Caco2-BBE Cells Research Articles

S1773 Lysosomes Direct Polarised Calcium Signalling in Human Colonic Crypts

BBE cells in the presence or absence of mature microRNAs and luciferase activity or GFP expression was analyzed. HT29-Cl.19A cells were infected with enteropathogenic E. coli (EPEC) and PepT1 and microRNAs levels were quantified by Northern blot and real-time RT-PCR. Results: Among the microRNAs tested, expression level of miR-619 was decreased following Caco2-BBE cell differentiation, and this is accompanied with an opposite pattern of PepT1 expression. This observation suggests that miR-619 is involved in PepT1 expression. Transfection of Caco2-BBE cells with miR-619 significantly decreased PepT1 mRNA and protein expression levels and transport activity, whereas other microRNA candidates did not exhibit any effects. This result demonstrates the specificity of miR-619 in the regulation of PepT1 expression. Importantly, miR-619 significantly decreased the luciferasse activity as well as GFP expression, indicating that miR-619 effectively binds PepT1 3'UTR mRNA. Furthermore, infection of HT29-Cl.19A with EPEC induced PepT1 expression and decreased miR-619 expression level, suggesting that this microRNA is implicated in the regulation of colonic PepT1 expression under pathological states. Conclusions: Here we demonstrate, for the first time, that PepT1 expression can be regulated by microRNAs. Our study reveals a novel mechanism underlying the regulation of this transporter, as well as open new insights to modulate epithelial transport.

S1771 Interferon-γ Selectively Modulates the Ability of EGF to Activate EGFR Signaling and Reverses Its Inhibitory Effects On K + Channel Activity in T84 Colonic Epithelial Cells

BBE cells in the presence or absence of mature microRNAs and luciferase activity or GFP expression was analyzed. HT29-Cl.19A cells were infected with enteropathogenic E. coli (EPEC) and PepT1 and microRNAs levels were quantified by Northern blot and real-time RT-PCR. Results: Among the microRNAs tested, expression level of miR-619 was decreased following Caco2-BBE cell differentiation, and this is accompanied with an opposite pattern of PepT1 expression. This observation suggests that miR-619 is involved in PepT1 expression. Transfection of Caco2-BBE cells with miR-619 significantly decreased PepT1 mRNA and protein expression levels and transport activity, whereas other microRNA candidates did not exhibit any effects. This result demonstrates the specificity of miR-619 in the regulation of PepT1 expression. Importantly, miR-619 significantly decreased the luciferasse activity as well as GFP expression, indicating that miR-619 effectively binds PepT1 3'UTR mRNA. Furthermore, infection of HT29-Cl.19A with EPEC induced PepT1 expression and decreased miR-619 expression level, suggesting that this microRNA is implicated in the regulation of colonic PepT1 expression under pathological states. Conclusions: Here we demonstrate, for the first time, that PepT1 expression can be regulated by microRNAs. Our study reveals a novel mechanism underlying the regulation of this transporter, as well as open new insights to modulate epithelial transport.

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function.

Angiotensin II directly regulates intestinal epithelial NHE3 in Caco2BBE cells

BackgroundAngiotensin II (AII) effects on intestinal Na+ transport may be multifactorial. To determine if AII might have a direct effect on intestinal epithelial Na+ transport, we investigated its actions on Na+ transport in human intestinal epithelial Caco2BBE cells.ResultsAII increased apical (brush border) sodium-hydrogen exchanger (NHE)-3, but not NHE2, activity within one hour. Similarly, only apical membrane NHE3 abundance increased at 1–2 hours without any change in total NHE3 protein abundance. From 4–48 hours, AII stimulated progressively larger increases in apical NHE3 activity and surface abundance, which was associated with increases in NHE3 protein expression. At 4–24 hours, NHE3 mRNA increases over baseline expression, suggesting increased gene transcription. This was supported by AII induced increases in rat NHE3 gene promoter-reporter activity. AII induction of NHE3 was blocked by the AII type I receptor antagonist losartan. Acute changes in AII-induced increases in NHE3 exocytosis were blocked by a phospholipase C inhibitor, an arachidonic acid cytochrome P450 epoxygenase inhibitor, as well as phosphatidylinositol 3 kinase (PI3K) inhibitors and Akt inhibitor, partially blocked by a metalloproteinase inhibitor and an EGF (epidermal growth factor) receptor kinase inhibitor, but not affected by an inhibitor of MEK-1 (MAPKK-1, mitogen activated protein kinase kinase-1).ConclusionWe conclude that angiotensin II has a direct role in regulating intestinal fluid and electrolyte absorption which may contribute to its overall effects in regulation systemic volume and blood pressure. AII activates several key signaling pathways that induce acute and chronic changes in NHE3 membrane trafficking and gene transcription.

Hepatocyte nuclear factor-4α regulates human cellular retinol-binding protein type II gene expression in intestinal cells

Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport, and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct repeat 1 (DR-1)-like nuclear receptor response element in the proximal promoter region and that endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) was a major transcription factor binding to the DR-1-like element. Cotransfection of HNF-4alpha expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4alpha significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4alpha protein levels by HNF-4alpha small interference RNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4alpha protein level and binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4alpha protein levels, HNF-4alpha protein binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4alpha is an important transcriptional factor that regulates human CRBPII gene expression and provide the possibility for a novel function of HNF-4alpha in the regulation of human intestinal vitamin A absorption and metabolism.

Bacterial Particle Endocytosis by Epithelial Cells Is Selective and Enhanced by Tumor Necrosis Factor Receptor Ligands

Bacterial pathogens use virulence strategies to invade epithelial barriers, but active processes of epithelial cells may also contribute to the endocytosis of microbial particles. To focus on the latter, we studied the uptake of fixed and fluorescently labeled bacterial particles in intestinal and bronchoepithelial cell cultures and found it to be enhanced in Caco-2BBe and NCI-H292 cells after treatment with tumor necrosis factor alpha and an agonist antibody against the lymphotoxin beta receptor. Confocal fluorescence microscopy, flow cytometry, and transmission electron microscopy revealed that Staphylococcus aureus and Yersinia enterocolitica were readily endocytosed, although there was scant uptake of Shigella sonnei, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae particles. Endocytosed Staphylococcus was often associated with cytoplasmic claudin-4 vesicles; this was not found for Yersinia, suggesting that cytokine treatment upregulated two distinct endocytosis pathways. Interestingly, when Staphylococcus and Yersinia were coincubated with epithelial monolayers, the cells were unlikely to take up Yersinia unless they had also endocytosed large numbers of Staphylococcus particles, although the two bacteria were apparently processed in distinct compartments. Cytokine treatment induced an upregulation and redistribution of beta1 integrin to the apical surface of NCI-H292 cells; consistent with this effect, treatment with anti-beta1 integrin antibody blocked uptake of both Yersinia and Staphylococcus in NCI-H292 and Caco-2BBe cells. Our results suggest that capture of bacterial particles by mucosal epithelial cells is selective and that different endocytic mechanisms are enhanced by proinflammatory cytokines.

Ecto-Phosphorylation of CD98 Regulates Cell-Cell Interactions

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.

Functional coupling of the downregulated in adenoma Cl−/base exchanger DRA and the apical Na+/H+exchangers NHE2 and NHE3

Non-nutrient-dependent salt absorption across the brush-border membrane of intestinal epithelial cells is primarily mediated by coupled apical Na(+)/H(+) (aNHE) and anion exchange transport, with the latter suspected of being mediated by DRA (downregulated in adenoma; SLC26A3) that is defective in congenital chloridorrhea. To investigate DRA in greater detail and determine whether DRA and NHE activities can be coupled, we measured (22)Na(+) and (36)Cl(-) uptake in Caco2BBE colon cells infected with the tet-off-inducible DRA transgene. Under basal conditions, DRA activity was low in normal and infected Caco2BBE cells in the presence of tetracycline, whereas NHE activities could be easily detected. When apical NHE activity was increased by transfection or serum-induced expression of the aNHE isoforms NHE2 and NHE3, increased (36)Cl(-) uptake was observed. Inhibition of DRA activity by niflumic acid was greater than that by DIDS as well as by the NHE inhibitor dimethylamiloride and the carbonic anhydrase inhibitor methazolamide. DRA activity was largely aNHE-dependent, whereas a component of DRA-independent aNHE uptake continued to be observed. Coupled aNHE and DRA activities were inhibited by increased cellular cAMP and calcium and were associated with synaptotagmin I-dependent, clathrin-mediated endocytosis. In summary, these data support the role of DRA in electroneutral NaCl absorption involving functional coupling of Cl(-)/base exchange and apical NHE.

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.

Nuclear Factor-κB Is a Critical Mediator of Ste20-Like Proline-/Alanine-Rich Kinase Regulation in Intestinal Inflammation

Inflammatory bowel disease (IBD) is thought to result from commensal flora, aberrant cellular stress, and genetic factors. Here we show that the expression of colonic Ste20-like proline-/alanine-rich kinase (SPAK) that lacks a PAPA box and an F-alpha helix loop is increased in patients with IBD. The same effects were observed in a mouse model of dextran sodium sulfate-induced colitis and in Caco2-BBE cells treated with the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha. The 5'-flanking region of the SPAK gene contains two transcriptional start sites, three transcription factor Sp1-binding sites, and one transcription factor nuclear factor (NF)-kappaB-binding site, but no TATA elements. The NF-kappaB-binding site was essential for stimulated SPAK promoter activity by TNF-alpha, whereas the Sp1-binding sites were important for basal promoter activity. siRNA-induced knockdown of NF-kappaB, but not of Sp1, reduced TNF-alpha-induced SPAK expression. Nuclear run-on and mRNA decay assays demonstrated that TNF-alpha directly increased SPAK mRNA transcription without affecting SPAK mRNA stability. Furthermore, up-regulation of NF-kappaB expression and demethylation of the CpG islands induced by TNF-alpha also played roles in the up-regulation of SPAK expression. In conclusion, our data indicate that during inflammatory conditions, TNF-alpha is a key regulator of SPAK expression. The development of compounds that can either modulate or disrupt the activity of SPAK-mediated pathways is therefore important for the control and attenuation of downstream pathological responses, particularly in IBD.

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.

CaSR stimulates secretion of Wnt5a from colonic myofibroblasts to stimulate CDX2 and sucrase-isomaltase using Ror2 on intestinal epithelia

To understand whether extracellular calcium-sensing receptor (CaSR) expression on colonic myofibroblast cells (18Co) contributed to epithelial homeostasis, we activated the CaSR with 5 mM Ca(2+), screened by RT-PCR Wnt family members, and measured their secretion. Transcripts for Wnt 1, 2, 2b, 3a, 4, and 7a were either absent or unchanged whereas Wnt3 decreased and Wnt5a increased. We assessed Wnt5a secretion by Western blot. High Ca(2+) (5 mM) substantially increased Wnt5a secretion; small interfering RNA (siRNA) against the CaSR reduced this to constitutive amounts. Expression of Wnt5a plasmid but not Wnt1 or Wnt3a increased caudal homeodomain factor CDX2 transcripts and protein in HT-29 adenocarcinoma cells. Wnt5a increased activity of a sucrase-isomaltase (SI) promoter in Caco-2BBE cells. Wnt5a protein stimulation of CDX2 transcripts and protein and SI reporter were increased by overexpression of wild-type Ror2, a Wnt5a receptor, and reduced with siRNA against Ror2. CaSR activation of HT-29 cells increased Ror2 protein expression. Ror2 protein was expressed in mouse jejunum from crypt base to villus tip and in the colon on surface epithelia. Our results show that activation of a G protein-coupled receptor, the CaSR, stimulates secretion of Wnt5a from myofibroblasts. Stimulation of epithelia by the CaSR increased the expression of a receptor for Wnt5a, the tyrosine kinase Ror2, suggesting existence of a unique paracrine relationship for CDX2 homoeostasis in the intestine and revealing new contributions of CaSR-activated myofibroblasts to intestinal stem cell niche microenvironments.

Butyrate Transcriptionally Enhances Peptide Transporter PepT1 Expression and Activity

BackgroundPepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte.ResultsWe found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by ∼2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles.ConclusionsCollectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

Ecto‐phosphorylation of CD98 regulates lymphocytes‐epithelia interactions

Ecto‐phosphorylation has been shown to play an important role in several cellular functions. Here we investigate the ecto‐phosphorylation of membrane glycoprotein CD98 by ecto‐protein kinases (ePK) and its functional consequences. Our results show that recombinant CD98 is phosphorylated by purified casein kinase 2 (CK2) and by ePK from Jurkat cells. Km and Vmax for the phosphorylation of CD98 by CK2 are 0.84 μM and 8.7e+6 μmol/min, respectively. In intestinal epithelial Caco2‐BBE cells, CD98 is also ecto‐phosphorylated by ePK from Jurkat cells. Interestingly, activation of CD98 in CHO cells expressing CD98 enhances the ecto‐phosphorylation of CD98 and increases the attachment of Jurkat cells to CHO cells, suggesting that the ecto‐phosphorylation of CD98 is important for cell‐cell interactions. To examine the role of CD98 ectodomain phosphorylation in cell‐cell interactions, we generated CD98‐CD69 and CD69‐CD98 constructs, as well as CD98 lacking PDZ‐binding domain or mutated at predicted serine phosphorylation sites and transfected these constructs into CHO cells. Cell adhesion assays show that mutation at S305‐S307‐S309 or deletion of extracellular C‐terminal or PDZ‐binding domain of CD98 significantly decreases the binding of Jurkat cells to CHO cells. In conclusion, we demonstrate, for the first time, that CD98 is ecto‐phosphorylated by ePK and its phosphorylation affects cell‐cell interactions.

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells

Vitamin A is derived from provitamin A carotenoids, mainly β-carotene, by β-carotene 15,15′-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T 3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T 3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T 3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRα1 and TRβ1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T 3 treatment in medium supplemented with fetal bovine serum. Furthermore, T 3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T 3 is an important hormone for the regulation of vitamin A and β-carotene metabolism-related gene expression in human small intestinal cells.

F.52. A Tight Junction Permeability Inhibitor Peptide AT-1001, Inhibits TNF-α Production from Human PBMC

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon γ, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.

Epithelial-derived Fibronectin Expression, Signaling, and Function in Intestinal Inflammation

Fibronectin (FN) is a multifunctional extracellular matrix protein that plays an important role in cell proliferation, adhesion, and migration. FN expression or its role in colitis is not known. The goal of this study is to characterize FN expression, regulation, and role during intestinal inflammation. Wild-type and transgenic mice expressing luciferase under the control of the human FN promoter, given water or 3% dextran sodium sulfate, were used as animal models of colitis. The Caco2-BBE model intestinal epithelial cell line was used for in vitro studies. FN protein is abundantly expressed by surface epithelial cells in the normal colon. Immunohistochemistry and luciferase assay in mice expressing the FN promoter linked to luciferase demonstrated that FN synthesis was up-regulated during colitis, during both the acute phase and the healing phase. In vitro experiments demonstrated that FN increased the expression of the FN integrin receptor alpha5beta1 in a dose- and time-dependent manner. FN also induced the expression and activation of NF-kappaB. Further, FN potentiated Caco2-BBE cell attachment and wound healing, which was inhibited by RGD peptide as well as NF-kappaB inhibitors MG-132 and 1-pyrrolidinecarbodithioic acid, ammonium salt. In conclusion, FN is abundantly expressed and synthesized by colonic epithelial cells. FN is transcriptionally up-regulated in epithelial cells during both the dextran sodium sulfate-induced colitic and the recovery phase. FN enhances cell attachment and wound healing, which is dependent on binding to the integrin receptor and the NF-kappaB signaling. Together our data show that epithelial-derived FN potentiates cell attachment and wound healing through epithelial-matrix interactions and that FN expression may have important implications for maintaining normal epithelial integrity as well as regulating epithelial response to injury during colitis.

Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells

The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-beta-cyclodextrin (MbetaCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MbetaCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.

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.

Extracellular Interaction between hCD98 and the PDZ Class II Domain of hCASK in Intestinal Epithelia

The extracellular domain of the glycoprotein-associated integrin hCD98 protrudes into the basolateral extracellular space of the intestine and contains a PDZ class II-binding domain (GLLLRFPYAA, amino acids 520-529). Protein-protein interaction studies in vitro as well as in human colonic sections and Caco2-BBE cells have revealed that hCD98 coimmunoprecipitated with the basolateral membrane-associated guanylate kinase hCASK and that this interaction occurred in a PDZ domain-dependent manner. These novel results, which provide the first evidence for a PDZ domain-dependent interaction between a membrane protein and an extracellular protein, open a new field of investigation related to extracellular signaling in cell biology.