Laetitia Charrier-Hisamuddin

ADAM-15: a metalloprotease that mediates inflammation

Cell‐cell and cell‐matrix interactions are of utmost importance in the pathogenesis of inflammatory diseases. For example, cell‐cell and cell‐matrix interactions are crucial for leukocyte homing and recruitment to inflammatory sites. The discovery of the disintegrin and metalloprotease (ADAM) proteins, which have both adhesive and proteolytic activities, raised the question of their involvement in inflammatory processes. More interestingly, the presence of the RGD integrin‐binding sequence in the disintegrin domain of ADAM‐15 (MDC‐15; metar‐gidin) highlighted ADAM‐15 as a protein particularly involved in cell‐cell interactions. These findings therefore prompted authors to investigate the roles of ADAM‐15 in inflammatory diseases. Because of the early description of ADAM‐15 expression in endothelial cells, work first focused on the roles of ADAM‐15 in vascular diseases, and ADAM‐15 was found to be associated with atherosclerosis. Other studies also pointed at ADAM‐15 as a mediator of rheumatoid arthritis and intestinal inflammation as well as inherent angiogenesis. The roles of ADAM‐15 in these diseases appear to involve mechanisms as different as cell‐cell interactions, cell‐extracellular matrix (ECM) interactions, and shedding activity. Here we review and discuss these recent discoveries pointing to ADAM‐15 as a mediator of mechanisms underlying inflammation and as a possible therapeutic target for prevention of inflammatory diseases.—Charrier‐Hisamuddin, L., Laboisse, C. L., Merlin, D. ADAM‐15: a metalloprotease that mediates inflammation. FASEB J. 22, 641–653 (2008)

Butyrate transcriptionally enhances peptide transporter PepT1 expression and activity.

Background PepT1, 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. Results We 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. Conclusions Collectively, 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.

PepT1 mediates transport of the proinflammatory bacterial tripeptide l-Ala-γ-d-Glu-meso-DAP in intestinal epithelial cells

PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-gamma-d-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-kappaB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.

PepT1 mediated tripeptide KPV uptake reduces intestinal inflammation: P-078.

and IKK , the catalytic subunits of the IKK signalosome that phosphorylates I B . DSS had greater effect on reduction of Hsp27 phosphorylation than CGN, and Tempol had less effect on CGN-induced activation of IL-8. In contrast to DSS, CGN exposure also activated an innate immune pathway of inflammation mediated by TLR4 and Bcl10. Bcl10 is a caspase-recruitment domain (CARD) containing protein, similar to NOD2, and is well-recognized to mediate a pathway of innate immune inflammatory response in lymphocytes and macrophages. In the IEC, Bcl10 co-immunoprecipitated with IKKã (also called Nemo), the regulatory domain of the IKK signalosome. CGN exposure produced an increase in the ubiquitination of Nemo; increased Nemo ubiquitination is associated with increased phospho-I B , leading to increased nuclear localization of NF B and increased secretion of IL-8. Bcl10 was not increased following exposure to DSS, and silencing of Bcl10 had no impact on DSS-induced activation of IL-8. The study results demonstrate that DSS generates greater increase in ROS than CGN at a comparable level of exposure, but that CGN also activates an immune responsive pathway through Bcl10 that is not induced by DSS. CGN appears to activate a pathway of innate immunity, due to its unusual á-1,3-galactosidic linkage that is immunogenic. In contrast to CGN, which is widely used as a food additive in the Western diet, DSS is not incorporated into the Western diet. Further elucidation of these mechanisms that model IBD may provide new insights into prevention and treatment of clinical IBD. Since the food additive CGN activates pathways of inflammation that involve innate immunity, as well as increased ROS, increased attention to its elimination from the diet is indicated.