title: Ascorbic acid regulates osterix expression in osteoblasts by activation of prolyl hydroxylase and ubiquitination-mediated proteosomal degradation pathway.
abstract: Mouse genetic studies reveal that ascorbic acid (AA) is essential for osteoblast (OB) differentiation and that osterix (Osx) was a key downstream target of AA action in OBs. To determine the molecular pathways for AA regulation of Osx expression, we evaluated if AA regulates Osx expression by regulating production and/or actions of local growth factors and extracellular matrix (ECM) proteins. Inhibition of actions of IGFs by inhibitory IGFBP-4, BMPs by noggin, and ECM-mediated integrin signaling by RGD did not block AA effects on Osx expression in OBs. Furthermore, blockade of components of MAPK signaling pathway had no effect on AA-induced Osx expression. Because AA is required for prolyl hydroxylase domain (PHD) activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome-mediated degradation of transcriptional repressor(s) of Osx gene. Treatment of OBs with dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate, known inhibitors of PHD, completely blocked AA effect on Osx expression and OB differentiation. Knockdown of PHD2 expression by Lentivirus-mediated shRNA abolished AA-induced Osx induction and alkaline phosphatase activity. Furthermore, treatment of OBs with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD2 and proteosomal degradation of a yet to be identified transcriptional repressor that is independent of BMP, IGF-I, or integrin-mediated signaling in mouse OBs.
authors.affiliation: Musculoskeletal Disease Center, Jerry L. Pettis VA Medical Center, California, USA.
authors.forename: Weirong
authors.initials: W
authors.lastname: Xing
authors.forename: Sheila
authors.initials: S
authors.lastname: Pourteymoor
authors.forename: Subburaman
authors.initials: S
authors.lastname: Mohan
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D017931:DNA Primers; D034741:RNA, Small Interfering; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D011393:Procollagen-Proline Dioxygenase; D046988:Proteasome Endopeptidase Complex; D001205:Ascorbic Acid