Identification of 1α,25-dihydroxyvitamin D3 response elements in the human transforming growth factor β2 gene

Yanhong Wu, Theodore A. Craig, Ward H. Lutz, Rajiv Kumar

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)2654-2660
Number of pages7
JournalBiochemistry
Volume38
Issue number9
DOIs
StatePublished - Mar 2 1999

Fingerprint

Calcitriol
Response Elements
Transforming Growth Factors
Genes
Osteoblasts
DNA
Calcitriol Receptors
Bone
Plasmids
Reporter Genes
Growth Hormone
Retinoic Acid Receptors
Nucleic Acid Repetitive Sequences
Transcription Initiation Site
Bone Development
Tretinoin
Bone and Bones
Vitamin D Response Element
DNA sequences
Electrophoretic Mobility Shift Assay

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of 1α,25-dihydroxyvitamin D3 response elements in the human transforming growth factor β2 gene. / Wu, Yanhong; Craig, Theodore A.; Lutz, Ward H.; Kumar, Rajiv.

In: Biochemistry, Vol. 38, No. 9, 02.03.1999, p. 2654-2660.

Research output: Contribution to journalArticle

Wu, Yanhong ; Craig, Theodore A. ; Lutz, Ward H. ; Kumar, Rajiv. / Identification of 1α,25-dihydroxyvitamin D3 response elements in the human transforming growth factor β2 gene. In: Biochemistry. 1999 ; Vol. 38, No. 9. pp. 2654-2660.
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AB - 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.

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