl-2 was in BSCs when compared to isolated Lrig1+ve SCs of the HF junctional zone (JZ) or hair germ (HG) progenitors (Appendix Fig S1B and C). Importantly, the expression of Bcl-2 was particularly high in sbBSCs when compared to bBSCs and non-bulge cells (non-BSCs) (Fig 2A). Further, analysis of Bcl-xL revealed that this anti-apoptotic factor is not differently expressed in sbBSCs compared with bBSCs and non-BSCs, suggesting that Bcl-xL does play a major role in this context (Appendix Fig S1A). Moreover, a number of previous studies already excluded a relevant alteration in platelets, shown to be dependent on Bcl-xL, both in patient and in mice after exposure to ABT-199 (Mason et al, 2007; Souers et al, 2013; Vandenberg & Cory, 2013; Vogler et al, 2013; Debrincat et al, 2015; Ganzel et al, 2020). We thus conclude that the intrinsically high level of Bcl-2 in CD34+/Itga6low sbBSCs may underlie their specific ABT-199 susceptibility. Figure 1. Differential requirement for Bcl-2 preventing suprabasal BSC loss A. Treatment scheme of experimental timeline and Immunofluorescence staining of telogen back skin hair follicles (HF) for Integrin α6 (Itgα6, red), active caspase-3 (aCas3, green) and DAPI (blue) in control (Ctr) and Bcl-2 inhibitor (ABT-199)-treated mice. B, bulge; CH, club hair; DP, dermal papilla; HF, hair follicle; HG, secondary hair germ. Scale bar, 50 µm. B. Quantification of back skin HF with aCas3+ cells in bulge, hair germ and dermal papilla of Ctr and ABT-199-treated mice (n = 4 mice biological replicates; mean ± standard deviation (SD); ***P < 0.001, ns, not significant, two-way ANOVA test). nd, not detected. C, D. Treatment scheme, FACS plot (C) and quantification (D) of keratinocytes stained for CD34 and Itgα6 after 6 days of treatment with vehicle solution (Ctr) or ABT-199 (n = 6 biological replicates; mean ± standard deviation [SD]; **P < 0.01, ns = not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs and n-B (non-bulge epidermal cells). **P < 0.005. Download figure Download PowerPoint Figure 2. Loss of suprabasal BSCs delays cyclic hair regeneration qRT–PCR for Bcl-2 mRNA expression in sorted n-B (non-Bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs; n = 3 biological replicates; mean ± standard deviation [SD]; ****P < 0.0001, ns=not significant, two-way ANOVA test). Treatment scheme of experimental timeline and histology of back skin sections of control (Ctr) and ABT-199-treated mice. Scale bar, 200 µm. Treatment scheme and hair-coat recovery monitored for 5 weeks following daily treatment (Pd50–Pd55) with vehicle solution (Ctr) and ABT-199. qRT–PCR for mRNA expression of the transcription factors Lef1 and Runx1 in epidermis of ABT-199-treated and control (Ctr) mice at P77 as shown in (B) (n = 3 biological replicates; mean ± standard error of mean (SEM); **P < 0.01, ns=not significant, two-tailed unpaired Student's t-test). Download figure Download PowerPoint Cyclic hair regeneration is regulated by Bcl-2 suprabasal bulge SCs Given that BSCs drive cyclic hair renewal by initiating a new anagen growth phase (Rompolas & Greco, 2014) (Fig EV1A), we next tested the role of sbBSCs in HF regeneration and investigated the physiological relevance of the sbBSC Bcl-2 dependency. Therefore, we treated mice with ABT-199 during the telogen resting phase to deplete sbBSCs (Pd50) and analysed skin samples during the following anagen phase (Pd77; Figs 2B and EV1B). Depletion of sbBSCs by ABT-199 significantly delayed hair growth for up to 3 weeks (Fig 2C; Appendix Fig S2A). In contrast to the normal hair growth observed in control mice at 3 weeks after treatment, ABT-199 treatment efficiently blocked telogen to anagen transition in the back skin and tail epidermis (Fig 2B; Appendix Fig S2B). This observation was supported by reduced expression of Lef1 and Runx1, transcription factors associated with hair growth, in mice treated with ABT-199 (Fig 2D) (Merrill et al, 2001; Hoi et al, 2010). Interestingly, ABT-199 treatment also resulted in hair greying (Fig 2C). This observation is in line with previous data from Bcl-2 knockout mouse models demonstrating the importance of Bcl-2 for melanocyte survival and skin pigmentation and most likely occurs independent from sbBSC loss (Yamamura et al, 1996; Mak et al, 2006). In this context, we detected a high number of melanocytes as shown by immunofluorescence staining for Trp2 (Fig EV1E) indicating that cell death detected in the HG may reflect apoptotic melanocytes. Together our data indicated that sbBSCs, and thus the composition and architecture of the BSC compartment, are important for anagen initiation and cyclic hair regeneration. The results also suggested that Bcl-2 dependency of a SC subpopulation is crucial for tissue homeostasis. To examine whether the Bcl-2 dependency of BSCs changes during the different stages of the hair cycle, mice were treated with ABT-199 in anagen (Pd70) and analysed during the same anagen phase 1 week later (Pd77; Fig EV1A, Appendix Fig S2C). Histological analysis of skin tissue sections revealed that progression through anagen was not affected. Although the expression of transcription factors Lef1 and Runx1 showed some variability between individual mice during the anagen phase after ABT-199 treatment, it was not significant (Appendix Fig S2D). In addition, ABT-199 treatment during anagen did not result in increased apoptosis (Appendix Fig S2E). Given that HF morphology, including the sbBSC compartment, is dramatically re-modelled during anagen (Hsu et al, 2011), the data support Bcl-2-mediated protection of sbBSCs during telogen but not anagen. To investigate whether Bcl-2 genetic deletion from the epidermis affects BSCs, we generated Bcl-2 epidermal knockout mice (Bcl-2EKO) by crossing K14Cre (Hafner et al, 2004) with Bcl-2fl/fl animals (Thorp et al, 2009). Similar to the ABT-199 experiments, apoptotic cells were increased in the HF bulge and the number of HFs showing aCas3+ BSCs rose from 10 to 60% in tail skin, while no change in the number of tail skin HFs with aCas3+ HG cells was detected (Appendix Fig S3A and B). However, no depletion of sbBSCs was detected in Bcl-2EKO mice (Appendix Fig S3C). This suggested that the acute inhibition of Bcl-2 by ABT-199 treatment induces a more severe sbBSC response than genetically and developmentally removing Bcl-2, which most likely allowed adaption and rewiring of the network to compensate for its absence. Alternatively, although established as highly selective Bcl-2 inhibitor (Souers et al, 2013), ABT-199 treatment may have interfered with other Bcl-2-family members or other anti-apoptotic proteins expressed by sbBSCs. Collectively, our data showed an anti-apoptotic Bcl-2 dependency of sbBSCs and identified an important function for Bcl-2 in HF homeostasis and regeneration. Suprabasal bulge SCs may be prone to detachment-induced apoptosis To investigate sbBSCs Bcl-2 dependency compared to their neighbouring bBSCs and to dissect the cell-intrinsic properties of the heterogeneous BSCs, we performed RNAseq on FACS sorted CD34+/Itga6low and CD34+/Itga6high BSCs. We focused on the differential regulation of cell death response mechanisms, where gene ontology (GO) and functional classification revealed two biological clusters of interest. The first cluster "positive regulation of apoptosis process" included upregulated pro-apoptotic genes, while the second cluster 'cell adhesion mediated by integrin' included downregulated genes for cell adhesion in sbBSCs (Fig 3A and B). An important cellular process that bridges both functional GO clusters is detachment-induced apoptosis, also known as anoikis (Taddei et al, 2012). In particular, expression of Bmf and Bim, two pro-apoptotic and anoikis-associated BH3-only members of the Bcl-2 protein family (Puthalakath et al, 2001; Taddei et al, 2012), was significantly upregulated in sbBSCs compared to bBSCs by both RNAseq data and qRT–PCR (Fig 3B and C). Our data suggested that the lack of integrin-mediated basal lamina attachment (Fig 3B) in sbBSCs and their expression of pro-apoptotic and anoikis-associated molecules, including Bim and Bmf (Fig 3B and C), may be counter-acted by an upregulation of Bcl-2 (Fig 2A), which protects these cells from undergoing apoptosis to ensure efficient cyclic HF regeneration (Fig EV1F). Figure 3. Suprabasal BSCs are prone to detachment-induced apoptosis GO classification of differentially regulated genes in P50 suprabasal BSCs (CD34+/Itgα6low) compared with basal BSCs (CD34+/Itgα6high) clustered for biological process (clustering and statistical analysis done with Metascape, according to Zhou et al, 2019). Columns of interest in red. Functional classification of selected differentially expressed genes in sorted suprabasal BSC vs basal BSC after RNAseq analysis (red: upregulated genes, green: downregulated genes; fold change > 2, P < 0.05; statistical analysis done according to Wagle et al, 2015). qRT–PCR for mRNA expression of the anoikis-related markers Bim and Bmf in sorted n-B (non-bulge), bBSC (basal BSCs) and sbBSC (suprabasal BSCs; n = 3 biological replicates; mean ± standard error of mean [SEM]; *P < 0.05, ns, not significant, two-way ANOVA test). Download figure Download PowerPoint Bcl-2 activity controls the size and architecture of the bulge SC compartment Next, we wanted to know whether Bcl-2 and apoptosis regulation play an instructive role for the composition of the BSC compartment and whether increased Bcl-2 expression affects SC function and cyclic hair regeneration. Therefore, we overexpressed Bcl-2 in the epidermis (Bcl-2EOE mice) by crossing K14Cre (Hafner et al, 2004) with Rosa26LSL.Bcl2.IRES.GFP mice (Knittel et al, 2016) (Appendix Fig S4A). Bcl-2EOE mice displayed a robust overexpression of Bcl-2 as shown by mRNA levels (˜ 17-fold increase in expression; Appendix Fig S4B) and BCL-2 and GFP levels as seen in epidermal keratinocytes, including the HF and IFE (Appendix Fig S4C–E). Next, we analysed whether Bcl-2 overexpression affected HF architecture and homeostasis in adult mice. Strikingly, the bulge area was enlarged and filled with densely packed keratinocytes in Bcl-2EOE HFs (Fig 4A and B; Appendix Fig S6A). Based on our results showing particular high Bcl-2 expression in sbBSCs (Fig 2A), we wondered whether Bcl-2 plays an instructive role by regulating the composition of the BSC compartment. However, no changes in the CD34+/Itga6high and CD34+/Itga6low BSC populations were detected in 8-week-old Bcl-2EOE mice, indicating that Bcl-2 expression does not affect the composition of the BSC pool, including the number of bBSCs and sbBSCs (Fig 4C). Figure 4. Bcl-2 expression regulates size and architecture of SC compartment and delays hair-coat recovery Histology of telogen back skin HF of Ctr and Bcl-2EOE mice (P60). B, bulge. Scale bar, 50 µm. Quantification of the bulge area in back skin at P60 (n = 4; multiple measurements/biological replicate; mean ± standard deviation [SD]; ****P < 0.0001, two-tailed unpaired Student's t-test). FACS plot and quantification of keratinocytes stained for CD34 and Itgα6 in 8-week-old control (Ctr) and Bcl-2EOE mice (n = 3 biological replicates; mean ± standard deviation [SD]; ns, not significant, two-tailed unpaired Student's t-test). suprabasal (sbBSC, CD34+/Itgα6low), basal (bBSC, CD34+/Itgα6high) BSCs. Immunofluorescence staining of telogen back skin HF for BrdU incorporation (1-h pulse, green) and DAPI (blue) in 8-week-old control (Ctr) and Bcl-2EOE mice. Scale bar, 50 µm. Quantification of BrdU+ bulge cells of Ctr and Bcl-2EOE mice. (n = 4 biological replicates; mean ± standard deviation [SD]; ns, not significant, two-tailed unpaired Student's t-test). Download figure Download PowerPoint More detailed analysis of known BSC marker, including Keratin 15, Sox 9 and Nfatc1 (Liu et al, 2003; Vidal et al, 2005; Horsley et al, 2008; Nowak et al, 2008), revealed that these SC markers were all expressed similar to controls within the enlarged HF bulge (Fig EV2, EV3). In addition, we did not observe cyst formation or abnormal expression of hair keratins in HFs of Bcl-2EOE mice (Appendix Fig S5A–D), indicating that differentiated hair keratinocytes did not contribute to the enlarged bulge. In order to check whether these cells belonged to the HG, immunostaining for P-cadherin was performed (Müller-Röver et al, 1999). The localization of the HG below the HF bulge was not altered; however, the increased BSC compartment affected the shape and architecture of the HG highlighting the cellular crosstalk between the two HF compartments (Fig EV2D). Further analysis of RNA expression of P-cadherin, Keratin14 and Lgr6 revealed that these cell populations were rather decreased in Bcl-2EOE mice and most likely did not contribute to the enlarged HF bulge area (Fig EV2E). Next, we investigated whether abnormal SC proliferation may be contributing to the enlarged HF bulge area in Bcl-2EOE mice. Importantly, no change in BrdU incorporation (one pulse BrdU and 1 h chase) was observed between Bcl-2EOE mice and control littermates at P60 (Fig 4D and E), indicating that increased proliferation might not contribute to the enlarged HF bulge area in Bcl-2EOE mice. Click here to expand this figure. Figure EV2. Hair follicle markers in Bcl-2EOE mice A–D. Immunofluorescence staining of telogen back skin hair follicle for Keratin 15 (Krt15, green) (A), Sox9 (white) (B), NfatC1 (red) (C) and hair germ marker P-cadherin (P-Cad, red) (D) in control (Ctr) and Bcl-2EOE mice. B: bulge, HG: secondary hair germ. Scale bars, 50 µm. E. qRT–PCR for HF markers K14, K24, Lgr6 and P-Cad mRNA expression in telogen back skin epidermis of control (Ctr) and Bcl-2EOE mice. (n = 4 biological replicates; mean ± standard error of mean (SEM); **P < 0.01, ns, not significant, two-tailed unpaired Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Increase in bulge and abnormal hair germ compartment impairs HF growth induction in Bcl-2EOE mice A, B. qRT–PCR for mRNA expression of SC quiescence-related markers NfatC1, Foxp1 Foxc1 (A) and HF activation-related markers Runx2, Lef1 (B) in epidermis of control (Ctr) and Bcl-2EOE mice (P27), (n = 4 biological replicates; mean ± standard error of mean [SEM]; *P < 0.05, **P < 0.01; ns, not significant, two-tailed unpaired Student's t-test). C. Immunofluorescence staining of back skin hair follicles for BrdU incorporation (1 h pulse) in control (Ctr) and Bcl-2EOE mice for hair growth initiation (n = 3 biological replicates). Dashed lines and DP, dermal papilla; HG, hair germ. Scale bar, 50 µm. D. Schematic model of hair follicle retraction in catagen hair follicles and bulge architecture comparing control and Bcl-2EOE mice. Download figure Download PowerPoint Bcl-2 overexpression blocks physiological apoptosis that is required for cyclic hair follicle regeneration Given that apoptosis is a physiological process during the regression phase (catagen) (Lindner et al, 1997; Mesa et al, 2015a), we hypothesized that the Bcl-2EOE phenotype was linked to impaired apoptosis during catagen. Therefore, we first focused on the process of HF transition through catagen (Fig EV1A). Strikingly, the column of keratinocytes forming the retracting epithelial strand was wider in Bcl-2EOE mice, suggesting that apoptosis was indeed impaired as expected (Fig 5A and B). Consistently, aCas3+ cells were not detectable in Bcl-2EOE mice compare
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