Title: iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia
Abstract: Article6 September 2011free access iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia Anissa Chikh Anissa Chikh Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Rubeta N H Matin Rubeta N H Matin Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Valentina Senatore Valentina Senatore Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Martin Hufbauer Martin Hufbauer Institute of Virology, University of Cologne, Cologne, Germany Search for more papers by this author Danielle Lavery Danielle Lavery Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Claudio Raimondi Claudio Raimondi Diabetes Department, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Paola Ostano Paola Ostano Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Maurizia Mello-Grand Maurizia Mello-Grand Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Chiara Ghimenti Chiara Ghimenti Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Adiam Bahta Adiam Bahta Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Sahira Khalaf Sahira Khalaf Surgery Department, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Baki Akgül Baki Akgül Institute of Virology, University of Cologne, Cologne, Germany Search for more papers by this author Kristin M Braun Kristin M Braun Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Giovanna Chiorino Giovanna Chiorino Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Michael P Philpott Michael P Philpott Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Catherine A Harwood Catherine A Harwood Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Daniele Bergamaschi Corresponding Author Daniele Bergamaschi Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Anissa Chikh Anissa Chikh Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Rubeta N H Matin Rubeta N H Matin Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Valentina Senatore Valentina Senatore Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Martin Hufbauer Martin Hufbauer Institute of Virology, University of Cologne, Cologne, Germany Search for more papers by this author Danielle Lavery Danielle Lavery Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Claudio Raimondi Claudio Raimondi Diabetes Department, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Paola Ostano Paola Ostano Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Maurizia Mello-Grand Maurizia Mello-Grand Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Chiara Ghimenti Chiara Ghimenti Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Adiam Bahta Adiam Bahta Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Sahira Khalaf Sahira Khalaf Surgery Department, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Baki Akgül Baki Akgül Institute of Virology, University of Cologne, Cologne, Germany Search for more papers by this author Kristin M Braun Kristin M Braun Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Giovanna Chiorino Giovanna Chiorino Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy Search for more papers by this author Michael P Philpott Michael P Philpott Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Catherine A Harwood Catherine A Harwood Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Daniele Bergamaschi Corresponding Author Daniele Bergamaschi Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK Search for more papers by this author Author Information Anissa Chikh1, Rubeta N H Matin1, Valentina Senatore1, Martin Hufbauer2, Danielle Lavery1, Claudio Raimondi3, Paola Ostano4, Maurizia Mello-Grand4, Chiara Ghimenti4, Adiam Bahta1, Sahira Khalaf5, Baki Akgül2, Kristin M Braun1, Giovanna Chiorino4, Michael P Philpott1, Catherine A Harwood1 and Daniele Bergamaschi 1 1Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK 2Institute of Virology, University of Cologne, Cologne, Germany 3Diabetes Department, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK 4Cancer Genomics Laboratory, Fondazione Edo ed Elvo Tempia Valenta, Biella, Italy 5Surgery Department, Blizard Institute, Barts and The London School of Medicine and Dentistry, London, UK *Corresponding author. Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK. Tel.: +44 207 882 2567; Fax: +44 207 882 7172; E-mail: [email protected] The EMBO Journal (2011)30:4261-4273https://doi.org/10.1038/emboj.2011.302 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 iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis. Introduction The epidermis is a stratified, self-renewing epithelium composed of keratinocytes that are continuously regenerated by the terminal differentiation pathway. Proliferative keratinocytes located in the basal layer periodically withdraw from the cell cycle, migrate upwards and commit to differentiation before ultimately being shed from the skin surface (Watt, 1989; Fuchs, 1990). During the differentiation process, epidermal keratinocytes progress through several stages, with the resulting layered architecture forming a barrier to protect against infection, dehydration and mechanical stress. As basal layer keratinocytes differentiate, cell junctional components are integrated in a polarized fashion (Green and Gaudry, 2000; Green et al, 2010), with adherens junctions most enriched in basal keratinocytes and expression of desmosomal components changing during the stratification process (Delva et al, 2009). In contrast, a tight junction barrier is sealed specifically within the upper layer of granular cells before these give rise to the stratum corneum, a dead cell layer which serves as a scaffold for the deposition of a lipid bilayer. Thus, in the epidermis, adhesion exerts a dynamic role in epidermal differentiation and stratification mediated by hemidesmosomes and focal adhesions (which function in cell–matrix adhesion), and desmosomes, adherens and tight junctions (which function in cell–cell adhesion). The ASPP (apoptosis stimulating protein of p53) proteins are a group of p53 co-activators (Trigiante and Lu, 2006). The apoptotic function of p53 is potentiated by ASPP1 and ASPP2, while a third family member iASPP, negatively modulates apoptosis. Inhibitory member of the ASPP (iASPP; encoded by PPP1R13L) is evolutionarily conserved from worm to human, and its expression is upregulated in human cancers (Bergamaschi et al, 2003, 2006; Zhang et al, 2005; Saebo et al, 2006). iASPP is expressed predominantly in epithelial cells, in the skin, testis, heart and stomach (Herron et al, 2005). Mutations in Ppp1r13l cause abnormalities of the heart and skin in both mice and cattle (Herron et al, 2005; Simpson et al, 2009). Mice which harbour a deletion mutation in PPP1R13L display wavy hair, open eyelids at birth and develop a rapidly progressive cardiomyopathy (Herron et al, 2005) while cattle which harbour a frame-shift mutation in bovine PPP1R13L exhibit cardiomyopathy and a woolly coat (Simpson et al, 2009). These changes are phenotypically similar to the human cardiocutaneous syndrome which is also characterized by cardiomyopathy, woolly hair and palmoplantar keratoderma (Protonotarios and Tsatsopoulou, 2004). Structural studies with p53 family members have recently shown that iASPP preferentially binds to p63, a homologue of p53 (Robinson et al, 2008), which plays a crucial role in epithelial development (Mills et al, 1999; Yang et al, 1999). TP63 is tissue specifically transcribed with two alternative promoters giving rise to TAp63 and ΔNp63 isoforms (Yang et al, 1998). During mouse embryonic development, TAp63 is expressed at the surface of the ectoderm prior to stratification and a shift in balance towards the truncated variant ΔNp63 is required for epidermal maturation (Koster et al, 2004; McKeon, 2004). In the mature epidermis, ΔNp63 is restricted to proliferative basal epidermal cells and is downregulated in more differentiated layers (Koster et al, 2004). Importantly, ΔNp63 maintains the stem cell population in the proliferative compartment of stratified epithelia (Mills et al, 1999; Yang et al, 1999; Pellegrini et al, 2001; Koster et al, 2005). Recent studies through conditional gene deletion show that p63 also regulates the proliferative potential of epidermal stem cells in adult skin and subsequently influences cell senescence and ageing in mice (Keyes et al, 2005; Senoo et al, 2007; Guo et al, 2009). The molecular mechanism underlying the regulation of iASPP is still poorly understood. Using mouse and human skin cultures, we report a determinant mechanism linking iASPP and p63 through the participation of two unreported microRNAs, which act as negative regulators of p63 protein. This controls the epithelial integrity program, affecting cell adhesion, proliferation and differentiation. Taken together, these findings uncover an essential role of iASPP for epithelial homeostasis. Results iASPP expression in skin development Detection of iASPP expression in mouse skin has previously been reported (Herron et al, 2005) although a functional role for iASPP in human skin has not yet been explored. In order to investigate a possible role for iASPP in vivo, the expression pattern of iASPP during mouse embryogenesis was established. Because p63 is the first keratinocyte-specific marker expressed in cells developing along a keratinocyte lineage pathway (Green et al, 2003), we compared expression of iASPP in relation to expression of p63 during epidermal morphogenesis. Immunohistochemical analysis (Figure 1A) showed iASPP expression in the developing epidermis of the early embryo (E12.5) which consists of two cell layers derived from the ectoderm, where p63 has initiated the epithelial stratification program (Koster and Roop, 2004). iASPP epithelial expression pattern is also maintained after commitment to differentiation (E15.5) where iASPP was expressed in the cytoplasm and demonstrated colocalization with p63 in the nuclei. These initial findings support the involvement of iASPP in the epidermal differentiation program. Figure 1.iASPP expression in skin development. (A) Observation of iASPP expression in developing skin during embryogenesis by immunofluorescence. Indirect immunofluorescence microscopy of skin sections taken from mice embryo during development ranging from 12.5 days to adult mice (7 weeks) revealed co-expression and colocalization of iASPP and p63. DAPI (blue) is used as a nuclear stain. Scale bar: 100 μm. (B) Detection of iASPP mRNA in the bulge stem cell population performed by quantitative PCR. Freshly isolated mouse keratinocytes were FACS sorted for the bulge stem cell population (CD34highCD49high), the basal cell population (CD49high) and all sorted. Analysis of CD34 expression confirms correct and clean sorting of the respective populations. Equivalent expression of iASPP expression was observed in the basal and bulge stem cell populations. Relative expression was determined by normalization with the β-actin housekeeping gene. (C) Observation of iASPP mRNA expression in cellular components of human epidermis. Semiquantitative RT–PCR reveals high expression of iASPP in keratinocytes. GAPDH is shown as a loading control. (D) iASPP localization in human adult skin. Immunofluorescence microscopy shows iASPP expression in the nuclei of the basal and spinous layers of the epidermis. Control staining with p63 (basal epidermal layer marker) confirms colocalization with iASPP in this compartment of the epidermis. Scale bar: 50 μm. (E) Induction of differentiation in keratinocytes using high serum calcium results in decrease of iASPP and p63 proteins, as shown by western blot analysis in both HaCaT cells and primary keratinocytes at the time points indicated. K14 and K10 were used as markers to confirm proliferation and differentiation, respectively. Actin is shown as a loading control. Download figure Download PowerPoint We next investigated whether iASPP was expressed in the epidermal stem cell compartment. Keratinocytes were isolated from adult mice between 7 and 8 weeks of age. The basal cell population of the skin was sorted by high CD49F and the bulge stem cell population by high CD49F and high CD34 expression (Trempus et al, 2003). qRT–PCR data showed that Ppp1r13l was expressed in the bulge stem cell compartment at a similar level to CD49F-positive basal keratinocytes (Figure 1B). Analysis of the constituent cell compartments of normal human skin demonstrated PPP1R13L expression in fibroblasts, keratinocytes and melanocytes, with the highest mRNA abundance in keratinocytes (Figure 1C). We observed colocalization of iASPP with p63 in the nuclei of the basal and suprabasal layers of human epidermis (Figure 1D). To investigate the physiological role of iASPP, we examined levels of endogenous iASPP in human primary keratinocytes and HaCaT cells (immortalized human keratinocytes) during differentiation. Cultured keratinocytes were able to differentiate ex vivo at a high calcium concentration confirmed by expression of specific keratinocyte differentiation markers including keratin 10 (K10) and concurrent downregulation of basal markers such as keratin 14 (K14) (Figure 1E). During keratinocyte differentiation, ΔNp63α levels decreased in both primary keratinocytes and HaCaT cells with a concomitant decrease observed in iASPP levels. These data were consistent with our data above, demonstrating the colocalization of iASPP and p63 expression in human skin. Thus, iASPP appears to be involved in the epidermal differentiation program. iASPP and p63 are linked in an autoregulatory feedback loop To determine the contribution of iASPP to regulation of epidermal formation, its interaction with p63 was explored. The human iASPP promoter contains three putative p53 binding sites that could be recognized by p63 (Supplementary Figure S1A). To establish whether p63 is a direct transcriptional regulator of PPP1R13L, chromatin immunoprecipitation (ChIP) was performed using HaCaT cells which endogenously express high levels of ΔNp63 (Figure 2A). Immunoprecipitation with specific anti-p63 antibodies (detecting p63 C′ and N′ terminus, respectively) demonstrated that p63 binds to the PPP1R13L promoter in vivo. To confirm that p63 expression affects iASPP protein expression, HEK293 cells (Human Embryonic Kidney cells, with undetectable p63 and low iASPP levels) were transiently transfected with TAp63 and ΔNp63 isoforms. Increased levels of endogenous iASPP expression were observed at both the protein and mRNA levels (Figure 2B; Supplementary Figure S1B). Moreover, depletion of p63 by small interfering RNA (siRNA) in keratinocytes significantly reduced endogenous iASPP protein and mRNA expression without affecting p53 (Figure 2C and D). Although at the protein level TAp63 is almost undetectable at the RNA level iASPP is clearly downregulated by both TA and ΔNp63 siRNA (Figure 2D; Supplementary Figure S1C). Similarly, upregulation of iASPP in HEK293 cells reactivates p63 expression (Figure 2E; Supplementary Figure S1D), while silencing of PPP1R13L by siRNA in primary keratinocytes as well as in a range of keratinocyte cell cultures drastically decreased ΔNp63 and TAp63 protein expression independently of p53 and without affecting IRF6, recently reported as having a regulatory feedback loop with ΔNp63 (Moretti et al, 2010; Figure 2F; Supplementary Figure S1E and F). To further assess the reciprocal regulation between iASPP and p63, in a more physiological context, primary keratinocytes were treated with UV-B and both iASPP and p63 proteins were similarly downregulated at 25 mJ/cm2 (Figure 2G; Supplementary Figure S1G). However, depletion of iASPP failed to alter TP63 mRNA expression, suggesting modulation of the protein by an intermediary mechanism (Figure 2H; Supplementary Figure S1H). Taken together, these findings suggest that iASPP and p63 (both ΔN and TA) are linked in an autoregulatory feedback loop that is not influenced by p53 expression. Figure 2.iASPP/p63 autoregulatory feedback loop. (A) ChIP assay showing binding of both the C′ and N′ terminus of p63 protein to iASPP promoter. Lane 1: isotope control antibody; lane 2: anti-p63 antibody H129 detecting the C′ terminus of p63; lane 3: anti-p63 antibody (H137) detecting the N′ terminus of p63; lane 4: input. Thymidine kinase (TK) promoter was used as a negative control while p21 was used as a positive control. (B) Western blots showing overexpression of p63 isoforms in HEK293 cells. All isoforms of TAp63 and ΔNp63 induce expression of iASPP at the protein level. GAPDH is shown as a loading control. (C) Western blot analysis showing depletion of p63 by siRNA in primary keratinocytes reflected in a concomitant downregulation of iASPP compared with control and siRNA-scramble (si-ctrl) cultures. (D) Upper panel: qRT–PCR performed on primary keratinocytes transfected with siRNA-scramble (si-ctrl), siRNA-ΔNp63 and siRNA-TAp63 showing the relative expression of iASPP mRNA. The lower panel corresponds at the qRT–PCR for the relative expression of p53 mRNA in the primary keratinocytes transfected with siRNA-scramble (si-ctrl) and siRNA-ΔNp63. (E) Western blot analysis of HEK293 cells overexpressing iASPP causes induction of ΔNp63. (F) Western blot analysis showing how depletion of iASPP by siRNA efficiently downregulates p63 protein levels in primary keratinocytes cells compared with control cultures and siRNA-scramble (si-ctrl). (G) Western blot analysis for p63 and iASPP expression in UV-B-irradiated primary keratinocytes with 5, 10, 25 and 50 mJ/cm2 of UV-B. GAPDH was used as an internal control. (H) qRT–PCR performed on primary keratinocytes transfected with siRNA-scramble (si-ctrl), and siRNA-iASPP reveals no statistical significant variation in relative expression of TA and ΔNp63 mRNA. Download figure Download PowerPoint iASPP controls p63 through microRNA regulation To explore the regulation of p63 by iASPP in human keratinocytes, a retroviral vector expressing short-hairpin RNA (shRNA) was used to stably knock down iASPP protein levels in keratinocytes. One unique shRNA construct specifically targeted and reduced endogenous iASPP at both protein and mRNA levels (Figure 3A and B). In keeping with earlier findings reported using siRNA-iASPP, TP63 mRNA expression was not affected in the shRNA-iASPP cells while p63 protein levels were significantly reduced. Treatment with a specific inhibitor of proteasome-mediated degradation, MG132, was not sufficient to restore ΔNp63 protein levels in iASPP-silenced keratinocytes indicating that iASPP does not affect p63 stability (Figure 3C). Therefore, in order to establish whether modulation of p63 by iASPP could alternatively occur via microRNAs (miRNA), an Agilent MicroRNA Profiling assay was performed. Three miRNAs were found to be upregulated as a consequence of iASPP silencing (Figure 3D). Target prediction, based on implementation of the miRanda algorithm, revealed that two of these three induced miRNAs, hsa-miR-574-3p and hsa-miR-720, were likely to control p63. Increased specific expression of the two identified miRNAs in the iASPP knockdown cells was demonstrated when compared with the control scrambled shRNA-treated cells or versus a miRNA (miR-193a-3p) unaffected by iASPP silencing, confirming the microRNA array data (Figure 3E). The efficiency of the specific antagomirs was also assessed by expression of them in the sh-iASPP-silenced cells. When miR-720 and miR-574-3p were, respectively, co-expressed with a luciferase reporter gene containing the 3′UTR of human p63, significant reduction of luciferase activity was observed in HEK293 cells (Figure 3G). Transduction only of specific antagomirs for miR-574-3p and miR-720 in keratinocytes restored ΔNp63 endogenous protein levels in sh-iASPP cells while an antagomir against miR-193a-3p fail to do so (Figure 3F). Furthermore, when primary keratinocytes are cultured in high calcium concentrations, increased expression of differentiation markers such as involucrin correlates with progressive upregulation of miR-720 and miR-574-3p (Figure 3H) while both anti-miR-720 and anti-miR-574-3p prevent the downregulation of ΔNp63 typically observed during differentiation (Figure 3I). Depletion of iASPP does not affect TP63 mRNA levels, suggesting that both miRNAs are acting through inhibition of p63 translation. Taken together, these data demonstrate that iASPP represses miR-720 and miR-574-3p which in turn could negatively regulate p63, providing a further mechanism through which iASPP can regulate p63 in the skin independently of p53. Moreover, this is the first evidence of two novel non-coding RNAs that control expression of p63 isoforms during skin differentiation. Figure 3.iASPP controls p63 through miRNA regulation. (A) Western blot analysis of several short-hairpin RNAs (shRNAs) that target iASPP gene in HaCaT cells. Construct 3 provides the best knockdown of iASPP protein and will be used from now on in the rest of the manuscript. (B) RT–PCR confirming specific targeting of iASPP sequence by shRNA. iASPP silencing fails again to inhibit mRNA levels of p63 in HaCaT cells. (C) Western blot analysis of iASPP-silenced keratinocytes treated with the proteosome inhibitor MG132 (5 mM, 6 h). Block of the proteosomal degradation is not sufficient to restore iASPP and ΔNp63 protein expression. (D) Log-fold change values of three miRNAs showing greatest overexpression in shRNA-iASPP versus shRNA-scramble HaCaT cells in the microRNA Array (Agilent platform). (E) Taqman qRT–PCR analysis validating miR expression array results confirming upregulation of both miR-720 and miR-574-3p in the iASPP knockdown cells. The efficiency of the specific antagomirs was also measured. As a negative control, the qRT–pCR with miR-193a-3p shows no induction by sh-iASPP. Analysis of miR expression was carried out using the cycle threshold (Ct) method with RNU48 as endogenous control. The quantification of the miRNA samples is evaluated with the ΔΔCt method. 2(−ΔΔCt) value is reported in the y coordinate in a linear scale. Data shown are mean values±s.e.m. of each sample run in triplicate. (F) Western blot analysis showing that antisense miRNA (anti-miR) in HaCaT cells restores expression of p63. No effect on ΔNp63 expression was observed as a result of anti-miR-720 and miR-574-3p in sh-ctrl cells while a negative control anti-miR-193a-3p fail to reinduce expression of ΔNp63. In contrast, both antagomirs restored expression of p63 in cells knocked down for iASPP. (G) Expression of human p63 3′UTR in a luciferase reporter gene (pGL3 luc) leads to diminished luciferase activity in the presence of miR-720 and miR-574-3p, respectively, in HEK293 cells. (*P<0.005, P-values were obtained by using an one-sided Student's t-test). (H) qRT–PCR performed in primary keratinocytes treated with calcium to induce differentiation and involucrin, an epithelial differentiation marker was quantified. In the same experimental conditions, the quantifications of miR-574-3p and miR-720 were performed in the same samples. (I) Western blot analysis showing how transduction of anti-miR-720 or anti-miR-574-3p can prevents decrease of ΔNp63 during keratinocytes differentiation (mimic in culture with high serum calcium). Download figure Download PowerPoint iASPP regulates epidermal adhesion and proliferation Microarray analysis was used to determine the function of iASPP in keratinocytes. The biological processes overrepresented within the list of genes modulated by PPP1R13L silencing (Supplementary Figure S2) are detailed in Supplementary Table S1. CD47, a plasma membrane protein, physically and functionally associated with integrins (Porter and Hogg, 1998) was significantly downregulated by iASPP depletion (Supplementary Figure S3A). Recent studies have highlighted the involvement of p63 proteins in cell adhesion, including integrin-mediated cell adhesion signalling (Carroll et al, 2006). Screening of various integrins using western blot analysis demonstrated significant modulation of these proteins by iASPP silencing, in particular β1 integrin (Figure 4A). These data support a role for iASPP in cell–matrix adhesion. Furthermore, p63 is an essential regulator of PERP, a critical component of the desmosome (Ihrie et al, 2005) and to support this we demonstrate that iASPP silencing significantly reduced PERP protein expression in keratinocytes (Figure 4B). Thus, we evaluated the effect of iASPP depletion on desmosomal proteins by western blot analysis and found significant dysregulation of desmosomal complexes (Figure 4B) which was confirmed at the mRNA level (Supplementary Figure S3B). Other adhesion complexes were also investigated and, for example, Claudin 1 (tight junction component) protein and Connexin 43 (gap junction component) protein confirmed the microarray analysis results. Overall, these data provide evidence that iASPP is crucial for maintaining the integrity of the same cell junction types in the epidermis as p63, thus strengthening our model of an autoregulatory feedback loop. Figure 4.iASPP regulates genes related to epidermal adhesion and proliferation. (A) Western blot analysis of the integrin-associated proteins of CD47 showing the downregulation of β1 integrin, αV integrin, an upregulation of α3 integrin in the cells sh-iASPP compared with the controls while β4 and β6 integrins are unaffected. GAPDH is shown as a loading control. (B) Western blot analysis of cell adhesion proteins affected by iASPP knockdown. In the desmosomal proteins, we observed a downregulation of Perp, desmocollin 3, Plakoglobin and Plakophilin proteins in the cells depleted for iASPP compared with the controls while desmoplakin is upregulated. In the adherens junction, β-catenin protein is downregulated and E-cadherin is unaffected. Connexin 43 from the GAP junction and Claudin 1 from the tight junct