Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.     

Human intestinal epithelial cell survival: differentiation state-specific control mechanisms

To investigate whether human intestinal epithelial cell survival involves distinct control mechanisms depending on the state of differentiation, we analyzed the in vitro effects of insulin, pharmacological inhibitors of Fak, MEK/Erk, and PI3-K/Akt, and integrin (beta1, beta4)-blocking antibodies on the survival of the well-established human Caco-2 enterocyte-like and HIEC-6 cryptlike cell models. In addition, relative expression levels of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, and Bad) and activation levels of Fak, Erk-2, and Akt were analyzed. Herein, we report that 1) the enterocytic differentiation process results in the establishment of distinct profiles of Bcl-2 homolog expression levels, as well as p125(Fak), p42(Erk-2), and p57(Akt) activated levels; 2) the inhibition of Fak, of the MEK/Erk pathway, or of PI3-K, have distinct impacts on enterocytic cell survival in undifferentiated (subconfluent Caco-2, confluent HIEC-6) and differentiated (30 days postconfluent Caco-2) cells; 3) exposure to insulin and the inhibition of Fak, MEK, and PI3-K resulted in differentiation state-distinct modulations in the expression of each Bcl-2 homolog analyzed; and 4) Fak, beta1 and beta4 integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of cell differentiation. Taken together, these data indicate that human intestinal epithelial cell survival is regulated according to differentiation state-specific control mechanisms.     

Down-regulation of MEK/ERK signaling by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells

In vitro experiments have shown that the establishment of cell-cell contacts in intestinal epithelial cell cultures is a critical step in initiating ERK inhibition, cell cycle arrest, and induction of the differentiation process. Herein, we determined the mechanisms through which E-cadherin-mediated cell-cell contacts modulate the ERK pathway in intestinal epithelial cells. We report that: (1) removal of calcium from the culture medium of newly confluent Caco-2/15 cells (30 min, 4 mM EGTA) results in the disruption of both adherens and tight junctions and clearly decreases Akt phosphorylation while increasing MEK and ERK activities. Akt, MEK, and ERK activation levels return to control levels 60 min after calcium restoration; (2) the use of E-cadherin blocking antibodies efficiently prevents Akt phosphorylation and MEK-ERK inhibition after 70 min of calcium restoration; (3) using the PI3K inhibitor LY294002 (15 microM) in calcium switch experiments, we demonstrate that the assembly of adherens junctions activates Akt activity and triggers the inhibition of ERK1/2 activities in a PI3K-dependent manner; (4) adenoviral infection of confluent Caco-2/15 cells with a constitutively active mutant of Akt1 strongly represses ERK1/2 activities; (5) inhibition of PI3K abolishes Akt activity but leads to a rapid and sustained activation of the MEK-ERK1/2 in confluent differentiating Caco-2/15 cells, but not in undifferentiated growing Caco-2/15 cells. Our data suggest that E-cadherin engagement leads to MEK/ERK inhibition in a PI3K/Akt-dependent pathway. This mechanism may account for the role of E-cadherin in proliferation/differentiation transition along the crypt-villus axis of the human intestinal epithelium.     

Gene expression of activin, activin receptors, and follistatin in intestinal epithelial cells

Gene expression of activin, activin receptors, and follistatin was investigated in vivo and in vitro using semiquantitative RT-PCR in intestinal epithelial cells. Rat jejunum and the intestinal epithelial cell line IEC-6 expressed mRNA encoding the betaA-subunit of activin, alpha-subunit of inhibin, activin receptors IB and IIA, and follistatin. An epithelial cell isolation study focused along the crypt-villus axis in rat jejunum showed that betaA mRNA levels were eight- to tenfold higher in villus cells than in crypt cells. Immunohistochemistry revealed the expression of activin A in upper villus cells. The human intestinal cell line Caco-2 was used as a differentiation model of enterocytes. Four- to fivefold induction of betaA mRNA was observed in postconfluent Caco-2 cells grown on filter but not in those cells grown on plastic. In contrast, follistatin mRNA was seen to be reduced after reaching confluence. Exogenous activin A dose-dependently suppressed the proliferation and stimulated the expression of apolipoprotein A-IV gene, a differentiation marker, in IEC-6 cells. These results suggest that the activin system is involved in the regulation of such cellular functions as proliferation and differentiation in intestinal epithelial cells.     

Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.     

Growth factor TGF-β induces intestinal epithelial cell (IEC-6) differentiation: miR-146b as a regulatory component in the negative feedback loop

TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TGF-β-induced intestinal cell growth. In this study, TGF-β caused a morphological change of IEC-6 cells and stimulated expression of the epithelial cell markers alkaline phosphatase, villin, and aminopeptidase N. By global microRNA profiling during TGF-β-induced intestinal crypt cell (IEC-6) differentiation, we identified 19 differentially expressed microRNAs. We showed by real-time Q-PCR that miR-146b expression increased rapidly after TGF-β treatment; sequence analysis and in vitro assays revealed that miR-146b targets SIAH2, an E3 ubiquitin ligase, with decreased protein expression upon IEC-6 cell differentiation. Transfection of miR-146b inhibitor before TGF-β treatment blocked the down-regulation of SIAH2 in response to TGF-β. Moreover, SIAH2 over-expression during TGF-β treatment caused a significant decrease in Smad7 protein expression in IEC-6 cells. Furthermore, activation of the ERK1/2 pathway is active in the up-regulation of miR-146b by TGF-β. These findings suggest a novel mechanism whereby TGF-β signaling during IEC-6 cell differentiation may be modulated in part by microRNAs, and we propose a key role for miR-146b in the homeostasis of growth factor TGF-β signaling through a negative feedback regulation involving down-regulation of SIAH2 repressed Smad7 activities.     

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 μM), and, less potently, by Caco-2 cells (IC(50) = 402 μM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 μM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.     

Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells.     

Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells

Constitutive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form cystlike structures in collagen gels and induces an invasive, myofibroblastlike phenotype. However, the reversibility of these cellular events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been investigated. We now report that loss of constitutively active MEK1 (caMEK1) and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with increased MEK2 protein expression, reexpression of ERK1 protein, and epithelial redifferentiation of these cells. The morphological changes toward an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7) are reflected by the upregulation of epithelial marker proteins, such as E-cadherin, -catenin, and cytokeratin, by the loss of -smooth muscle actin expression, and by the ability of these epithelial revertants to form well-organized spherical cysts when grown in three-dimensional collagen gels. Further evidence for a role of the MEK1-ERK1/2 module in epithelial-mesenchymal transition was obtained from the analysis of two novel, spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells). In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein expression, and loss of ERK1 protein expression is associated with phenotypic alterations similar to those observed in transdifferentiated C7caMEK1 cells. C7e1 cells at least partially regained some of their epithelial characteristics at higher passages. In contrast, C7e2 cells maintained a transdifferentiated phenotype at high passage, were unable to generate cystlike epithelial structures, and retained invasive properties when grown on a three-dimensional collagen matrix. We conclude that in renal epithelial MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated with loss of ERK1 protein expression, reduced MEK2 protein expression, and increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2 results in increased MEK2 protein expression and reexpression of ERK1 protein, concomitant with the restoration of epithelial phenotype and the ability to form cystic structures. mitogen-activated protein kinase; extracellular signal-regulated kinase; epithelial differentiation; epithelial-to-mesenchymal transition; invasion; Madin-Darby canine kidney cells     

TNF-α induces autophagy through ERK1/2 pathway to regulate apoptosis in neonatal necrotizing enterocolitis model cells IEC-6

Necrotizing enterocolitis (NEC) is a potentially fatal illness in premature neonates. Tumor necrosis factor-α (TNF-α) and autophagy are associated with the pathogenesis of NEC. This study aimed to explore whether TNF-α might regulate apoptosis in neonatal NEC model cells IEC-6 via regulation of autophagy. NEC rat model was induced by hand feeding and exposure to asphyxia/cold-stress for histologic examination. The NEC in vitro model (IEC-6/NEC cells) was established by stimulating the intestinal epithelial cell line IEC-6 with lipopolysaccharide (LPS, 100 μg/mL) for 3 h to investigate the effects of TNF-α on IEC-6 proliferation and apoptosis. In this study, NEC rats showed decreased proliferating cell nuclear antigen (PCNA) expression, increased TUNEL-positive cells, higher expression of TNF-α, p-ERK1/2, and autophagy-related proteins in rat small intestine compared with their controls. Additionally, the LPS-stimulated IEC-6/NEC cells showed a significantly decreased proliferation and increased apoptosis compared with the control cells. Furthermore, the LPS-stimulated IEC-6/NEC cells exhibited enhanced autophagy level, as evidenced by a dose-dependent increase in Beclin-1 protein expression, LC3II/LC3I ratio and accumulation of MDC-positive autophagic vacuoles. Moreover, inhibition of autophagy by wortmannin or LY294002 significantly abolished the LPS-mediated decreased proliferation and increased apoptosis of IEC-6/NEC cells. Results also showed that inhibition of ERK1/2 pathway using U0126 significantly inhibited TNF-α-induced autophagy. Furthermore, the TNF-α-mediated inhibition of IEC-6 proliferation and promotion of IEC-6 apoptosis was abolished by U0126. Our findings demonstrated that TNF-α might induce autophagy through ERK1/2 pathway to regulate apoptosis in neonatal NEC cells IEC-6. Our study enhances our understanding of neonatal NEC pathogenesis.