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SMALL PROLINE RICH PROTEIN-2 EXPRESSION IN DIFFERENTIATED SMALL INTESTINAL EPITHELIUM AND IN MODELS OF INTESTINAL EPITHELIAL DIFFERENTIATION

P Hui, M Ropeleski
Gastrointestinal Diseases Research Unit (GIDRU), Departments of Medicine and Anatomy/Cell Biology, Queen's University, Kingston, Ontario

Small proline-rich protein-2 (SPRR2) may contribute to intestinal function by mediating commensal flora signaling to differentiated epithelia of the small intestine and is up-regulated during intestinal adaptation and during Th2 inflammation in a STAT-6-dependent manner. SPRR2 function however, is largely unknown. Using the human Caco-2 model of differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and in pre-confluent day 3 cells in response to treatment with inducers of differentiation such as short chain fatty acids (SCFA).
SPRR2 protein expression was examined by immunoblotting and its seven isoforms(SPRR2a-g) were detected by semi-quantitative RT-PCR over a time course of 15 days, as were the time and dose-dependent effects of butyrate and propionate. T84 and HT-29 cells were also examined. SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting as well as in human intestinal crypt (HIEC) cells and 18-20 week fetal villus cells.
SPRR2 expression increased at day 11 achieving a maximal increase at 15 days. The SPRR2 isoforms which rose most significantly at day 11-15 were SPRR2d, SPRR2e and SPRR2g. This was not observed in T84 or HT-29 cells. SCFAs induced SPRR2 expression maximally at 24 hours at a concentration of 5 mM. SPRR2 was predominantly cytoplasmic and did not accumulate in the Triton X-100-insoluble cytoskeletal fraction; however, it was present in the membrane- and nuclei-enriched fractions. SPRR2 was detected in human fetal epithelial villus cells but was not detected in HIEC cells.
We conclude that SPRR2 protein expression is associated with the differentiated state of the intestinal epithelium and have established an in vitro model to study the physiology and regulation of SPRR2 as well as the implications of SPRR2 gene knockdown on intestinal epithelial function.
Supported by the Crohn's and Colitis Foundation of Canada and the GIDRU CIHR GI Training Grant

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