Identification of 1 alpha,25-dihydroxyvitamin D3 response elements in the human transforming growth factor beta 2 gene
Transforming growth factor-beta (TGF-beta) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) i...
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| Veröffentlicht in: | Biochemistry (Easton) 1999-03, Vol.38 (9), p.2654-2660 |
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| creator | Wu, Y Craig, T A Lutz, W H Kumar, R |
| description | Transforming growth factor-beta (TGF-beta) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH)2D3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segments of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,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 1 alpha,25(OH)2D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,25(OH)2D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta 2 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 alpha (RXR alpha) 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-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,25(OH)2D3. |
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Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH)2D3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segments of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,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 1 alpha,25(OH)2D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,25(OH)2D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta 2 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 alpha (RXR alpha) 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-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,25(OH)2D3.</description><identifier>ISSN: 0006-2960</identifier><identifier>PMID: 10052935</identifier><language>eng</language><publisher>United States</publisher><subject>Base Sequence ; Calcitriol - pharmacology ; Cells, Cultured ; Dimerization ; Fetus ; Human Growth Hormone - genetics ; Human Growth Hormone - metabolism ; Humans ; Molecular Sequence Data ; Osteoblasts ; Promoter Regions, Genetic - drug effects ; Receptors, Calcitriol - genetics ; Receptors, Calcitriol - metabolism ; Receptors, Retinoic Acid - genetics ; Receptors, Retinoic Acid - metabolism ; Repetitive Sequences, Nucleic Acid - physiology ; Response Elements - drug effects ; Response Elements - physiology ; Retinoid X Receptors ; Transcription Factors - genetics ; Transcription Factors - metabolism ; Transcription, Genetic - drug effects ; Transfection ; Transforming Growth Factor beta - genetics</subject><ispartof>Biochemistry (Easton), 1999-03, Vol.38 (9), p.2654-2660</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10052935$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wu, Y</creatorcontrib><creatorcontrib>Craig, T A</creatorcontrib><creatorcontrib>Lutz, W H</creatorcontrib><creatorcontrib>Kumar, R</creatorcontrib><title>Identification of 1 alpha,25-dihydroxyvitamin D3 response elements in the human transforming growth factor beta 2 gene</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Transforming growth factor-beta (TGF-beta) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH)2D3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segments of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,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 1 alpha,25(OH)2D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,25(OH)2D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta 2 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 alpha (RXR alpha) 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-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,25(OH)2D3.</description><subject>Base Sequence</subject><subject>Calcitriol - pharmacology</subject><subject>Cells, Cultured</subject><subject>Dimerization</subject><subject>Fetus</subject><subject>Human Growth Hormone - genetics</subject><subject>Human Growth Hormone - metabolism</subject><subject>Humans</subject><subject>Molecular Sequence Data</subject><subject>Osteoblasts</subject><subject>Promoter Regions, Genetic - drug effects</subject><subject>Receptors, Calcitriol - genetics</subject><subject>Receptors, Calcitriol - metabolism</subject><subject>Receptors, Retinoic Acid - genetics</subject><subject>Receptors, Retinoic Acid - metabolism</subject><subject>Repetitive Sequences, Nucleic Acid - physiology</subject><subject>Response Elements - drug effects</subject><subject>Response Elements - physiology</subject><subject>Retinoid X Receptors</subject><subject>Transcription Factors - genetics</subject><subject>Transcription Factors - metabolism</subject><subject>Transcription, Genetic - drug effects</subject><subject>Transfection</subject><subject>Transforming Growth Factor beta - genetics</subject><issn>0006-2960</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo1kEtPwzAQhHMA0VL4C8gnTkRy_EpzROXRSpW49B5t4nVjlMTBdgr991iiSCPtzOrbPcxVtqSUqpxVii6y2xA-UxS0FDfZoqBUsorLZXbaaRyjNbaFaN1InCEFgX7q4InJXNvurL37OZ9shMGO5IUTj2FyY0CCPQ7pNpC0jx2Sbh4gOQ9jMM4n-kiO3n3Hjhhoo_OkwQiEkSOOeJddG-gD3l_mKju8vR4223z_8b7bPO_zSQqZF7rFgjLga1E0Tam0BImcAQNUDTVU6YpWHI1MWktDUdNKiLJEoQqlDeOr7PHv7eTd14wh1oMNLfY9jOjmUKvUDVVcJPDhAs7NgLqevB3An-v_ovgvGV9kkQ</recordid><startdate>19990302</startdate><enddate>19990302</enddate><creator>Wu, Y</creator><creator>Craig, T A</creator><creator>Lutz, W H</creator><creator>Kumar, R</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>19990302</creationdate><title>Identification of 1 alpha,25-dihydroxyvitamin D3 response elements in the human transforming growth factor beta 2 gene</title><author>Wu, Y ; Craig, T A ; Lutz, W H ; Kumar, R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p545-1dce102a3841bb76d5a5e32a2ae6b0f06d9093ef5ef585f0ed094477e4616df23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Base Sequence</topic><topic>Calcitriol - pharmacology</topic><topic>Cells, Cultured</topic><topic>Dimerization</topic><topic>Fetus</topic><topic>Human Growth Hormone - genetics</topic><topic>Human Growth Hormone - metabolism</topic><topic>Humans</topic><topic>Molecular Sequence Data</topic><topic>Osteoblasts</topic><topic>Promoter Regions, Genetic - drug effects</topic><topic>Receptors, Calcitriol - genetics</topic><topic>Receptors, Calcitriol - metabolism</topic><topic>Receptors, Retinoic Acid - genetics</topic><topic>Receptors, Retinoic Acid - metabolism</topic><topic>Repetitive Sequences, Nucleic Acid - physiology</topic><topic>Response Elements - drug effects</topic><topic>Response Elements - physiology</topic><topic>Retinoid X Receptors</topic><topic>Transcription Factors - genetics</topic><topic>Transcription Factors - metabolism</topic><topic>Transcription, Genetic - drug effects</topic><topic>Transfection</topic><topic>Transforming Growth Factor beta - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wu, Y</creatorcontrib><creatorcontrib>Craig, T A</creatorcontrib><creatorcontrib>Lutz, W H</creatorcontrib><creatorcontrib>Kumar, R</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wu, Y</au><au>Craig, T A</au><au>Lutz, W H</au><au>Kumar, R</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of 1 alpha,25-dihydroxyvitamin D3 response elements in the human transforming growth factor beta 2 gene</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1999-03-02</date><risdate>1999</risdate><volume>38</volume><issue>9</issue><spage>2654</spage><epage>2660</epage><pages>2654-2660</pages><issn>0006-2960</issn><abstract>Transforming growth factor-beta (TGF-beta) is one of the most abundant growth factors secreted by bone cells, and regulation of TGF-beta expression is crucial for bone development and growth. Previous studies from our laboratory demonstrated that 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) inhibits human osteoblast and keratinocyte growth by increasing TGF-beta2 secretion and synthesis. To examine the mechanism by which 1 alpha,25(OH)2D3 regulates TGF-beta 2 transcription in osteoblasts, we ligated segments of the human TGF-beta 2 promoter 5' of a growth hormone reporter gene in a growth hormone reporter plasmid and examined the effects of 1 alpha,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 1 alpha,25(OH)2D3 treatment, whereas the region from -553 to +68 bp of the transcription start site showed no response following 1 alpha,25(OH)2D3 treatment. Transfection of osteoblasts with reporter plasmids containing TGF-beta 2 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 alpha (RXR alpha) 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-RXR alpha-vitamin D response element complex formation. The TGF-beta 2 promoter contains two imperfect direct repeat DNA sequences: TGTAGAACAAGTAGA and AATGAAGTTGGTGGA that mediate the effect of 1 alpha,25(OH)2D3.</abstract><cop>United States</cop><pmid>10052935</pmid><tpages>7</tpages></addata></record> |
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| subjects | Base Sequence Calcitriol - pharmacology Cells, Cultured Dimerization Fetus Human Growth Hormone - genetics Human Growth Hormone - metabolism Humans Molecular Sequence Data Osteoblasts Promoter Regions, Genetic - drug effects Receptors, Calcitriol - genetics Receptors, Calcitriol - metabolism Receptors, Retinoic Acid - genetics Receptors, Retinoic Acid - metabolism Repetitive Sequences, Nucleic Acid - physiology Response Elements - drug effects Response Elements - physiology Retinoid X Receptors Transcription Factors - genetics Transcription Factors - metabolism Transcription, Genetic - drug effects Transfection Transforming Growth Factor beta - genetics |
| title | Identification of 1 alpha,25-dihydroxyvitamin D3 response elements in the human transforming growth factor beta 2 gene |
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