Regulation of the 5′-flanking region of the human CYP27B1 gene in osteoblast cells

Synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is catalysed by the enzyme 25-hydroxyvitamin D3-1α-hydroxylase (CYP27B1). Regulation of CYP27B1 gene expression is poorly understood, particularly in non-renal tissues including bone where 1,25(OH)2D3 is hypothesised to serve autocrine/paracrine roles. Transient transfection of ROS 17/2.8 osteoblast-like cells with reporter gene constructs containing deletions of the 5′-flanking region of the human CYP27B1 gene revealed a proximal promoter, enhancer region and strong upstream repressive region. Putative CCAAT and GC boxes, as well as Ets protein binding sites were shown to contribute to promoter and enhancer activities respectively in common with kidney and prostate cells. Inhibition of basal expression was largely attributed to a palindrome 5′-GTCTCAGAC-3′ (−1015/−1007 bp) that contains two putative canonical Smad binding elements. We conclude that repression of CYP27B1 gene expression may be a common event but the novel inhibitory elements we have identified may be unique to osteoblasts.