Matrix metalloproteinase 11 protects from diabesity and promotes metabolic switch.

MMP11 overexpression is a bad prognostic factor in various human carcinomas. Interestingly, this proteinase is not expressed in malignant cells themselves but is secreted by adjacent non-malignant mesenchymal/stromal cells, such as cancer associated fibroblasts (CAFs) and adipocytes (CAAs), which favors cancer cell survival and progression. As MMP11 negatively regulates adipogenesis in vitro, we hypothesized that it may play a role in whole body metabolism and energy homeostasis. We used an in vivo gain- (Mmp11-Tg mice) and loss- (Mmp11-/- mice) of-function approach to address the systemic function of MMP11. Strikingly, MMP11 overexpression protects against type 2 diabetes while Mmp11-/- mice exhibit hallmarks of metabolic syndrome. Moreover, Mmp11-Tg mice were protected from diet-induced obesity and display mitochondrial dysfunction, due to oxidative stress, and metabolic switch from oxidative phosphorylation to aerobic glycolysis. This Warburg-like effect observed in adipose tissues might provide a rationale for the deleterious impact of CAA-secreted MMP11, favouring tumor progression. MMP11 overexpression also leads to increased circulating IGF1 levels and the activation of the IGF1/AKT/FOXO1 cascade, an important metabolic signalling pathway. Our data reveal a major role for MMP11 in controlling energy metabolism, and provide new clues for understanding the relationship between metabolism, cancer progression and patient outcome.

Local adipocyte cancer cell paracrine loop: can "sick fat" be more detrimental?

This review article focuses on the emerging role of tumor resident adipocytes. It provides in vitro and in vivo evidence that they are essential for cancer development/progression. In addition to systemic effects, their tumor-promoting impact is dependent on local functions, notably via a complex adipocyte cancer cell paracrine loop (ACCPL). Indeed, this event leads to dramatic phenotypic and/or functional modifications of both cell types as well as of the extracellular matrix. Adipocytes undergo delipidation leading to adipocytes/cancer-associated adipocytes/cancer-associated fibroblasts de-differentiation processes. In turn, cancer cell aggressiveness is exacerbated through increased proliferation, migration, and invasion properties. This is accompanied by intense tissue remodeling, conducting to the occurrence of the tumor stroma. The molecular pathways involved in ACCPL remain largely unknown. Nevertheless, several clues are starting to emerge. Moreover, obesity is currently a sign of increased risk and poor prognosis in human carcinomas. How adiposopathy might impact tumors and specifically the ACCPL is still under investigation. However, available experimental, epidemiological, and clinical data allow to draw some directions. Interestingly, there are numerous similarities between the ACCPL-induced and obesity-related molecular alterations. It might, therefore, be hypothesized that obesity provides a "constitutively active" local permissive environment for cancer cells. Improving our knowledge about ACCPL in both lean and obese patients remains a challenging task. Indeed, deciphering the cellular and molecular mechanisms behind ACCPL might provide new targets for improving diagnosis/prognosis and the design of innovative therapeutic strategies, and even, in case of obesity, for preventing cancer.

Functional relationship between matrix metalloproteinase-11 and matrix metalloproteinase-14.

MMP-11 is a key factor in physiopathological tissue remodeling. As an active form is secreted, its activity must be tightly regulated to avoid detrimental effects. Although TIMP-1 and TIMP-2 reversibly inhibit MMP-11, another more drastic scenario, presumably via hydrolysis, could be hypothesized. In this context, we have investigated the possible implication of MMP-14, since it exhibits a spatiotemporal localization similar to MMP-11. Using native HFL1-produced MMP-11 and HT-1080-produced MMP-14 as well as recombinant proteins, we show that MMP-11 is a MMP-14 substrate. MMP-14 cleaves MMP-11 catalytic domain at the PGG(P1)-I(P1')LA and V/IQH(P1)-L(P1')YG scissile bonds, two new cleavage sites. Interestingly, a functional test showed a dramatical reduction in MMP-11 enzymatic activity when incubated with active MMP-14, whereas inactive point-mutated MMP-14 had no effect. This function is conserved between human and mouse. Thus, in addition to the canonical reversible TIMP-dependent inhibitory system, irreversible MMP proteolytic inactivation might occur by cleavage of the catalytic domain in a MMP-dependent manner. Since MMP-14 is produced by HT-1080 cancer cells, whereas MMP-11 is secreted by HFL1 stromal cells, our findings support the emerging importance of tumor-stroma interaction/cross-talk. Moreover, they highlight a Janus-faced MMP-14 function in the MMP cascade, favoring activation of several pro-MMPs, but limiting MMP-11 activity. Finally, both MMPs are active at the cell periphery. Since MMP-14 is present at the cell membrane, whereas MMP-11 is soluble into the cellular microenvironment, this MMP-14 function might represent one critical regulatory mechanism to control the extent of pericellular MMP-11 bioavailability and protect cells from excessive/inappropriate MMP-11 function.

[The tumoral stroma, a breeding ground for cancer cells]. / Le stroma tumoral - un terreau fertile pour la cellule cancéreuse.

Carcinomas are constituted by malignant epithelial cells and the tumor microenvironment also called tumoral stroma. The present non-exhaustive review will focus on cellular and molecular key events implicating cancer-associated fibroblasts (CAF) and the extracellular matrix (ECM) in the stroma remodeling processes occuring during tumor invasive steps. We will also discuss the place of the stroma in the cancerology today, the recent progresses done and its usefulness to design new therapies.

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration.

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration.

STARD3 or STARD3NL and VAP form a novel molecular tether between late endosomes and the ER.

Inter-organelle membrane contacts sites (MCSs) are specific subcellular regions favoring the exchange of metabolites and information. We investigated the potential role of the late-endosomal membrane-anchored proteins StAR related lipid transfer domain-3 (STARD3) and STARD3 N-terminal like (STARD3NL) in the formation of MCSs involving late-endosomes (LEs). We demonstrate that both STARD3 and STARD3NL create MCSs between LEs and the endoplasmic reticulum (ER). STARD3 and STARD3NL use a conserved two phenylalanines in an acidic tract (FFAT)-motif to interact with ER-anchored VAP proteins. Together, they form an LE-ER tethering complex allowing heterologous membrane apposition. This LE-ER tethering complex affects organelle dynamics by altering the formation of endosomal tubules. An in situ proximity ligation assay between STARD3, STARD3NL and VAP proteins identified endogenous LE-ER MCS. Thus, we report here the identification of proteins involved in inter-organellar interaction.

Perispeckles are major assembly sites for the exon junction core complex.

The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites.

In vivo evidence that TRAF4 is required for central nervous system myelin homeostasis.

Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies. However, TRAF4 does not fit the paradigm of TRAF function in immune and inflammatory responses. Its physiological and molecular functions remain poorly understood. Behavorial analyses show that TRAF4-deficient mice (TRAF4-KO) exhibit altered locomotion coordination typical of ataxia. TRAF4-KO central nervous system (CNS) ultrastructure shows strong myelin perturbation including disorganized layers and disturbances in paranode organization. TRAF4 was previously reported to be expressed by CNS neurons. Using primary cell culture, we now show that TRAF4 is also expressed by oligodendrocytes, at all stages of their differentiation. Moreover, histology and electron microscopy show degeneration of a high number of Purkinje cells in TRAF4-KO mice, that was confirmed by increased expression of the Bax pro-apoptotic marker (immunofluorescence), TUNEL analysis, and caspase-3 activation and PARP1 cleavage (western blotting). Consistent with this phenotype, MAG and NogoA, two myelin-induced neurite outgrowth inhibitors, and their neuron partners, NgR and p75NTR were overexpressed (Q-RT-PCR and western blotting). The strong increased phosphorylation of Rock2, a RhoA downstream target, indicated that the NgR/p75NTR/RhoA signaling pathway, known to induce actin cytoskeleton rearrangement that favors axon regeneration inhibition and neuron apoptosis, is activated in the absence of TRAF4 (western blotting). Altogether, these results provide conclusive evidence for the pivotal contribution of TRAF4 to myelination and to cerebellar homeostasis, and link the loss of TRAF4 function to demyelinating or neurodegenerative diseases.

Inhibition of cyclooxygenase-2 causes regression of gastric adenomas in trefoil factor 1 deficient mice.

Cyclooxygenase-2 (Cox-2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox-2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox-2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox-2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox-2 gene [also known as prostaglandin-endoperoxide synthase 2 (Ptgs2)] and a Cox-2 selective drug celecoxib. Our results show that genetic deletion of Cox-2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox-2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8-14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox-2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox-2 is sufficient to reversibly disrupt gastric adenomas in mice.

Adipocyte is a non-trivial, dynamic partner of breast cancer cells.

While the participation of adipocytes is well known in tissue architecture, energy supply and endocrine processes, their implication during natural cancer history is just beginning to unfold. An extensive review of the literature concerning the impact of resident adipocytes on breast cancer development/progression was performed. This review provides in vitro and in vivo evidence that adipocytes located close to invasive cancer cells, referred to as cancer-associated adipocytes (CAAs), are essential for breast tumor development/progression. Their deleterious function is dependent, at least partly, on their crosstalk with invasive cancer cells. Indeed, this event leads to dramatic phenotypic and/or functional modifications of both cell types. Adipocytes exhibit delipidation and acquire a fibroblast-like shape. In parallel, cancer cell aggressiveness is exacerbated through increased migratory and invasive properties. Moreover, obesity is currently a sign of poor prognosis in human carcinomas. In this context, a high number of "obese" resident adipocytes might be predicted to be detrimental. Accordingly, there are some similarities between the molecular alterations observed in hypertrophied adipocytes and in CAAs. How adipocytes function to favor tumorigenesis at the molecular level remains largely unknown. Nevertheless, progress has been made recently and molecular clues are starting to emerge. Deciphering the cellular and molecular mechanisms behind the adipocyte-cancer cell heterotypic crosstalk is of great interest since it might provide new targets for improving diagnosis/prognosis and for the design of innovative therapeutic strategies. They might also improve our understanding of the relationship between obesity/metabolic disorders and cancer risk and/or poor patient outcome.

Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.

BACKGROUND & AIMS: Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS: N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS: Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS: Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.

TRAF4, at the Crossroad between Morphogenesis and Cancer.

Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) is a gene whose expression is altered in cancers. It is overexpressed in a variety of carcinomas of different origins, often as a consequence of amplification. TRAF4 encodes an adaptor protein that belongs to the TRAF protein family. While most TRAF proteins influence immune and inflammation processes, TRAF4 is mainly involved in developmental and morphogenic processes. Interestingly, this protein has been shown to be linked to crucial cellular functions such as cell polarity and the regulation of reactive oxygen species production.

Intake of grape-derived polyphenols reduces C26 tumor growth by inhibiting angiogenesis and inducing apoptosis.

This study evaluated the in vivo antitumor activity of grape-derived polyphenols. BALB/c mice were subcutaneously implanted with C26 colon carcinoma cells, and 2 d later they received either solvent or red wine polyphenols (RWPs) (100 mg/kg/d, human equivalent dose approximately 500 mg/d) in the drinking water for 25 d. Wistar rats received either solvent or RWPs (100 mg/kg/d, human equivalent dose approximately 1000 mg/d) in the drinking water 1 wk before injection of azoxymethane and were studied 10 wk later. In mice, RWPs inhibited tumor growth by 31%, reduced tumor vascularization and the number of lung metastases, decreased proliferation as indicated by down-regulation of Ki67, cyclin D1, and UHRF1, and increased apoptosis as indicated by TUNEL staining and active caspase-3 levels in tumor cells. RWPs reduced expression of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, and cyclooxygenase-2 and increased expression of tumor suppressor genes p16(INK4A), p53, and p73 in tumor cells. In rats, RWPs reduced by 49% the number of azoxymethane-induced aberrant crypt foci (preneoplastic lesions) in colon. Thus, RWPs effectively reduced the development of colon carcinoma tumors in vivo by blunting tumor vascularization and by inhibiting proliferation and promoting apoptosis of tumor cells subsequent to an up-regulation of tumor suppressor genes.

Matrix metalloproteinase 11/stromelysin-3 exerts both activator and repressor functions during the hematogenous metastatic process in mice.

MMP11 expression is a poor prognosis factor in human carcinomas. Although it has been shown to favor primary tumor development, its role in metastatic processes remains unclear. We studied the hematogenous metastatic activity of C26 mouse colon cancer cells injected into the tail vain of wild-type or MMP11-deficient mice during 2 months. Using X-ray computed tomography to image metastasis development in recipient living mice, lung metastases were found to occur earlier and to grow faster in wild-type mice. Histological analyses of the lung, liver, kidney, adrenal gland, mammary gland, ovary and salivary gland, performed at the end of experiment, also showed lower numbers of metastases in wild-type mice, regardless of organ. Lung metastases showed similar Factor VIII-positive vascular networks regardless of the mouse MMP11 status. However, those found in MMP11-deficient mice also exhibited vessel-like structures that did not express Factor VIII, Lyve-1 and vimentin, and were not stained with PAS. Consequently, they did not correspond to vascular or lymphatic vessels or to vascular mimicry channels. Collectively, these results revealed significant spatio-temporal variability that is dependent on host MMP11 status. Furthermore, they point-out the paradoxical role of MMP11 in favoring the onset and growth of lung metastases but limiting lung foci number, and inhibiting the cancer cell dissemination to other organs. These data highlight the complexity of the metastatic process in which the same factor can play activator or repressor functions depending on the metastatic step.

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity.

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Methyl donor deficiency affects fetal programming of gastric ghrelin cell organization and function in the rat.

Methyl donor deficiency (MDD) during pregnancy influences intrauterine development. Ghrelin is expressed in the stomach of fetuses and influences fetal growth, but MDD influence on gastric ghrelin is unknown. We examined the gastric ghrelin system in MDD-induced intrauterine growth retardation. By using specific markers and approaches (such as periodic acid-Schiff, bromodeoxyuridine, homocysteine, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunostaining, reverse transcription-polymerase chain reaction), we studied the gastric oxyntic mucosa cellular organization and ghrelin gene expression in the mucosa in 20-day-old fetuses and weanling pups, and plasma ghrelin concentration in weanling rat pups of dams either normally fed or deprived of choline, folate, vitamin B6, and vitamin B12 during gestation and suckling periods. MDD fetuses weighed less than controls; the weight deficit reached 57% at weaning (P < 0.001). Both at the end of gestation and at weaning, they presented with an aberrant gastric oxyntic mucosa formation with loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Ghrelin cells were abnormally located in the pit region of oxyntic glands. At weaning, plasma ghrelin levels were decreased (-28%; P < 0.001) despite unchanged mRNA expression in the stomach. This decrease was associated with lower body weight. Taken together, these data indicate that one mechanism through which MDD influences fetal programming is the remodeling of gastric cellular organization, leading to dysfunction of the ghrelin system and dramatic effects on growth.

Caveolin-1 (P132L), a common breast cancer mutation, confers mammary cell invasiveness and defines a novel stem cell/metastasis-associated gene signature.

Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-alpha as predicted, because only estrogen receptor-alpha-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis.

Cancer cells, adipocytes and matrix metalloproteinase 11: a vicious tumor progression cycle.

This brief review focuses on the emerging role of matrix metalloproteinase 11 (MMP-11) in cancer progression. It has recently been shown that MMP-11 is induced in adipose tissue by cancer cells as they invade their surrounding environment. MMP-11 negatively regulates adipogenesis by reducing pre-adipocyte differentiation and reversing mature adipocyte differentiation. Adipocyte dedifferentiation in turn leads to the accumulation of nonmalignant peritumoral fibroblast-like cells, which favor cancer cell survival and tumor progression. This MMP-11-mediated bi-directional cross-talk between invading cancer cells and adjacent adipocytes/pre-adipocytes highlights the central role that MMP-11 plays during tumor desmoplasia and represents a molecular link between obesity and cancer.

Tumor necrosis factor receptor-associated factor 4 is a dynamic tight junction-related shuttle protein involved in epithelium homeostasis.

BACKGROUND: Despite numerous in vivo evidences that Tumor Necrosis Factor Receptor-Associated Factor 4 (TRAF4) plays a key biological function, how it works at the cellular and molecular level remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we show using immunofluorescence and immuohistochemistry that TRAF4 is a novel player at the tight junctions (TJs). TRAF4 is connected to assembled TJs in confluent epithelial cells, but accumulates in the cytoplasm and/or nucleus when TJs are open in isolated cells or EGTA-treated confluent cells. In vivo, TRAF4 is consistently found at TJs in normal human mammary epithelia as well as in well-differentiated in situ carcinomas. In contrast, TRAF4 is never localized at the plasma membrane of poorly-differentiated invasive carcinomas devoid of correct TJs, but is observed in the cytoplasm and/or nucleus of the cancer cells. Moreover, TRAF4 TJ subcellular localization is remarkably dynamic. Fluorescence recovery after photobleaching (FRAP) experiments show that TRAF4 is highly mobile and shuttles between TJs and the cytoplasm. Finally, we show that intracellular TRAF4 potentiates ERK1/2 phosphorylation in proliferating HeLa cells, an epithelial cell line known to be devoid of TJs. CONCLUSIONS/SIGNIFICANCE: Collectively, our data strongly support the new concept of TJs as a dynamic structure. Moreover, our results implicate TRAF4 in one of the emerging TJ-dependent signaling pathways that responds to cell polarity by regulating the cell proliferation/differentiation balance, and subsequently epithelium homeostasis. Drastic phenotypes or lethality in TRAF4-deficient mice and drosophila strongly argue in favor of such a function.