A crucial role of WW45 in developing epithelial tissues in the mouse
2008; Springer Nature; Volume: 27; Issue: 8 Linguagem: Inglês
10.1038/emboj.2008.63
ISSN1460-2075
AutoresJoo‐Hyeon Lee, Tae-Shin Kim, Tae-Hong Yang, Bon‐Kyoung Koo, Sang-Phil Oh, Kwang‐Pyo Lee, Hyun-Jung Oh, Sang‐Hee Lee, Young–Yun Kong, Jin‐Man Kim, Dae‐Sik Lim,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoArticle27 March 2008free access A crucial role of WW45 in developing epithelial tissues in the mouse Joo-Hyeon Lee Joo-Hyeon Lee National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Tae-Shin Kim Tae-Shin Kim National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Tae-Hong Yang Tae-Hong Yang National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Korea Search for more papers by this author Sang-Phil Oh Sang-Phil Oh National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Kwang-Pyo Lee Kwang-Pyo Lee National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Hyun-Jung Oh Hyun-Jung Oh National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Sang-Hee Lee Sang-Hee Lee Division of Electron microscopic Research, Korea Basic Science Institute, Daejeon, Korea Search for more papers by this author Young-Yun Kong Young-Yun Kong Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Korea Search for more papers by this author Jin-Man Kim Jin-Man Kim Department of Pathology, College of Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Dae-Sik Lim Corresponding Author Dae-Sik Lim National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Joo-Hyeon Lee Joo-Hyeon Lee National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Tae-Shin Kim Tae-Shin Kim National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Tae-Hong Yang Tae-Hong Yang National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Korea Search for more papers by this author Sang-Phil Oh Sang-Phil Oh National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Kwang-Pyo Lee Kwang-Pyo Lee National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Hyun-Jung Oh Hyun-Jung Oh National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Sang-Hee Lee Sang-Hee Lee Division of Electron microscopic Research, Korea Basic Science Institute, Daejeon, Korea Search for more papers by this author Young-Yun Kong Young-Yun Kong Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Korea Search for more papers by this author Jin-Man Kim Jin-Man Kim Department of Pathology, College of Medicine, Chungnam National University, Daejeon, Korea Search for more papers by this author Dae-Sik Lim Corresponding Author Dae-Sik Lim National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Author Information Joo-Hyeon Lee1, Tae-Shin Kim1, Tae-Hong Yang1, Bon-Kyoung Koo2, Sang-Phil Oh1, Kwang-Pyo Lee1, Hyun-Jung Oh1, Sang-Hee Lee3, Young-Yun Kong2, Jin-Man Kim4 and Dae-Sik Lim 1 1National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea 2Division of Molecular and Life Sciences, Pohang University of Science and Technology, Kyungbuk, Korea 3Division of Electron microscopic Research, Korea Basic Science Institute, Daejeon, Korea 4Department of Pathology, College of Medicine, Chungnam National University, Daejeon, Korea *Corresponding author. National Research Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseoung-D, Yuseong-G, Daejeon 305-701, Korea. Tel.: +82 42 869 2635; Fax: +82 42 869 2610; E-mail: [email protected] The EMBO Journal (2008)27:1231-1242https://doi.org/10.1038/emboj.2008.63 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The role and molecular mechanisms of a new Hippo signalling pathway are not fully understood in mammals. Here, we generated mice that lack WW45 and revealed a crucial role for WW45 in cell-cycle exit and epithelial terminal differentiation. Many organs in the mutant mouse embryos displayed hyperplasia accompanied by defects in terminal differentiation of epithelial progenitor cells owing to impaired proliferation arrest rather than intrinsic acceleration of proliferation during differentiation. Importantly, the MST1 signalling pathway is specifically activated in differentiating epithelial cells. Moreover, WW45 is required for MST1 activation and translocation to the nucleus for subsequent LATS1/2 activation upon differentiation signal. LATS1/2 phosphorylates YAP, which, in turn, translocates from the nucleus into the cytoplasm, resulting in cell-cycle exit and terminal differentiation of epithelial progenitor cells. Collectively, these data provide compelling evidence that WW45 is a key mediator of MST1 signalling in the coordinate coupling of proliferation arrest with terminal differentiation for proper epithelial tissue development in mammals. Introduction Homeostasis of regenerative epithelial tissues such as skin and intestine is maintained through a tightly balanced process of proliferation and terminal differentiation. During normal epithelial development, proliferating progenitor cells, often referred to as transiently amplifying cells, actively divide a limited number of times before they undergo cell-cycle exit and terminally differentiate into postmitotic cells (Blanpain et al, 2007). Cancer can develop as a result of inappropriate proliferation of progenitor cells accompanied by a partial or complete loss of differentiation (Reya et al, 2001). Therefore, understanding the signalling networks that control cell-cycle exit and terminal differentiation in epithelial tissues will provide insights into the mechanisms underlying tumorigenesis. A new signalling network, known as the ‘Hippo pathway’ in Drosophila, seems to be a key developmental programme in controlling proliferation and apoptosis for proper organ development in Drosophila (Edgar, 2006; Harvey and Tapon, 2007; Pan, 2007; Saucedo and Edgar, 2007). The Ste-20 family kinase Hippo (Harvey et al, 2003; Jia et al, 2003; Pantalacci et al, 2003; Udan et al, 2003; Wu et al, 2003), WW adaptor protein Salvador (Kango-Singh et al, 2002; Tapon et al, 2002) and NDR kinase Warts (Justice et al, 1995; Xu et al, 1995) are the key components of the Hippo pathway that restrict cell proliferation and promote apoptosis in differentiating epithelial cells by regulating expression of cyclin E and Diap1. The Hippo kinase phosphorylates and activates the Warts kinase, and this process is facilitated by the scaffolding protein Salvador or Mats (Wu et al, 2003; Wei et al, 2007). Warts, together with Mats, then phosphorylates and inhibits the transcription coactivator Yorkie (Huang et al, 2005; Lai et al, 2005). Expanded, Merlin and Fat, all of which localize to the plasma membrane, function upstream of the Hippo pathway (Bennett and Harvey, 2006; Cho et al, 2006; Hamaratoglu et al, 2006; Silva et al, 2006; Willecke et al, 2006). In flies, mutations of these factors lead to increased cell proliferation and decreased cell death. The phenotypes of flies with mutations in the Hippo pathway can be rescued with their respective human counterparts (Tao et al, 1999; Wu et al, 2003; Huang et al, 2005; Lai et al, 2005), indicating that the Hippo pathway may have an analogous role in epithelial tissue development in mammals. Several reports on each mammalian component of the Hippo pathway have shown that the pathway is involved in cell death and cell-cycle regulation. MST1/2 kinases (Hippo homologues) were originally reported to be involved in apoptosis with caspase-3-mediated proteolytic activation (Lee et al, 1998). LATS1/2 (Warts homologues) have been implicated in the regulation of cell-cycle progression, apoptosis (Tao et al, 1999; Xia et al, 2002), mitotic exit and cytokinesis (McPherson et al, 2004; Yang et al, 2004). YAP (a Yorkie homologue) is involved in apoptosis by interacting with p73 (Matallanas et al, 2007). Although mutation of WW45 (a Salvador homologue) has been reported in several cancer cell lines (Tapon et al, 2002), little is known about the functional significance of WW45 in mammals. So far, only limited biochemical interactions have been reported, including the phosphorylation of LATS1/2 by MST1/2, the associations of WW45 with MST1/2 and LATS1/2, binding of LATS1 to MOB1 (a MATS homologue) and formation of a complex comprising RASSF1A, MST2, WW45 and LATS1 (Chan et al, 2005; Hergovich et al, 2006; Guo et al, 2007). The Hippo pathway has also been implicated in mammalian tumorigenesis. Mice lacking LATS1 develop some types of tumour, and hWW45 and Mats are mutated in several cancer cell lines (St John et al, 1999; Tapon et al, 2002; Lai et al, 2005). NF2, the human orthologue of Merlin, is a tumour suppressor gene, mutations of which lead to neurofibromatosis (McClatchey and Giovannini, 2005). YAP is overexpressed in mammalian cancers and transgenic mice overexpressing YAP have an increased liver size and dysplasia with expanded undifferentiated progenitor cells in the intestine (Zender et al, 2006; Camargo et al, 2007; Dong et al, 2007). Of the Hippo pathway proteins, only LATS1-, LATS2-, NF2- and YAP-null mice have been generated; however, these mice are either early embryonic lethal or fail to recapitulate defects seen in the respective Drosophila mutants (McClatchey et al, 1997; St John et al, 1999; McPherson et al, 2004, Morin-kensicki et al, 2006). Therefore, compared with Drosophila, much less is known about the physiological function of the Hippo pathway in mammalian epithelial development. Furthermore, the molecular mechanisms by which this pathway is regulated during development are not fully understood in mammals. In the present study, we generated mice lacking WW45 to examine the role of the Hippo pathway in mammals. Mutant embryos displayed unchecked proliferation and defects in terminal differentiation of epithelial cells. We also revealed the molecular mechanism by which MST1 signalling is spatiotemporally regulated to allow cell-cycle exit and activation of terminal differentiation in epithelial cells. Results Retarded growth and perinatal lethality of the WW45-deficient mice To identify the role of WW45 in vivo, we generated WW45 mutant mice using embryonic stem (ES) cell technology. The targeted mutation replaced a 2.4-kb genomic region containing WW45 exon 2 with a puromycin cassette, leading to premature stop in WW45. After electroporation, the targeted clone was identified and transmitted through the germ line (Figure 1A). The absence of WW45 protein was confirmed by immunoblot assay (Figure 1B). Heterozygous mice were born healthy and fertile, and developed normally. However, only three dwarf homozygotes were found among 954 littermates generated from heterozygous intercrosses, indicating that most of the null mice were embryonic lethal. Viable WW45−/− embryos were found at embryonic days 17.5 (E17.5) and E18.5 (Supplementary Table 1). WW45−/− embryos up to E11.5 were morphologically indistinguishable from their control littermates. However, from about E13.5 onwards, WW45−/− embryos were slightly smaller than the controls, indicating a slower gain in body weight (Figure 1C). Despite growth retardation, we observed no consistent overt defects that would cause the embryonic lethality of mutants. This result prompted us to examine placentas from mutants and their littermates. WW45-null placentas displayed immature development with poor growth and vascularization of the labyrinth layers (Figure 1D). Defective intermingling of fetal and maternal vessels was confirmed by staining with anti-PECAM1 and anti-laminin (Supplementary Figure S1). Thus, the malfunctional labyrinth layer may affect the growth and viability of WW45−/− embryos. Figure 1.Targeted disruption of the WW45 gene by homologous recombination. (A) Schematic representation of the WW45 targeting strategy, showing the mouse WW45 genomic locus, targeting vectors and targeted locus. Exon 2 was replaced with a puro cassette, and diphtheria toxin A was used for negative selection. Five exons are indicated by black boxes. Also indicated are the 5′-external probe for Southern hybridization of genomic DNA, predicted sizes of fragments with restriction sites and primer pairs for PCR. Arrowheads represent primers used for genotyping of the WT (1 and 2) or MT (1 and 3) alleles. Bg, BglII restriction site. (B) Southern blot analysis of genomic DNA digested with the restriction enzyme BglII using the 5′ probe, and PCR genotype analysis of embryos. Western blot analysis (WB) shows the presence of WW45 in cultured primary fibroblasts. The arrow indicates bands of WW45. (C) Growth retardation of mutant (−/−) WW45 embryos. Appearance of wild-type and mutant embryos at E17.5 (top). Growth curves of wild-type (+/+) and mutant (−/−) WW45 embryos (bottom). (D) Haematoxylin and eosin (H&E)-stained sections of placentas from E17.5 embryos. Note that the major layers had defective maturations with reduced and disordered vasculature in mutant placentas. de, decidua; sp, spongiotrophoblast layer; la, labyrinth layer. Scale bar: 500 μm. Boxed regions are shown at high magnification. Download figure Download PowerPoint Hyperplasia and immature differentiation of epithelial cells in the WW45-deficient embryos Previous Drosophila studies have proposed important roles for SAV1 in the regulation of proliferation and apoptosis in epithelial tissues. Thus, we performed histological analyses of WW45−/− embryos at various embryonic stages. Interestingly, hyperproliferation of epithelial cells was clearly observed in the skin and intestine (Figures 2 and 3) and other organs (Supplementary Figure S2) of the WW45−/− embryos at E17.5. Figure 2.Hyperproliferation and immature differentiation in WW45−/− epidermis at E17.5. (A) H&E-stained sections of wild-type (a) and mutant (a′) epidermis. Evaluation of cellular proliferation was conducted by co-immunohistochemistry analysis with anti-Ki67 and anti-E-cadherin (b, b′). Note the increased numbers of Ki67-positive cells, including in multiple cell layers, in the mutant epidermis. Quantitation of the percentage of proliferating cells per 1 mm2 area in mutant versus control epithelium from three independent experiments (±s.d.). Boxed regions are shown at high magnification in the right panel of each genotype. WT, wild type; MT, mutant. (B) Differentiation marker expression in the skin of wild-type or mutant embryos. Immunohistochemistry analysis was performed with antibodies against K14, K10, loricrin and filaggrin, in addition to TUNEL staining for analysis of apoptosis. 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are shown in blue. Note the lack of stratification and differentiation and the increased numbers of progenitor cells in the mutant epidermis. (C) Electron microscopy analysis of the wild-type (upper panel) and mutant (lower panel) epidermis. Note the loss of columnar morphology in basal cells and the disorganized suprabasal cells, as well as the loss of flattened granular and cornified layers in the mutant skin. Scale bar: 100 μm (A, B), 2 μm (C). Download figure Download PowerPoint Figure 3.Hyperplasia and immature differentiation of WW45−/− intestinal epithelium at E17.5. (A) H&E-stained sections of wild-type (a) and mutant (a′) intestine. Immunostaining analysis with anti-Ki67 and anti-E-cadherin (b, b′). Note the increased numbers of Ki67-positive cells, including in multiple cell layers, in the mutant epithelium. Quantitation of the percentage of proliferating cells per 1 mm2 area in epithelium from three independent experiments (±s.d.). Boxed regions are shown at high magnification in the right panel of each genotype. WT, wild type; MT, mutant. (B) Differentiation marker expression in the intestine of wild-type or mutant embryos. Immunohistochemistry analysis was performed with indicated antibodies, in addition to Alcian blue staining. Note the significant reductions in the levels of terminal differentiating cells, goblet cells and entero-endocrine cells in mutants compared with wild-type littermates. (C) Electron microscopy analysis of the wild-type (upper panel) and mutant (lower panel) intestines. Note the reduced density of brush-border microvilli of mutant enterocytes compared with the densely compacted, uniformly distributed microvilli of wild-type enterocytes. Scale bar: 100 μm (A, B), 2 μm (C). Download figure Download PowerPoint We first characterized skin development in these WW45−/− mice. Dividing keratinocytes are normally restricted to the basal layer of wild-type epidermis, and as cells exit from the cell cycle, these keratinocytes move outwards and differentiate to form the spinous layers, the granular layers and the dead enucleated stratum corneum layers at the skin surface (Figure 2Aa). By contrast, the mutant epidermis had a more dense basal layer and the expanded suprabasal layers were less differentiated, with reduced enucleation and compaction of the developing granular cells (Figure 2Aa′). Development of hair follicles was rarely seen, and only small premature hair follicles were seen in null embryos at this stage. Co-staining for E-cadherin and Ki67 revealed that the mutant skin contained increased numbers of proliferative epithelial cells, compared with wild-type skin (Figure 2Ab, b′). Almost all the basal cells and several suprabasal cells expressed Ki67 in the mutant epidermis, whereas proliferation was restricted to the basal layer in the wild-type epidermis. TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling)-positive cells were also seen in wild-type epidermis but not in mutant epidermis (Figure 2Be, e′). Thus, increased proliferation in the suprabasal layer and repressed apoptosis of terminally differentiated keratinocytes contribute to hyperplasia in the epidermis of mutant embryos. Epithelia of WW45−/− embryos were hyperproliferative but did not seem to undergo normal differentiation; therefore, we investigated whether epithelial differentiation was delayed and/or defective in mutant epithelia using a panel of antibodies against proteins that are expressed at defined stages of differentiation. Keratin 14 was normally expressed in one or two layers of basal cells in wild-type embryos, whereas it was strongly expressed in the multilayered basal cells in mutant embryos (Figure 2Ba, a′). Moreover, there were increased numbers of keratin-10-expressing cells in the suprabasal layers of the WW45−/− epidermis compared with wild-type embryos (Figure 2Bb′). Expression levels of loricrin and filaggrin, which are markers of late keratinocyte differentiation, were significantly downregulated in mutant epidermis, indicating defects in late differentiation (Figure 2Bc′, d′). Skin-barrier development with X-gal staining further confirmed the absence of terminally differentiated layers (Supplementary Figure S3). Electron microscopy analysis clearly showed that the epidermis of WW45-null embryos was thicker than the wild-type epidermis. Moreover, the granular and cornified layers present dysmaturation in the mutant epidermis, with nucleated cells reaching the epidermal surface (Figure 2C). These data indicate that the suprabasal mutant keratinocytes fail to stop proliferating and terminally differentiate. We also examined intestinal development in mutant embryos. Wild-type intestinal epithelium consists of a monolayer of polarized epithelial cells organized into crypts. By contrast, the mutant epithelium was multilayered and displayed hypercellularity with pseudostratified and enlarged nuclei, perturbed differentiation with loss of goblet cells and increased numbers of mitotic cells (Figure 3Aa′ and Supplementary Figure S2a, a′). Furthermore, Ki67 staining revealed extensive proliferation throughout the villus epithelium in the small intestine, whereas proliferative cells were restricted to the crypt bases in the control epithelium (Figure 2Ab, b′). Indeed, all epithelial cells in the mutant colons were Ki67-positive, indicating dysplasia (Supplementary Figure S4A). In agreement with these results, bromodeoxyuridine (BrdU) pulse experiments further confirmed significantly increased numbers of dividing cells in mutant epithelia of many organs (Supplementary Figure S4B). We then examined differentiation of the various intestinal epithelial cell lineages. During differentiation of enterocytes, the FABP protein was detected at normal levels in the villi of wild-type embryos but at markedly reduced levels in the mutant embryos (Figure 3Ba, a′). Similarly, chromogranin labelling, which detects differentiation along the entero-endocrine lineage, was rarely detected in mutant embryos (Figure 3Bb, b′). Staining with Alcian blue, a marker for goblet cells, revealed a complete absence of muco-secreting goblet cells in all mutant intestinal tracts (Figure 3Bc, c′). Ultrastructural analysis also revealed poorly developed microvillus brush borders on the apical surfaces of the villous enterocytes, indicating defective enterocyte differentiation in the mutant epithelium of the small intestine (Figure 3C). Interestingly, these mutant cells had enlarged nuclei located close to the apical region, indicating a loss of apical–basal polarity. In addition to immature differentiation of the mutant skin and intestine, immature differentiation was also detected in the lungs of mutant embryos (Lee and Lim, personal observation). Taken together, these results indicate that WW45 deficiency induces hyperplasia and immature differentiation in epithelial tissues. WW45 regulates cell-cycle exit in epithelial progenitor cells during differentiation We tested whether excessive proliferation of mutant cells was due to increased proliferation rates or failure of cell-cycle exit for terminal differentiation. First, we examined the cell-cycle duration in epithelial progenitor cells by analysing the proportion of cycling cells (Ki67+) in S-phase 1 h after injection of BrdU (Schmahl, 1983). Although the percentage of BrdU-labelled cells was increased in mutant embryos, the BrdU+Ki67+/Ki67+ labelling index was approximately the same in wild-type and mutant embryos, indicating similar proliferation rates in wild-type and mutant embryos (Figure 4A). Second, we determined the frequency of cell-cycle re-entry by assessing the proportion of dividing cells (BrdU+) 24 h after BrdU injection (Chenn and Walsh, 2002). During the time interval between BrdU application and analysis, cells can leave (Ki67−) or re-enter the cell cycle (Ki67+). The mean ratio of BrdU+Ki67+/BrdU+ cells was significantly increased by 49% in the mutant small intestine and by 58% in the mutant colon compared with wild-type controls (Figure 4B), indicating that WW45 promotes exit from the cell cycle in epithelial progenitors during embryonic development. Figure 4.Increased numbers of cycling cells in the developing WW45−/− epithelium resulted from inefficient growth arrest. (A) The proportion of progenitor cells (Ki67+) labelled with BrdU after a 1 h pulse. There is no difference in cell-cycle length between wild-type and mutant embryos. (B) Analysis of immunoreactivity for Ki67 in BrdU-positive cells 24 h after BrdU injection. The fraction of cells re-entering the cell cycle (BrdU+Ki67+) is significantly increased in mutant embryos. (C) Growth curves of primary keratinocytes isolated from wild-type and mutant epidermis show similar proliferation rates. (D) Induction of calcium-stimulated differentiation in primary keratinocytes. The level of BrdU incorporation in keratinocytes cultured in the absence or presence of Ca2+ for the times indicated is shown. The mutant keratinocytes show inefficient growth arrest under differentiation conditions. (A–D) Data represent triplicate independent experiments (±s.d.). Download figure Download PowerPoint Failure of cell-cycle exit of WW45−/− keratinocytes during in vitro differentiation To further analyse the rates of proliferation and differentiation of epithelial cells, we isolated primary keratinocytes from the skin of embryos. Consistent with in vivo data (Figure 4A), WW45−/− keratinocytes had normal cell-cycle distribution and the rate of proliferation was not significantly increased compared with control cells (Figure 4C and data not shown). Again, this suggests that WW45 is unlikely to regulate the rate of proliferation. We then examined the ability of WW45 to regulate proliferation arrest and differentiation of developing epidermal cells by adding calcium, transforming growth factor (TGF)-β or LiCl, which have been shown to induce proliferation exit, and possibly terminal differentiation, of keratinocytes (Hennings et al, 1980; Shipley et al, 1986; Olmeda et al, 2003). With Ca2+, TGF-β or LiCl treatment, the wild-type cells showed efficient growth arrest and the numbers of BrdU-labelled cells were reduced. By contrast, mutant cells continued to proliferate and were BrdU-positive 24 h after Ca2+, TGF-β or LiCl treatment (Figure 4D and Supplementary Figure S5). Consistently, the expression of filaggrin was evident in wild-type but not in mutant keratinocytes after induction of differentiation (Figure 5A). These data indicate that increased proliferation in WW45−/− epithelial tissues results from impaired growth arrest of progenitor cells during differentiation rather than from an increased rate of proliferation. Figure 5.Activation of MST1 signalling pathway during differentiation. (A) Protein status of components of the MST1 pathway under differentiation conditions. Primary keratinocytes were incubated with or without Ca2+ for 24 h and analysed by western blot analysis with the indicated antibodies. Note the activation of MST1, as shown by pMST1 blotting, and the mobility shift of YAP and LATS1/2 in the control keratinocytes, but not in mutant keratinocytes, under differentiation conditions. Keratinocyte differentiation was confirmed by western blot analysis of filaggrin expression. (B) Wild-type and mutant primary keratinocytes cultured with or without Ca2+ for 24 h were analysed by immunoprecipitation with anti-MST1 and by western blot assays. Note that complex formation is only detected with Ca2+ treatment in the control samples. (C) Phosphorylation of YAP by activated LATS2 through MST1/WW45. WW45−/− primary keratinocytes were co-transfected with the indicated plasmids and probed with the indicated antibodies. Note the mobility shift of YAP and LATS2 in the presence of MST1 and intact WW45. Download figure Download PowerPoint Activation of the MST signalling pathway during keratinocyte differentiation in vitro To further characterize the molecular mechanisms by which WW45 regulates cell-cycle exit during differentiation, we investigated the phosphorylation and localization of MST, LATS and YAP in keratinocytes during differentiation. Interestingly, autophosphorylation of MST1 was induced upon differentiation, as shown by phospho-MST1 immunoblotting, indicating MST1 activation (Figure 5A). By contrast, this autophosphorylation of MST1 was not detected in mutant cells. Therefore, WW45 is required for MST1 activation after induction of differentiation with calcium treatment. In addition, the mobility patterns of LATS1/2 and YAP differ after induction of differentiation with calcium treatment (Figure 5A). Phosphorylation of LATS1/2 and YAP was detected in differentiated wild-type keratinocytes, but not in differentiated mutant keratinocytes. Thus, phosphorylation of YAP seems to be dependent on the MST1 signalling pathway, in particular on LATS1/2, during epithelial differentiation in mammals. We then investigated whether the formation of components of the MST1 signalling pathway might be associated with and affect differentiation of keratinocytes. We first transfected WW45−/− keratinocytes with LATS1/2, MST1 and YAP with or without WW45, and then induced differentiation before immunoprecipitation. In mutant keratinocytes, LATS1and LATS2 co-precipitated with YAP but not with MST1. By contrast, these proteins form a stable complex in the presence of WW45 (Supplementary Figure S6). Moreover, endog
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