Regulation of the CYP27B1 5′-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

Abstract

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)2D3 is catalysed by the enzyme 25-hydroxyvitamin D3-1α-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)2D3 by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)2D3, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24h later. The results showed that 1,25(OH)2D3 did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.