Factors involved in the duodenal expression of the human calbindin-D9k gene

Calbindin-D9k is expressed in the cytoplasm of intestinal cells, where it i s critical for dietary calcium absorption. Two striking aspects of the expr ession of this gene are its vitamin-D dependency and regional differences i n expression, with high levels only in duodenum. We report studies of the h uman calbindin-D9k promoter. Differences between the reported sequences of the human calbindin-D9k promoter were first clarified before undertaking a functional analysis of this sequence. Studies of the rat gene have indicate d that several transcription factors, including the caudal-related homeobox factor (CDX-2), hepatic nuclear factor-4 and CCAAT-enhancer-binding protei n ct. (C/EBP alpha), could interact with elements in the promoter. Although these elements are conserved in the human gene, we show here that their in testinal distribution makes them unlikely to be critical positive factors. The calbindin-D9k gene contains multiple potential binding sites for homeob ox transcription factors; one of these, known as IPF-1 or PDX-1, co-localiz es in the intestine with calbindin-D9k. We show in gel-shift assays that th e sequence within a putative vitamin-D-response element in the human calbin din-D9k promoter can bind expressed IPF-1/PDX-1 protein, although we cannot confirm binding of the vitamin-D-receptor protein. CDX-2 binds to the regi on around the TATA box, as in the rat gene, and may act as a negative facto r in the distal intestine. Transfection studies in Caco-2 and MCF-7 cells w ith heterologous reporter vectors containing up to 1303 bp of the gene show ed that this functioned as a weak promoter and indicated the presence of su ppressor sequences, but did not show vitamin-D responsiveness. This indicat es that other elements are also needed for the control of human calbindin-D 9k expression.