Cloning and characterization of a new intestinal inflammation-associated colonic epithelial Ste20-related protein kinase isoform

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 gamma, 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.     

Overexpression of Ste20-related proline/alanine-rich kinase exacerbates experimental colitis in mice

Inflammatory bowel disease, mainly Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in 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.     

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.     

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.     

The TNF receptor, RELT, binds SPAK and uses it to mediate p38 and JNK activation

Receptor expressed in lymphoid tissues (RELT) is a new member of the TNFR family with little known regarding its signaling. Typically, TNFRs engage TRAFs for activation of NF-kappaB and MAPK cascades. We found that RELT does not use the standard signaling pathways characteristic of other TNFRs. While overexpression of RELT in 293 cells induced p38 and JNK activation, it did not activate NF-kappaB. In addition, no binding of RELT to TRAF1,2,3,5, or 6 was detected. Using a yeast two-hybrid system, we identified a Ste20-related proline-alanine-rich kinase (SPAK) that binds RELT. Disruption of the SPAK binding motif, 349RFRV, in RELT inhibited RELT activation of p38 and JNK. In addition, a kinase-dead SPAK acted as an inhibitor of RELT signaling. Thus, we conclude that RELT does not rely on the canonical TRAF pathways for its function, but instead uses a kinase, SPAK, to mediate p38 and JNK activation.     

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.     

Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE

In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na(+)-independent, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V(max)) without any modification in the apparent Michaelis-Menten constant (K(m)). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers.     

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.     

IL-6 induces NF-kappa B activation in the intestinal epithelia

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.     

Mast cell modulates tumorigenesis caused by repeated bowel inflammation condition in azoxymethane/dextran sodium sulfate-induced colon cancer mouse model

Mast cells infiltrate the inflammatory microenvironment and regulate the production of many pro-inflammatory cytokines and mediators of inflammatory cell production to promote tumor development and growth in intestinal lesions. Currently, there are insufficient studies of the mediators and signaling pathways regulated by mast cells that influence the pathogenesis of colon cancer in inflamed colon tissue. This study aimed to confirm the role of mast cells in the incidence and growth of colitis-associated colon cancer (CAC) and to identify inflammation-mediated factors and signaling pathways related to tumor development. CAC was induced by the administration of azoxymethane (AOM) and dextran sodium sulfate (DSS) in mast cell-deficient (WBB6F1/J-W/W^V) and mast cell–sufficient control (WBB6F1_+/+) mice. The results confirmed that mast cell-deficient mice exhibited less tumor formation than normal mice under the same conditions, and down-regulated expression of pro-inflammatory cytokines and mediators. Mast cells play an important role in tumor formation by regulating pro-inflammatory cytokines and inflammatory mediators in CAC, indicating that they can act as new targets for the prevention and treatment of CAC.     

PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells

Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a protein kinase C (PKC) delta-mediated intracellular signaling pathway. Down-regulation of PKCdelta prevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signaling pathway identified coimmunoprecipitation of PKCdelta with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCdelta activates SPAK, which subsequently activates NKCC1 through phosphorylation. Double-stranded silencing RNA directed against SPAK reduced SPAK protein expression by 65.8% and prevented increased phosphorylation of NKCC1 and functional activation of NKCC1 during hyperosmotic stress, measured as bumetanide-sensitive basolateral to apical (86)Rb flux. Using recombinant proteins, we demonstrate direct binding of PKCdelta to SPAK, PKCdelta-mediated activation of SPAK, binding of SPAK to the amino terminus of NKCC1 (NT-NKCC1, amino acids 1-286), and competitive inhibition of SPAK-NKCC1 binding by a peptide encoding a SPAK binding site on NT-NKCC1. The carboxyl terminus of SPAK (amino acids 316-548) pulls down endogenous NKCC1 from Calu-3 total cell lysates and glutathione S-transferase-tagged NT-NKCC1 pulls down endogenous SPAK. In intact cells, hyperosmotic stress increased phosphorylated PKCdelta, indicating activation of PKCdelta, and activity of endogenous SPAK kinase. Inhibition of PKCdelta activity with rottlerin blocked the increase in SPAK kinase activity. The results indicate that PKCdelta acts upstream of SPAK to increase activity of NKCC1 during hyperosmotic stress.     

Peanut lectin stimulates proliferation of colon cancer cells by interaction with glycosylated CD44v6 isoforms and consequential activation of c-Met and MAPK: functional implications for disease-associated glycosylation changes

Peanut agglutinin lectin (PNA) binds the Thomsen-Friedenreich (TF) oncofetal carbohydrate antigen (galactose beta1-3N-acetylgalactosamine alpha) that shows increased expression in colon cancer, adenomas, and inflammatory bowel disease. PNA is mitogenic, both in vitro and in vivo, for colon epithelial cells. In these cells, PNA binds predominantly to cell-surface TF antigen expressed by high molecular weight isoforms of the transmembrane glycoprotein CD44 that are generated in inflamed and neoplastic colonic epithelia by altered RNA splicing. Our aim was to identify the signaling mechanism underlying the proliferative response to PNA. This was investigated in HT29, T84, and Caco2 colon cancer cells. Parallel lectin and immunoblotting of PNA affinity-purified HT29 cell membrane extracts showed PNA binding to high molecular weight CD44v6 isoforms. Within 5 min, PNA (25 microg/mL) caused a 6-fold increase in phosphorylation of hepatocyte growth factor receptor c-Met, known to co-associate with CD44v6. This was followed by the downstream activation of p44/p42 mitogen-activated protein kinase (MAPK) over 15-20 min. The presence of 100 microg/mL asialofetuin, a TF antigen-expressing glycoprotein, blocked both PNA-induced c-Met and MAPK activation. A similar PNA-induced c-Met and MAPK phosphorylation was also seen in T84 cells that express CD44v6 but not in Caco2 cells that lack CD44v6. PNA-induced cell proliferation was completely blocked by 1 microM PD98059, an inhibitor of MAPK activation (p < 0.0001). The expression of TF antigen by CD44 isoforms in colonic epithelial cells allows lectin-induced mitogenesis that is mediated by phosphorylation of c-Met and MAPK. It provides a mechanism by which dietary, microbial, or endogenous galactose-binding lectins could affect epithelial proliferation in the cancerous and precancerous colon.