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.

Prolyl hydroxylase domain proteins regulate bone mass through their expression in osteoblasts

“…A previous study showed that knockdown of PHD2 expression was able to abolish ascorbic acid-induced osterix (Osx) expression and alkaline phosphatase activity in MC3T3-E1 or primary mouse calvarial osteoblasts (Xing et al, 2011). In a recent study, Cheng et al generated the conditional Phd2 knockout mice by crossing floxed Phd2 mice with a Col1a2-Cre line, and found the mutant mice displayed short stature, as well as impaired bone mineral content, bone area, and bone mineral density in long bones, but not in vertebrae.…”

Thyroid hormone receptor-β1 signaling is critically involved in regulating secondary ossification via promoting transcription of the Ihh gene in the epiphysis

“…Trabecular bone microarchitecture of the tibia epiphyses (i.e., the SOC) isolated from 21-day-old mice were assessed by CT (viva CT40; Scanco Medical, Switzerland) as described previously (49,51). The tibias were fixed in 10% formalin overnight, washed with PBS, and immersed in PBS to prevent them from drying.…”

“…To analyze SOC formation, the proximal tibia epiphyses were used for measurement of newly formed bone. Parameters such as bone volume (BV, mm 3 ), bone volume fraction (BV/TV, %), trabecular number (Tb.N, mm Ϫ1 ), trabecular thickness (Tb.Th, m) and trabecular space (Tb.Sp, m) were evaluated as described previously (6,49,51).…”

The Roles and Mechanisms of Actions of Vitamin C in Bone: New Developments

“…Under normoxia, and in the presence of Fe2+, 2-oxoglutarate and AA, PHD proteins are activated to induce hydroxylation of certain conserved proline residues in HIFs. The hydroxylated HIFs interact with the β-domain of von Hippel-Lindau tumor suppressor protein (pVHL), and are subsequently ubiquitinated by the pVHL-E3 ligase complex, thereby marking HIFs for degradation by the 26S proteasome (88,89) . Thus, AA-induced activation of PHDs may modulate functions of bone cells in part by regulating the protein level of HIFs and other transcription factors.…”