MOLECULAR BASIS OF ENTEROCYTE REGULATION DURING ONTOGENY

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In this grant I propose to study the regulation of both the neonatal intestinal immunoglobulin receptors (fcRn) and polymeric receptor (pIgR) genes, as a model to understand small intestinal epithelial ontogeny and adaptation. The Fc receptor transports IgG from breast milk and is essential for establishing humoral immunity in the neonate. Located on the enterocytes apical membrane, FcRn transport IgG transcellulary to the basolateral membrane where its released into the systemic circulation. In contrast, pIgR transports IgA from the basolateral membrane to the lumen. In rodents, I have shown that both FcRn and pIgR regulation have a unique tissue, developmental and hormonal-pattern of expression. FcRn's expression is developmentally restricted to the suckling phase, during which time the transcript is produced in a proximodistal gradient, and is inhibited by corticosteroids administration. In contrast, pIgR mRNA levels are induced after warning, and may be further enhanced by corticosteroids. To begin addressing at a molecular level the mechanism underlying enterocyte regulation during development, the rat FcRn and murine pIgR genes have been cloned. We have done transient-transfection studies with an intestinal cell line, using chimeric constructs of either the 5' - flanking regions of the FcRn or pIgR genes fused to the reporter luciferase. Transfection of clones derived by nested deletions has been done, and has led to the identification of several activator and repressor regions that appear to modulate basal activity. Moreover, I will assess the response of each gene to corticosteroids using in vitro transfection experiments with cells containing chimeric constructs. Experiments with homologous and heterologous promoters will be used to define enhancer repressors elements, and DNase I footprint and electrophoretic gel mobility shift assays will be done to define the DNA-protein interaction. To enhance our ability to draw more physiologically relevant conclusions from our in vitro experiments, a transgenic mouse model is presented that appears to reflect FcRns normal pattern of expression. Similar lines will be developed with the pIgR 5'-flanking region to define the elements needed to control the genes in vivo expression. The projects long term objectives is to evaluate the transcriptional factor that regulates the intestinal and developmental-specific pattern of expression. Analysis of the FcRn and pIgR genes offers a unique model that may lead to a better understanding of the molecular mechanism of intestinal ontogeny, and specifically the intrinsic clock which controls its regulation.