Abstract
We have recently identified and characterized the human osteoclast (OCL) inhibitory peptide-1 (OIP-1/hSca), a member of Ly-6 gene family. OIP-1 is an important physiologic regulator of OCL development and bone resorption activity. To determine the molecular mechanisms that regulate OIP-1 gene expression in OCL precursor cells, we isolated and characterized the OIP-1/hSca gene (2 Kb) promoter sequence. IFN-gamma (50 ng/ml) treatment of RAW 264.7 macrophage cells transfected with OIP-1 gene (-1 to -1988 bp relative to transcription start site) promoter-luciferase reporter plasmid demonstrated a significant (4 fold) increase in OIP-1 gene promoter activity. Sequence analysis of OIP-1 gene promoter region further identified a potential Stat-1 binding motif at -1629 to -1639 bp position. Stat-1 specific inhibitor, fludarabine (50 muM) abolished IFN-gamma stimulated OIP-1 gene promoter activity. Electrophoretic mobility shift assay (EMSA) further confirmed activated Stat-1 binding to the OIP-1 gene promoter sequence suggesting that IFN-gamma regulates OIP-1 gene expression in OCL precursor cells through a Stat-1 dependent signaling pathway. We further show that knock-down of TRADD enhances IFN-gamma signaling to increase OIP-1 gene expression in OCL precursor cells. These results should provide insights into the molecular control of OIP-1 gene expression and inhibition of OCL activity in the bone microenvironment.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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ATPases Associated with Diverse Cellular Activities
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Adaptor Proteins, Signal Transducing / biosynthesis*
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Adaptor Proteins, Signal Transducing / genetics
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Animals
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Antineoplastic Agents / metabolism
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Antineoplastic Agents / pharmacology
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Bone Marrow / metabolism
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Bone Resorption / genetics
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Bone Resorption / metabolism*
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Cell Differentiation / physiology*
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Cell Line
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Electrophoretic Mobility Shift Assay / methods
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Gene Expression Regulation / drug effects
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Gene Expression Regulation / physiology*
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Interferon-gamma / metabolism
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Interferon-gamma / pharmacology
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LIM Domain Proteins
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Mice
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Osteoclasts / cytology
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Osteoclasts / metabolism*
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Proteasome Endopeptidase Complex
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Response Elements / physiology*
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STAT1 Transcription Factor / antagonists & inhibitors
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STAT1 Transcription Factor / genetics
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STAT1 Transcription Factor / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology
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Transcription Factors / biosynthesis*
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Transcription Factors / genetics
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Vidarabine / analogs & derivatives
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Vidarabine / pharmacology
Substances
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Adaptor Proteins, Signal Transducing
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Antineoplastic Agents
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LIM Domain Proteins
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PSMC5 protein, human
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STAT1 Transcription Factor
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STAT1 protein, human
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Transcription Factors
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Interferon-gamma
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Proteasome Endopeptidase Complex
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ATPases Associated with Diverse Cellular Activities
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Vidarabine
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fludarabine