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dc.contributor.authorBanerjee, Chaitali
dc.date2022-08-11T08:08:39.000
dc.date.accessioned2022-08-23T16:03:16Z
dc.date.available2022-08-23T16:03:16Z
dc.date.issued1998-04-16
dc.date.submitted2006-09-18
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31442
dc.description<p>In the process of seeking author's permission to provide full text.</p>
dc.description.abstractOsteocalcin (OC) is a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix. The Osteocalcin gene is transcriptionally regulated by several basal, hormone- and cytokine-responsive elements. To address the potential role of TGF-β1 regulation and tissue-specific expression of the OC gene, we defined regulatory elements and factors mediating the transcriptional activity of the rat OC (rOC) promoter. TGF-β1 modulates the differentiation of cells of the osteogenic lineage and downregulates the osteoblast-specific expression of OC. By promoter deletion and mutational analyses, a TGF-β1 responsive element at nt -146 to -139 and a contiguous tissue-specific enhancer element at nt -136 to -130 on the rOC promoter were identified. These studies show that Fra-2, a member of the AP-1 family of proteins, binds to the TGF-β1 responsive element and activates basal OC expression. TGF-β1 induced phosphorylation of Fra-2 inhibits this activation, resulting in repression of OC gene transcription. The tissue-specific enhancer element contiguous to the TGF-β1 responsive element contains an AML (Cbfa) binding sequence. This element, designated OC Box II, contributes to 75% of the basal OC promoter activity and forms an osteoblast-specific protein-DNA complex in in-vitro assays. The activation potential of this binding sequence was established by overexpressing AML (Cbfa) transcription activator proteins in osteoblastic as well as in non-osseous cell lines. Interestingly, overexpression not only enhances rOC promoter activity in osteoblasts but also mediates promoter activity in a non-osseous human fibroblastic cell-line. Subsequently, we identified AML-3 (Cbfa1) as the major AML family member present in the osteoblast specific complex and demonstrate that AML-3 (Cbfa1) is expressed predominantly as a 5.4 kb transcript in rat bone tissues. Finally, to establish the functional involvement of AML (Cbfa) transcription factors in osteoblast differentiation, we utilized antisense strategies to demonstrate that blocking expression of all AML (Cbfa) related genes in primary osteoblast cultures significantly decreased several parameters which are linked to differentiation of normal diploid osteoblasts. These results indicate that AML-3 (Cbfa1) is a key transcription factor in bone cells and that the activity of the AML (Cbfa) family of proteins is required for completion of osteoblast differentiation.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectOsteocalcin
dc.subjectGenes, Regulator
dc.subjectTransforming Growth Factor beta
dc.subjectDissertations, UMMS
dc.subjectCell Biology
dc.subjectMolecular Biology
dc.titleThe Osteocalcin Gene: Transcriptional Elements and Factors Regulating TGF-β1 Responsiveness and Tissue-Specific Expression in Bone Cells: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/145
dc.legacy.embargo2017-04-24T00:00:00-07:00
dc.identifier.contextkey207314
html.description.abstract<p>Osteocalcin (OC) is a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix. The <em>Osteocalcin</em> gene is transcriptionally regulated by several basal, hormone- and cytokine-responsive elements. To address the potential role of TGF-β1 regulation and tissue-specific expression of the <em>OC</em> gene, we defined regulatory elements and factors mediating the transcriptional activity of the rat OC (rOC) promoter. TGF-β1 modulates the differentiation of cells of the osteogenic lineage and downregulates the osteoblast-specific expression of <em>OC</em>. By promoter deletion and mutational analyses, a TGF-β1 responsive element at nt -146 to -139 and a contiguous tissue-specific enhancer element at nt -136 to -130 on the rOC promoter were identified. These studies show that Fra-2, a member of the AP-1 family of proteins, binds to the TGF-β1 responsive element and activates basal <em>OC</em> expression. TGF-β1 induced phosphorylation of Fra-2 inhibits this activation, resulting in repression of <em>OC</em> gene transcription. The tissue-specific enhancer element contiguous to the TGF-β1 responsive element contains an AML (Cbfa) binding sequence. This element, designated OC Box II, contributes to 75% of the basal OC promoter activity and forms an osteoblast-specific protein-DNA complex in <em>in-vitro</em> assays. The activation potential of this binding sequence was established by overexpressing AML (Cbfa) transcription activator proteins in osteoblastic as well as in non-osseous cell lines. Interestingly, overexpression not only enhances rOC promoter activity in osteoblasts but also mediates promoter activity in a non-osseous human fibroblastic cell-line. Subsequently, we identified AML-3 (Cbfa1) as the major AML family member present in the osteoblast specific complex and demonstrate that <em>AML-3 (Cbfa1)</em> is expressed predominantly as a 5.4 kb transcript in rat bone tissues. Finally, to establish the functional involvement of AML (Cbfa) transcription factors in osteoblast differentiation, we utilized antisense strategies to demonstrate that blocking expression of all AML (Cbfa) related genes in primary osteoblast cultures significantly decreased several parameters which are linked to differentiation of normal diploid osteoblasts. These results indicate that AML-3 (Cbfa1) is a key transcription factor in bone cells and that the activity of the AML (Cbfa) family of proteins is required for completion of osteoblast differentiation.</p>
dc.identifier.submissionpathgsbs_diss/145
dc.contributor.departmentGraduate School of Biomedical Sciences, Molecular Cell Biology


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