Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells.

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-kappaB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-kappaB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-kappaB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-kappaB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-alpha and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells.

Growth phase-dependent expression of ICAD-L/DFF45 modulates the pattern of apoptosis in human colonic cancer cells.

The inhibitor of caspase-3-activated DNase (ICAD) is a caspase-3 substrate that controls nuclear apoptosis. ICAD has two isoforms: a functional isoform of M(r) 45,000, ICAD-L/DNA fragmentation factor (DFF) 45; and a M(r) 35,000 isoform, ICAD-S/DFF35. ICAD-deficient murine cells display resistance to apoptotic stimuli and absence of typical nuclear changes of apoptosis. Our aim was to: (a) characterize the ICAD expression in several human colonic cancer cell lines compared with human normal colonocytes; and (b) correlate the phenotypic features of apoptosis to the level of ICAD expression. ICAD expression was assessed by immunoblot analysis. Early markers of apoptosis of cultured cells included lactate dehydrogenase retention in dying cells, cytokeratin 18 cleavage, and caspase-3 activation. Nuclear markers of apoptosis were assessed by Hoechst staining of nuclei, electron microscopy, and DNA electrophoresis. Inhibition of caspases was performed using a broad-spectrum caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone. ICAD expression was restricted to the functional ICAD-L/DFF45 isoform in colonic cancer cells as well as in human normal colonocytes. In a clonal derivative of HT29 cells (HT29-Cl.16E cells), ICAD expression was found to be down-regulated during the exponential phase of growth, and the cell death triggered by IFN-gamma, anti-Fas antibody plus Adriamycin was characterized by the expression of early markers of apoptosis, whereas the key nuclear features of apoptosis were absent. In contrast, exposure of confluent cells to this treatment led to a typical apoptotic nuclear fragmentation. Both forms of apoptosis, in exponentially growing and confluent cells, were sensitive to the broad spectrum inhibitor of caspases, z-Val-Ala-Asp-fluoromethyl ketone. Our findings support the concept that the expression of ICAD is essential to the execution of full-blown apoptosis in colonic cancer cells. Altogether, our results point to ICAD as a potential target for restoring a normal apoptotic signal transduction pathway in colonic cancer cells.

Position in cell cycle controls the sensitivity of colon cancer cells to nitric oxide-dependent programmed cell death.

Mounting evidence suggests that the position in the cell cycle of cells exposed to an oxidative stress could determine their survival or apoptotic cell death. This study aimed at determining whether nitric oxide (NO)-induced cell death in colon cancer cells might depend on their position in the cell cycle, based on a clone of the cancer cell line HT29 exposed to an NO donor, in combination with the manipulation of the cell entry into the cell cycle. We show that PAPA NONOate (pNO), from 10(-4) m to 10(-3) m, exerted early and reversible cytostatic effects through ribonucleotide reductase inhibition, followed by late resumption of cell growth at 5 x 10(-4) m pNO. In contrast, 10(-3) m pNO led to late programmed cell death that was accounted for by the progression of cells into the cell cycle as shown by (a) the accumulation of apoptotic cells in the G(2)-M phase at 10(-3) m pNO treatment; and (b) the prevention of cell death by inhibiting the entry of cells into the cell cycle. The entry of pNO-treated cells into the G(2)-M phase was associated with actin depolymerization and its S-glutathionylation in the same way as in control cells. However, the pNO treatment interfered with the build-up of a high reducing power, associated in control cells with a dramatic increase in reduced glutathione biosynthesis in the G(2)-M phase. This oxidative stress prevented the exit from the G(2)-M phase, which requires a high reducing power for actin deglutathionylation and its repolymerization. Finally, our demonstration that programmed cell death occurred through a caspase-independent pathway is in line with the context of a nitrosative/oxidative stress. In conclusion, this work, which deciphers the connection between the position of colonic cancer cells in the cell cycle and their sensitivity to NO-induced stress and their programmed cell death, could help optimize anticancer protocols based on NO-donating compounds.

Colonic leptin: source of a novel proinflammatory cytokine involved in IBD.

Leptin, a peptide encoded by the obese (ob) gene, is primarily secreted by adipocytes and is a critical hormone that controls body weight due to its central effects. Recently, additional roles for leptin in the gastrointestinal tract have been suggested because gastric lining cells also produce and release leptin in response to meal-related stimuli. While gastric epithelia might thus directly contribute to circulating leptin following a meal, here we show that inflamed colonic epithelial cells express and release leptin apically into the intestinal lumen. In addition, we demonstrate leptin expression and secretion in vitro in epithelial cells. In response to luminal leptin, model intestinal epithelia critically activate the NF-kappaB, a key signaling system to pro-inflammatory stimuli. The inflammatory effect of luminal leptin was characterized in vivo in mice administered intrarectal leptin. Leptin induced epithelial wall damage and neutrophil infiltration that represent characteristic histological findings in acute intestinal inflammation. These observations provide evidence for an intraluminal biological signaling of leptin and a new pathophysiological role for intraluminal leptin during states of intestinal inflammation such as inflammatory bowel disease.

ADAM-15/metargidin mediates homotypic aggregation of human T lymphocytes and heterotypic interactions of T lymphocytes with intestinal epithelial cells.

Intestinal epithelial cells (IEC) play an immunoregulatory role in the intestine. This role involves cell-cell interactions with intraepithelial lymphocytes that may also play a role in some enteropathies. The discovery of the RGD motif-containing Protein ADAM-15 (a disintegrin and metalloprotease-15) raises the question of its involvement in these cell-cell interactions. Cell adhesion assays were performed using the Jurkat E6.1 T cell line as a model of T lymphocytes and Caco2-BBE monolayers as a model of intestinal epithelia. Our results show that an anti-ADAM-15 ectodomain antibody inhibited the attachment of Jurkat cells on Caco2-BBE monolayers. Overexpression of ADAM-15 in Caco2-BBE cells enhanced Jurkat cell binding, and overexpression of ADAM-15 in Jurkat cells enhanced their aggregation. Mutagenesis experiments showed that both the mutation of ADAM-15 RGD domain or the deletion of its cytoplasmic tail decreased these cell-cell interactions. Moreover, wound-healing experiments showed that epithelial ADAM-15-mediated Jurkat cell adhesion to Caco2-BBE cells enhances the mechanisms of wound repair. We also found that ADAM-15-mediated aggregation of Jurkat cells increases the expression of tumor necrosis factor-alpha mRNA. These results demonstrate the following: 1) ADAM-15 is involved in heterotypic adhesion of intraepithelial lymphocytes to IEC as well as in homotypic aggregation of T cells; 2) both the RGD motif and the cytoplasmic tail of ADAM-15 are involved for these cell-cell interactions; and 3) ADAM-15-mediated cell-cell interactions are involved in mechanisms of epithelial restitution and production of pro-inflammatory mediators. Altogether these findings point to ADAM-15 as a possible therapeutic target for prevention of inappropriate T cell activation involved in some pathologies.

The oligopeptide transporter hPepT1: gateway to the innate immune response.

Bacterial products that are normally present in the lumen of the colon, such as N-formylated peptides and muramyl-dipeptide, are important for inducing the development of mucosal inflammation. The intestinal dipeptide transporter, hPepT1, which is expressed in inflamed but not in noninflamed colonic epithelial cells, mediates the transport of these bacterial products into the cytosol of colonic epithelial cells. The small bacterial peptides subsequently induce an inflammatory response, including the induction of MHC class I molecules expression and cytokines secretion, via the activation of nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins, for example NOD2, and activation of NF-kappaB. Subsequent secretion of chemoattractants by colonic epithelial cells induces the movement of neutrophils through the underlying matrix, as well as across the epithelium. These bacterial products can also reach the lamina propria through the paracellular pathway and across the basolateral membrane of epithelial cells. As a consequence, small formylated peptides can interact directly with immune cells through specific membrane receptors. Since immune cells, including macrophages, also express hPepT1, they can transport small bacterial peptides into the cytosol where these may interact with the NBS-LRR family of intracellular receptors. As in intestinal epithelial cells, the presence of these small bacterial peptides in immune cells may trigger immune response activation.

Dystroglycan receptor is involved in integrin activation in intestinal epithelia.

The dystroglycans (alpha-DG and beta-DG), which play important roles in the formation of basement membranes, have been well studied in skeletal muscle and nerve, but their expression and localization in intestinal epithelial cells has not been previously investigated. Here, we demonstrated that the DG complex, composed of alpha-DG, beta-DG, and utrophin, is specifically expressed in the basolateral membrane of the Caco-2-BBE monolayer. The DG complex coprecipitated with beta(1)-integrin, suggesting a possible interaction among these proteins. In addition, we observed that activation of DG receptors by laminin-1 enhanced the interaction between beta(1)-integrin and laminin-1, whereas activation of DG receptors by laminin-2 reduced the interaction between beta(1)-integrin and laminin-2. Finally, we demonstrated that the intracellular COOH-terminal tail of beta-DG and its binding to the DG binding domain of utrophin are crucial for the interactions between laminin-1/-2 and beta(1)-integrin. Collectively, these novel results indicate that dystroglycans play important roles in the regulation of interactions between intestinal epithelial cells and the extracellular matrix.

hPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes.

Here, we examined hPepT1 expression in the monocytic cell line, KG-1. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that hPepT1 is expressed in KG-1 cells, while cDNA cloning and direct sequencing confirmed the sequence of KG-1 hPepT1 (accession number, AY634368). Immunoblotting of cell lysates from KG-1 cells or macrophages isolated from human peripheral blood revealed a approximately 100 kDa immunoreactive band mainly present in the membrane fraction. Uptake experiments showed that the transport of 20 microM radiolabeled Gly-Sarcosine ([14C]Gly-Sar) in KG-1 cells was Na+, Cl- dependent and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS)-sensitive. In addition, hPepT1 activity was likely to be coupled to a Na+/H+ exchanger, as evidenced by the fact that [14C]Gly-Sar uptake was not affected by the absence of Na+ when cells were incubated at low pH (5.2). Interestingly, hPepT1-mediated transport was reduced in KG-1 cells incubated at low pH as it was also observed in nonpolarized Caco2-BBE cells. This pattern of pH-dependence is due to a disruption of the driving force of hPepT1-mediated transport events. This was supported by our finding that nonpolarized cells, Caco2-BBE cells and KG-1 cells, have an increased permeability to H+ when compared to polarized Caco2-BBE cells. Finally, we showed that hPepT1 is responsible for transporting fMLP into undifferentiated and differentiated (macrophage-like) KG-1 cells. Together, these results show that hPepT1 is expressed in nonpolarized immune cells, such as macrophages, where the transporter functions best at the physiological pH 7.2. Furthermore, we provide evidence for hPepT1-mediated fMLP transport, which might constitute a novel immune cell activation pathway during intestinal inflammation.

Activation of epithelial CD98 glycoprotein perpetuates colonic inflammation.

Anomalies in the regulation and function of integrins have been implicated in the etiology of various pathologic conditions, including inflammatory disorders such as irritable bowel disease. Several classes of cell surface glycoproteins such as CD98 have been shown to play roles in integrins-mediated events. Here, we investigated the role of CD98 in intestinal inflammation using both in vivo and in vitro approaches. We found that in Caco2-BBE monolayers and colonic tissues, expression of CD98 was upregulated by the proinflammatory cytokine, interferon gamma (INF gamma). Furthermore, CD98 was highly upregulated in colonic tissues from mice with active colitis induced by dextran sodium sulfate (DSS), but not in DSS-treated INF gamma -/- mice. Administration of an anti-CD98 antibody worsened DSS-induced colitis in mice but had no effect on untreated control mice. Finally, we used Caco2-BBE cell monolayers to model intestinal epithelial wound healing, and found that activation of epithelial CD98 in DSS-treated monolayers inhibited monolayer reconstitution, but had no affect on untreated control monolayers. Our data collectively indicate that (i) CD98 upregulation is mediated by INF gamma during intestinal inflammation and (ii) activation of epithelial CD98 protein aggravates intestinal inflammation by reducing intestinal epithelial reconstitution. Overall, our data suggest that epithelial CD98 plays an important role in the perpetuation of intestinal inflammation.

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers.

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.

CD98 and intracellular adhesion molecule I regulate the activity of amino acid transporter LAT-2 in polarized intestinal epithelia.

We have previously shown that the heterodimer CD98/LAT-2 (LAT-2: amino acid transporter) is expressed in the basolateral membrane of intestinal epithelia and is associated with beta1 integrin (Merlin, D., Sitaraman, S., Liu, X., Easterburn, K., Sun, J., Kucharzik, T., Lewis, B., and Madara, J. L. (2001) J. Biol. Chem. 276, 39282-39289). In the present study we examined the interaction of CD98/LAT2 with intracellular adhesion molecule I (ICAM-1) and the potential of such interaction on the activation of intracellular signal in Caco2-BBE cell monolayers. ICAM-1 was found to be expressed to the basolateral domain and to selectively coimmunoprecipitate with CD98/LAT-2 in Caco2-BBE monolayers. Using antibodies as ligands to CD98 and ICAM-1, we demonstrate that the basolateral cross-linking of CD98 and ICAM-1 differentially affects the intrinsic activity of the LAT-2 transporter. Whereas CD98 ligation decreases the Km and Vm of the LAT-2 transporter, ICAM-1 ligation increases Km and Vm of the amino acid transporter LAT-2. In addition, basolateral cross-linking of CD98 or ICAM-1 induces threonine phosphorylation of an approximately 160-kDa supramolecular complex that is consistent with CD98/LAT-2-ICAM-1 complex. Together these findings demonstrate that (i). CD98/LAT-2 interacts with ICAM-1 in Caco2-BBE cell monolayers, and (ii). CD98 and ICAM-1 ligands generate intracellular signals that regulate the amino acids transporter (LAT-2) activity. Our data provide a novel mechanism by which events such as adhesion may be integrated by amino acid transport activity resulting from the direct interaction of cell surface molecules such as CD98 and ICAM-1.

Interferon-gamma increases hPepT1-mediated uptake of di-tripeptides including the bacterial tripeptide fMLP in polarized intestinal epithelia.

Interferon-gamma causes a global phenotypic switch in intestinal epithelial function, in which enterocytes become immune accessory cells. The phenotypic switch is characterized by a down-regulation of membrane transporters and up-regulation of immune accessory molecules in intestinal epithelial cells. However, the effect of interferon-gamma on the intestinal epithelia di-tripeptide hPepT1 transporter has not been investigated. In this study we demonstrate that 1) interferon-gamma increases di-tripeptide uptake in dose- and time-dependent manner in model intestinal epithelia (Caco-2 BBE cell monolayers), 2) the increase in di-tripeptides induced by interferon-gamma is hPepT1 mediated, 3) interferon-gamma does not affect the hPept1 expression at the mRNA and protein levels 4) interferon-gamma increases the intracellular pH and consequently enhances the H+-electrochemical gradient across apical plasma membrane in model intestinal epithelia (Caco2-BBE monolayers). We suggest that interferon-gamma could increase the hPepT1 mediated di-tripeptides uptake in inflamed epithelial cells. Under these conditions, interferon-gamma will increase the intracellular amount of such diverse prokaryotic and eucaryotic small di-tripeptides in inflamed epithelial cells. The intracellular accumulation of such di-tripeptides may be important in enterocytes becoming immune accessory cells.

Metabolic control of resistance of human epithelial cells to H2O2 and NO stresses.

The carbon flux through the oxidative branch of the pentose phosphate pathway (PPP) can be viewed as an integrator of the antioxidant mechanisms via the generation of NADPH. It could therefore be used as a control point of the cellular response to an oxidative stress. Replacement of glucose by galactose sensitized the human epithelial cell line HGT-1 to H2O2 stress. Here we demonstrate that, due to the restricted galactose flux into the PPP, the H2O2 stress led to early cellular blebbing followed by cell necrosis, these changes being associated with a fall in the NADPH/NADP+ ratio and GSH depletion. H2O2 cytotoxicity was prevented by adding 2-deoxyglucose (2dGlc). This protection was associated with an increased flow of 2-deoxyglucose 6-phosphate into the oxidative branch of the PPP together with the prevention of the NADPH/NADP+ fall and the maintenance of intracellular GSH redox homoeostasis. Inhibitors of enzyme pathways connecting the PPP to GSH recycling abolished the 2dGlc protection. In carbohydrate-free culture conditions, 2dGlc dose-dependent protective effect was paralleled by a dose-dependent influx of 2dGlc into the PPP leading to the maintenance of the intracellular redox status. By contrast, in Glc-fed cells, the PPP was not a control point of the cellular resistance to H2O2 stress as they maintained a high NADPH/NADP+ ratio. Both 2dGlc and Glc inhibited, through the maintenance of GSH redox status, NO cytotoxicity on galactose-containing Dulbecco's modified Eagle's medium (Gal-DMEM)-fed cells. 2dGlc did not prevent the fall of ATP content in NO-treated Gal-DMEM-fed cells, indicating that NO cytotoxicity was essentially due to the disruption of GSH redox homoeostasis and not to the alteration of ATP production by the mitochondrial respiratory chain. The maintenance of ATP content in NO-treated glucose-fed cells was due to their ability to derive their energy from anaerobic glycolysis. In conclusion, Gal-DMEM and 2dGlc-supplemented Gal-DMEM provide a useful system to decipher and organize into a hierarchy the targets of several stresses at the level of intact barrier epithelial cells.