Identification of 1α,25-Dihydroxyvitamin D3Response Elements in the Human Transforming Growth Factor β2 Gene

Abstract

Transforming growth factor-β (TGF-β) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-β expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) inhibits human osteoblast and keratinocyte growth by increasing TGF-β2 secretion and synthesis. To examine the mechanism by which 1α,25(OH)₂D₃ regulates TGF-β2 transcription in osteoblasts, we ligated segments of the human TGF-β2 promoter 5‘ of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1α,25(OH)₂D₃ administration on the expression of growth hormone in cells transfected with such chimeric promoter−reporter plasmids. The promoter region extending from −973 to +68 bp of the transcription start site elicited a 7-fold increase in reporter gene activity in transiently transfected osteoblasts after 1α,25(OH)₂D₃ treatment, whereas the region from −553 to +68 bp of the transcription start site showed no response following 1α,25(OH)₂D₃ treatment. Transfection of osteoblasts with reporter plasmids containing TGF-β2 promoter DNA from −869 to −658 bp revealed a 3.8-fold increase in reporter gene activity. DNA fragments from this region (−743 to −676 bp and −786 to −728 bp) ligated into reporter plasmids also showed increases in reporter activity. Gel retardation assay experiments showed that DNA fragments from −774 to −735 bp and −714 to −675 bp both formed a DNA−protein complex with bacterially expressed human retinoic acid X receptor α (RXRα) and vitamin D receptor (VDR) and with nuclear extracts from human bone cells. Addition of a vitamin D receptor antibody to reactions containing the aforesaid DNA fragments and bone cell nuclear extract resulted in further retardation of the mobility of the DNA−protein complex (super-shifting). Removal of two putative direct repeat DNA fragments in this region abolished VDR−RXRα−vitamin D response element complex formation. The TGF-β2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1α,25(OH)₂D₃.