Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.     

IL-1beta causes an increase in intestinal epithelial tight junction permeability

IL-1beta is a prototypical proinflammatory cytokine that plays a central role in the intestinal inflammation amplification cascade. Recent studies have indicated that a TNF-alpha- and IFN-gamma-induced increase in intestinal epithelial paracellular permeability may be an important mechanism contributing to intestinal inflammation. Despite its central role in promoting intestinal inflammation, the role of IL-1beta on intestinal epithelial tight junction (TJ) barrier function remains unclear. The major aims of this study were to determine the effect of IL-1beta on intestinal epithelial TJ permeability and to elucidate the mechanisms involved in this process, using a well-established in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. IL-1beta (0-100 ng/ml) produced a concentration- and time-dependent decrease in Caco-2 transepithelial resistance. Conversely, IL-1beta caused a progressive time-dependent increase in transepithelial permeability to paracellular marker inulin. IL-1beta-induced increase in Caco-2 TJ permeability was accompanied by a rapid activation of NF-kappaB. NF-kappaB inhibitors, pyrrolidine dithiocarbamate and curcumin, prevented the IL-1beta-induced increase in Caco-2 TJ permeability. To further confirm the role of NF-kappaB in the IL-1beta-induced increase in Caco-2 TJ permeability, NF-kappaB p65 expression was silenced by small interfering RNA transfection. NF-kappaB p65 depletion completely inhibited the IL-1beta-induced increase in Caco-2 TJ permeability. IL-1beta did not induce apoptosis in the Caco-2 cell. In conclusion, our findings show for the first time that IL-1beta at physiologically relevant concentrations causes an increase in intestinal epithelial TJ permeability. The IL-1beta-induced increase in Caco-2 TJ permeability was mediated in part by the activation of NF-kappaB pathways but not apoptosis.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Mediators of interleukin-1 beta action Na(+)-K(+)ATPase in Caco-2 cells

Interleukin1-beta has been demonstrated previously to reduce the activity and expression of the Na(+)-K(+) pump in the rat jejunum and colon. This work attempts to elucidate the signal transduction pathway underlying its effect using Caco-2 cells. IL-1beta reduced, in these cells also, the activity and expression of ATPase, in a dose and time-dependent manner. The down-regulatory effect of the cytokine on the ATPase was not evident, when p38 MAP kinase was inhibited, but appeared in presence of inhibitors of MEK and NFkappaB, although activation of NF-kappaB was demonstrated by western blot analysis. The effect of IL-1beta on the pump disappeared in the presence of indomethacin, a COX inhibitor. Exogenous PGE2 reduced the expression of the pump within 15 minutes, and this effect was still apparent when p38MAPK was inhibited. Curcumin, a JNK/AP-1 inhibitor, partially abolished the effect of IL-1beta on ATPase expression but did not interfere with the effect of PGE2. These results indicate that IL-1beta reduces the expression of ATPase independently of NFkB but, through a major pathway involving p38 and COX-2/PGE2, and another pathway involving JNK/AP1.     

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

Genistein is an isoflavonic phyto-oestrogen contained in soya beans. It is thought to display anti-cancer effects. This study was designed to investigate its effect on human intestinal colon cancer Caco-2 cells. MTT assay, flow cytometric analysis and western blotting were used to investigate the effect of genistein on cell proliferation, cell cycle progression and protein alterations of selected cell cycle-related proteins in Caco-2 cells. Our results showed that genistein and daidzein significantly suppressed cell proliferation. Genistein treatment was demonstrated to modulate cell cycle distribution through accumulation of cells at G2/M phase, with a significant decreasing effect of Cyclin B1 and Serine/threonine-protein kinase 2 (Chk2) proteins expression. However, daidzein did not alter the cell cycle progression in Caco-2 cells. All these observation strongly indicate that genistein has anti-proliferative effect in human intestinal colon cancer Caco-2 cells through the down-regulation of cell cycle check point proteins, Cyclin B1 and Chk2.     

Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2

Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: -3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4-7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of alpha-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase alpha (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis.     

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPARγ is expressed at considerable levels in human colon cancer cells. This suggests that PPARγ expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPARγ expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPARγ mRNA and protein in these cells were in the order HT-29>LOVO>Caco-2>DLD-1. We also found that PPARγ overexpression promoted cell growth inhibition in PPARγ lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPARγ expression and the cells' sensitivity for proliferation.     

Induction of interleukin-1 beta production in human dermal fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha. Involvement of protein kinase-dependent and adenylate cyclase-dependent regulatory pathways.

It has previously been demonstrated that interleukin-1 (IL-1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL-1 beta production by cultured human dermal fibroblasts. We have shown that IL-1 beta is constitutively expressed as a cell-associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) exerted a dose-dependent stimulation on the production of the cell-associated IL-1 beta, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL-1 beta production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 microgram/ml) of PGE2. In contrast, higher concentrations (0.1 and 1 micrograms/ml) of PGE2, as well as exogenous dibutyryl-cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL-1 alpha and TNF-alpha. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL-1 beta expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2-induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL-1 alpha and TNF-alpha, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL-1 beta induction by the two cytokines studied.     

Picroside III,an Active Ingredient of Picrorhiza scrophulariiflora,Ameliorates Experimental Colitis by Protecting Intestinal Barrier Integrity

This study aims to explore the protective effects of Picroside III, an active ingredient of Picrorhiza scrophulariiflora, on the intestinal epithelial barrier in tumor necrosis factor-α (TNF-α) induced Caco-2 cells and dextran sulfate sodium (DSS) induced colitis in mice. Results show that Picroside III significantly alleviated clinical signs of colitis including body weight loss, disease activity index increase, colon shortening, and colon tissue damage. It also increased claudin-3, ZO-1 and occludin expressions and decreased claudin-2 expression in the colon tissues of mice with colitis. In vitro, Picroside III also significantly promoted wound healing, decreased the permeability of cell monolayer, upregulated the expressions of claudin-3, ZO-1 and occludin and downregulated the expression of claudin-2 in TNF-α treated Caco-2 cells. Mechanism studies show that Picroside III significantly promoted AMP-activated protein kinase (AMPK) phosphorylation in vitro and in vivo, and blockade with AMPK could significantly attenuate the upregulation of Picroside III in ZO-1 and occludin expressions and the downregulation of claudin-2 expression in TNF-α treated Caco-2 cells. In conclusion, this study demonstrates that Picroside III attenuated DSS-induced colitis by promoting colonic mucosal wound healing and epithelial barrier function recovery via the activation of AMPK.     

alpha3beta1 integrin induced suppression of the Caco-2 epithelial cell IL-1 signaling pathway leading to NF-(kappa)B activation

Intestinal epithelial cells (IECs) produce several potent cytokines in response to interleukin-1 (IL-1) and may play a role in the inflammatory response. Previously, we determined that treatment of the Caco-2 cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced cytokine secretion and mRNA levels, suggesting that the alpha3beta1 integrin may play a role in the regulation of IEC cytokine responses to IL-1. In this report, treatment of the Caco-2 cells with the anti-alpha3 integrin antibody resulted in a suppression of IL-1 induced levels of NF-kappaB binding activity in nuclear extracts, as determined by EMSA, as well as phosphorylation and degradation of the inhibitor, I(kappa)B(alpha). The anti-integrin antibody treatment was also found to suppress I(kappa)B kinase (IKK) activity and IKK(beta) phosphorylation. Culture of the Caco-2 cells on purified laminin-5, the ligand for the alpha3beta1 integrin, also resulted in suppression of IL-1 induced phosphorylation of I(kappa)B(alpha) and IKK(beta). Together with our previous findings, these results suggest that alpha3beta1 integrin binding results in a suppression of the IL-1 signaling pathway leading to the activation of NF-(kappa)B and ultimately IEC cytokine responses. These studies define a novel regulatory mechanism which may be important in the control of IEC cytokine responses during inflammation.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.