Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4a (HNF4a) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4a recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4a depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4a. HNF4a, in cooperation with its target HNF1a, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4a-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. Conclusion: The pivotal role of HNF4a in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4a activator and repressor functions are necessary for the identity of hepatocytes. (HEPATOLOGY 2011;53:2063-2074 E pithelial-to-mesenchymal transition (EMT) is the process by which polarized cells of the epithelium lose cell-cell connections and acquire the mesenchymal characteristics of motility and invasiveness. The reverse process, mesenchymal-to-epithelial transition (MET), often occurs at a site secondary to the original EMT population. The dynamic EMT/MET processes are essential for embryonic development and wound repair and initiate the pathological states of fibrosis and metastatic cancer.

We previously demonstrated that ␤ 4 integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large ␤ 4 cytoplasmic domain that are involved in the interaction of ␣ 6 ␤ 4 with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of ␤ 4 that lacked either all or portions of the ␤ 4 cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of ␤ 4 was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of ␣ 6 ␤ 4 to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the ␤ 4 cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of ␣ 6 ␤ 4 integrin to increase invasion. Interestingly, the extracellular domain of ␤ 4 is not necessary for ␣ 6 ␤ 4 to stimulate invasion. The association of ␣ 6 ␤ 4 with ErbB-2 is dependent upon the ␤ 4 cytoplasmic domain and can occur in the absence of ␣ 6 ␤ 4 heterodimerization. Finally, we observed strong activation of PI3K with ␤ 4 wild type and with those ␤ 4 deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between ␣ 6 ␤ 4 and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the ␤ 4 cytoplasmic domain (amino acids 854 -1183) in this event.Integrins are the major family of cell surface receptors that mediate attachment to the extracellular matrix. The interaction between integrins and their ligands is involved in the regulation of many cellular functions, including embryonic development, cell proliferation, as well as tumor growth and metastasis. Integrins are composed of ␣ and ␤ transmembrane subunits that heterodimerize to form different receptors. A single ␣ subunit (e.g. ␣ v or ␣ 6 ) can associate with different ␤ subunits, (␤ 1 , ␤ 3 , ␤ 5 , or ␤ 1 , ␤ 4 , respectively), promoting different ligand binding specificity (1-3). The ␣ 6 ␤ 4 integrin is a receptor for various isoforms of the basement membrane component laminin (4 -6), and its expression is restricted to epithelia, endothelia and peripheral nerves (7-9). In many epithelia, ␣ 6 ␤ 4 is found in hemidesmosomes where it plays an essential role in their organization (10,11). This integrin can also interact with F-actin and promote the migration of invasive carcinoma cells (12, 13).The intracellular portion of the ␤ 4 subunit is much larger (1,000 amino acids) than that of all the other known ␤ subunits, and it does not exhibit apparent sequence homology with them (14 -16). Increasing evidence indicates that the ability of ␣ 6 ␤ 4 to regulate cell proliferation, motility, and invasion is dependent upon signal transduction events that are mediated by the ␤ 4 cytoplasmic domain (17-19)...

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the α6β4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733–20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of α6β4 in carcinoma cells that express wild-type p53. In contrast, α6β4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the α6β4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of α6β4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

Peritoneal dialysis is a form of renal replacement alternative to the hemodialysis. During this treatment, the peritoneal membrane acts as a permeable barrier for exchange of solutes and water. Continual exposure to dialysis solutions, as well as episodes of peritonitis and hemoperitoneum, can cause acute/chronic inflammation and injury to the peritoneal membrane, which undergoes progressive fibrosis, angiogenesis, and vasculopathy, eventually leading to discontinuation of the peritoneal dialysis. Among the different events controlling this pathological process, epithelial to mesenchymal transition of mesothelial cells plays a main role in the induction of fibrosis and in subsequent functional deterioration of the peritoneal membrane. Here, the main extracellular inducers and cellular players are described. Moreover, signaling pathways acting during this process are elucidated, with emphasis on signals delivered by TGF-β family members and by Toll-like/IL-1β receptors. The understanding of molecular mechanisms underlying fibrosis of the peritoneal membrane has both a basic and a translational relevance, since it may be useful for setup of therapies aimed at counteracting the deterioration as well as restoring the homeostasis of the peritoneal membrane.

Abstract. The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant-negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21 (wAF1/cIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity.

Epithelial-to-mesenchymal transition (EMT) is a coordinated process, occurring both during morphogenesis and tumor progression, that allows epithelial cells to dissociate from initial contacts and migrate to secondary sites. The transcriptional repressors of the Snail family induce EMT in different epithelial cell lines and their expression is strictly correlated with EMT during the development and progression of carcinomas. We have previously shown that EMT in hepatocytes correlates with the downregulation of hepatic differentiation key factors HNFs (hepatocyte nuclear factors), and in particular of HNF4alpha. Here, we demonstrate that Snail overexpression is sufficient (i) to induce EMT in hepatocytes with conversion of morphology, downregulation of several epithelial adhesion molecules, reduction of proliferation and induction of matrix metalloproteinase 2 expression and, (ii) most relevantly, to repress the transcription of the HNF4alpha gene through a direct binding to its promoter. These finding demonstrate that Snail is at the crossroads of the regulation of EMT in hepatocytes by a dual control of epithelial morphogenesis and differentiation.

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the ␣ 6 ␤ 4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of ␣ 6 ␤ 4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that ␣ 6 ␤ 4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these ␣ 6 ␤ 4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of ␣ 6 ␤ 4 with a ␤ 4 integrin-specific antibody promotes p53-dependent apoptosis in cells that express both ␣ 6 ␤ 4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of ␣ 6 ␤ 4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949 -960), these studies suggest that the ability of ␣ 6 ␤ 4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.Integrins are the primary receptors used by cells to interact with extracellular matrices. Although initial studies had emphasized the functional contribution of integrins to cell adhesion and migration, a significant finding was the observation that integrins are essential for cell survival (2, 3). Specifically, epithelial cells, endothelial cells, and fibroblasts are prone to growth arrest and apoptosis when deprived of integrin-mediated contact with the extracellular matrix (4, 5). To date, several integrins including ␣ 5 ␤ 1 (6 -7), ␣ v ␤ 3 (8 -10), and ␣ 6 ␤ 1 (11) have been implicated in the promotion of cell growth and survival.Arguably, one of the most complex integrins in terms of both structure and function is ␣ 6 ␤ 4 . This integrin is distinguished structurally from other integrins on the basis of the unusually large cytoplasmic domain of its ␤ 4 subunit (12-14). Aside from its involvement in cell adhesion and migration (1,(15)(16)(17)(18)(19)(20), the ␣ 6 ␤ 4 integrin can promote growth arrest and apoptosis in some carcinoma cells. Specifically, we reported that ␣ 6 ␤ 4 expression induces the growth arrest and apoptosis of the RKO colon carcinoma cell line (21), a finding that has been substantiated in other carcinoma cell lines (22-23), as well as in endothelial cells (24). These findings, however, conflict with considerable evidence that supports a role for ␣ 6 ␤ 4 in promoting carcinoma invasion and progression (1,19). In order to understand how ␣ 6 ␤ 4 can deliver these apparently conflicting signals, we analyzed the mechanism by which ␣ 6 ␤ 4 promotes apoptosis in more detail. S...