Evaluation of a Putative Vitamin D Response Element in the Avian Calcium Binding Protein Gene

A primary response of the avian intestine to 1,25-dihydroxyvitamin D3 [1,25-(OH)₂D₃] is increased synthesis of a 28-kD calcium-binding protein, calbindin-D_28k (CaBP). This study examined whether 1,25-(OH)₂D₃ regulates CaBP gene transcription by an interaction of the vitamin D receptor (VDR) with a vitamin D-responsive element (VDRE) in the CaBP promoter. A genomic clone of CaBP containing about 1 kb of 5′-flanking DNA and 13 kb of the structural gene was isolated. 5′-Flanking DNA from −320 to −306 had considerable sequence similarity to VDREs identified in other genes. Indeed, a portion of the CaBP gene containing this region (−743 to +47) linked to a growth hormone reporter construct elicited a 1,25-(OH)₂D₃-dependent, VDR-dependent increase in reporter expression in transiently transfected chicken embryo fibroblasts. However, deletion analysis demonstrated that the sequences responsible for this induction reside 3′ to −133 and the putative VDRE at −320 to −306 was not involved in the response. Furthermore, transfection of heterologous promoter constructs consisting of a Ban I fragment (−354 to −252) linked to the Herpes simplex thymidine kinase promoter revealed no effect of this region on reporter expression. Gel mobility shift analysis confirmed that this putative VDRE in the CaBP promoter was not a high-affinity binding site for VDR. Consequently, functional significance with respect to the primary induction of CaBP by 1,25-(OH)₂D₃ cannot be ascribed to this region of the CaBP promoter.