Control of the SOST bone enhancer by PTH using MEF2 transcription factors.
Expression of the osteocyte-derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH.


BACKGROUND
Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52-kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness.


METHODS
Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast-like UMR-106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody-induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT-PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA-mediated RNA knockdown.


RESULTS
PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co-localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co-expression of a dominant negative MEF2C mutant. Finally, siRNA-mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR-106 osteoblast-like cells.


CONCLUSIONS
These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.
Leupin
Olivier
O
Bone and Cartilage Unit, Musculoskeletal Disease Area, Novartis Institutes for BioMedical Research, Basel, Switzerland.
Kramer
Ina
I
Collette
Nicole M
NM
Loots
Gabriela G
GG
Natt
François
F
Kneissel
Michaela
M
Keller
Hansjoerg
H
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001846:Bone Development / Q000187:drug effects* / Q000235:genetics; D001847:Bone Diseases / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis* / Q000235:genetics; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004622:Embryo, Mammalian / Q000378:metabolism / Q000473:pathology; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D006023:Glycoproteins; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D064326:MEF2 Transcription Factors; D051379:Mice; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D051381:Rats; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D010281:Parathyroid Hormone; C486030:Sost protein, mouse; C486031:Sost protein, rat
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