Inhibition of basal p38 or JNK activity enhances epithelial barrier function through differential modulation of claudin expression.

Tight junctions (TJs) form a barrier to the paracellular diffusion of ions and solutes across epithelia. Although transmembrane proteins of the claudin family have emerged as critical determinants of TJ permeability, little is known about the signaling pathways that control their expression. The aim of this study was to assess the role of three mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinase-1/2 (ERK1/2), c-Jun NH(2)-terminal kinases (JNKs), and p38 kinases, in the regulation of epithelial barrier function and claudin expression in mammary epithelial cells. Addition of either PD169316 (a p38 inhibitor) or SP600125 (a JNK inhibitor) induced formation of domes (a phenomenon dependent on TJ barrier function) and enhanced transepithelial electrical resistance, whereas U0126 (an inhibitor of the ERK1/2 activators MEK1/MEK2) had no significant effect. Similar results were obtained using mechanistically unrelated p38 or JNK inhibitors. PD169316 increased the expression of claudin-4 and -8, whereas SP600125 increased claudin-4 and -9 and downregulated claudin-8. Silencing of p38alpha by isoform-specific small interfering RNAs increased claudin-4 and -8 mRNAs, whereas silencing of p38beta only increased claudin-4 mRNA. Silencing of either JNK1 or JNK2 increased claudin-9 mRNA expression while decreasing claudin-8 mRNA. Moreover, selective silencing of JNK2 increased claudin-4 and -7 mRNAs. Finally, both PD169316 and SP600125 inhibited the paracellular diffusion of Na(+) and Cl(-) across epithelial monolayers. Collectively, these results provide evidence that inhibition of either p38 or JNK enhances epithelial barrier function by selectively modulating claudin expression, implying that the basal activity of these MAPKs exerts a tonic effect on TJ ionic permeability.

Unsaturated fatty acids promote hepatoma proliferation and progression through downregulation of the tumor suppressor PTEN.

BACKGROUND/AIMS: The impact of dietary fatty acids on the development of cancers is highly controversial. We recently demonstrated that unsaturated fatty acids trigger the downregulation of the tumor suppressor PTEN through an mTOR/NF-kappaB-dependent mechanism in hepatocytes. In this study, we investigated whether unsaturated fatty acids promote hepatoma progression by downregulating PTEN expression. METHODS: The effects of fatty acids and PTEN-specific siRNAs on proliferation, invasiveness and gene expression were assessed using HepG2 hepatoma cells. The tumor promoting activity of unsaturated fatty acids was evaluated in vivo using HepG2 xenografts in nude mice. RESULTS: Incubation of HepG2 cells with unsaturated fatty acids, or PTEN-specific siRNAs, increased cell proliferation, cell migration and invasiveness, and altered the expression of genes involved in inflammation, epithelial-to-mesenchymal transition and carcinogenesis. These effects were dependent on PTEN expression levels and were prevented by mTOR and NF-kappaB inhibitors. Consistent with these data, the development and size of subcutaneous HepG2-derived tumors in nude mice xenografts were dramatically increased when mice were fed with an oleic acid-enriched diet, even in the absence of weight gain. CONCLUSIONS: These data demonstrate that dietary unsaturated fatty acids promote hepatoma progression by reducing the expression of the tumor suppressor PTEN.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Induction of CXCL1 by extracellular matrix and autocrine enhancement by interleukin-1 in rat pancreatic beta-cells.

As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix. Four cytokines were overexpressed on 804G-ECM compared with poly-L-lysine: C-X-C motif ligand 1 (CXCL1), CXCL2, interferon-inducible protein-10, and IL-1beta. A time-course experiment indicated that maximal induction by 804G-ECM of CXCL1/2 and interferon-inducible protein-10 occurred at 4 h. Stimulation of CXCL1 release by beta-cells on 804G-ECM was confirmed at the protein level. Moreover, secreted CXCL1 was shown to be functionally active by attracting rat granulocytes. Preventing the interaction of beta1 integrins and laminin-5 (a major component of 804G-ECM) with specific antibodies resulted in a 40-50% inhibition of CXCL1 expression. Using the nuclear factor-kappaB pathway inhibitor Bay 11-7082 it is demonstrated that CXCL1 expression and secretion are dependent on nuclear factor-kappaB activation. IL-1 secreted by beta-cells plated on 804G-ECM was found to be a key soluble mediator because treatment of cells with the IL-1 receptor antagonist significantly reduced both CXCL1 gene expression and secretion. It is concluded that ECM induces expression of cytokines including CXCL1 with amplification by IL-1 acting via a positive autocrine feedback loop.

Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells.

BACKGROUND: Formation of branching tubes is a fundamental step in the development of glandular organs. To identify extracellular cues that orchestrate epithelial tubulogenesis, we employed an in vitro assay in which EpH4-J3B1A mammary epithelial cells form spheroidal cysts when grown in collagen gels under serum-free conditions, but form branching tubules in the presence of fetal calf serum (FCS). RESULTS: Initial experiments showed that the tubulogenesis-inducing activity of FCS was markedly increased by heating (70 degrees C) or transient acidification to pH3. We therefore hypothesized that the tubulogenic agent was transforming growth factor-beta (TGF-beta), a cytokine that is present in serum in latent form and can be activated by heat or acid treatment. We found indeed that the tubulogenic activity of acidified FCS is abrogated by addition of either SB-431542, a selective inhibitor of the TGF-beta type I receptor, or a neutralizing antibody to TGF-beta-1. On the other hand, addition of low concentrations (20-100 pg/ml) of exogenous TGF-beta-1 recapitulated the effect of acidified FCS in inducing morphogenesis of hollow tubes. In contrast, higher concentrations of TGF-beta-1 induced the formation of thin cellular cords devoid of a detectable lumen. To gain insight into the mechanisms underlying TGF-beta-1-induced tube formation, we assessed the potential role of matrix metalloproteinases (MMPs). By western blot and gelatin zymography, we observed a dose-dependent increase in MMP-9 upon TGF-beta-1 treatment. Tube formation was suppressed by a synthetic broad-spectrum metalloproteinase inhibitor, by recombinant tissue inhibitor of metalloproteinases-2 (TIMP-2) and by a selective inhibitor of MMP-9, indicating that this morphogenetic process requires the activity of MMP-9. CONCLUSION: Altogether, our results provide evidence that, at low concentrations, TGF-beta-1 promotes MMP-dependent branching tubulogenesis by mammary epithelial cells in vitro, and suggest that it plays a similar role during mammary gland development in vivo.

Tumour necrosis factor alpha confers an invasive, transformed phenotype on mammary epithelial cells.

Although loss of cell-cell adhesion and gain of invasive properties play a crucial role in the malignant progression of epithelial tumours, the molecular signals that trigger these processes have not been fully elucidated. In light of the well-established relationship between chronic inflammation and cancer, we hypothesized that pro-inflammatory cytokines disrupt epithelial-cell adhesion and promote cell migration. To test this hypothesis, we used an in vitro model in which 31EG4-2A4 mouse mammary epithelial cells grown in a collagen gel form compact spheroidal colonies. Among the several cytokines examined, tumour necrosis factor alpha (TNF-alpha) caused a pronounced 3D scattering of preformed epithelial-cell colonies and induced 31EG4-2A4 cells grown on top of a collagen gel to invade the underlying matrix. In addition, TNF-alpha abolished contact-mediated inhibition of cell proliferation and stimulated cell growth both in the absence of exogenous mitogens and under anchorage-independent conditions. TNF-alpha induced the expression of matrix metalloproteinase 9 (MMP-9). Addition of the MMP inhibitor BB-94 abrogated TNF-alpha-induced 3D scattering. TNF-alpha also enhanced the attachment of 31EG4-2A4 cells to type-I collagen and markedly increased the expression of the alpha2 integrin subunit. Addition of a blocking antibody to beta1-integrin or of rhodocetin (a specific alpha2beta1 antagonist) to collagen-gel cultures abrogated 3D scattering. Collectively, these results demonstrate an essential role for MMPs and alpha2beta1 integrin in the invasive response of 31EG4-2A4 cells to TNF-alpha. We propose that the biological activities described in this study contribute to the ability of TNF-alpha to promote tumour progression and cancer-cell dissemination.

Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins.

Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells.

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.

Neutrophils as a key cellular target for angiostatin: implications for regulation of angiogenesis and inflammation.

Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis-associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL-8, MIP-2, and GROalpha), formyl-Met-Leu-Phe (fMLP), and 12-O-tetradecanoylphorbol 13-acetate, and repressed fMLP-induced mitochondrial activity. Two different angiostatin forms (kringles 1-4 and 1-3) were effective, whereas whole plasminogen had no effect. IL-8, MIP-2, and GROalpha induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine-induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil-mediated angiogenesis and indicate that angiostatin might inhibit inflammation.