Regulation of calbindin D9k gene expression by cellular differentiation in Caco -2 cells
Abstract
Calbindin D9k is a cytosolic calcium binding protein required for intestinal calcium absorption. In mammals, calbindin D9k is mainly found in duodenum and its intestinal expression is restricted to differentiated enterocytes. The homeodomain protein cdx-2 has been proposed to play an important role in intestine-specific gene expression; the calbindin D9k gene promoter contains two cdx-2 response elements. Initial experiments showed that calbindin D9k gene expression was not completely dependent on cdx-2 mRNA levels in Caco-2 cells. We hypothesized that other factors associated with intestinal cell differentiation contribute to the regulation of calbindin D9k gene expression. In silico analysis of the calbindin D9k promoter identified three evolutionarily conserved regions (cluster I, II, III, distal to proximal, respectively). Promoter mapping with reporter gene analysis indicated that cluster I and III contained positive regulatory elements while cluster II contained a repressor. Using site-directed mutagenesis of elements identified in clusters I and III by in silico analysis or others, we demonstrated that two HNF-1 sites were essential for differentiation-induced calbindin D9k promoter activity. While mutation of the distal cdx-2 site reduced basal promoter activity (suggesting a permissive role) it did not alter the fold change due to differentiation. Mutation of the distal HNF-1 site and the proximal HNF-1 site had the same impact on differentiation-induced promoter activity as deletion of cluster I (80% suppression) and cluster III (50% suppression), respectively. Mutation the proximal cdx-2 and the C/EBP site had no effects on promoter activity. We verified that the distal HNF-1 response element was a functional binding site by electrophoretic mobility shift assay. Increasing binding of nuclear extract to this site was observed as Caco-2 cell differentiated, suggesting it could account for the differentiation-induced calbindin D9k promoter activity. Exogenous HNF-1α transactivated calbindin D 9k promoter dramatically in proliferating Caco-2 cells. However, the stimulation was due exclusively to the proximal HNF-1 site but not the distal HNF-1 site. We propose that this HNF-1 binding site plays a pivotal role by interacting with HNF-1α protein, which in turn recruits other cofactors to allow the maximal expression of calbindin D9k gene in Caco-2 cells.
Degree
Ph.D.
Advisors
Fleet, Purdue University.
Subject Area
Molecular biology
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