The binding of PTH to its receptor induces G␣ s -dependent cyclic AMP (cAMP) accumulation to turn on effector kinases, including protein kinase A (PKA). The phenotype of mice with osteoblasts specifically deficient for G␣ s is mimicked by a mutation leading to cytoplasmic retention of the transcriptional coregulator ␣NAC, suggesting that G␣ s and ␣NAC form part of a common genetic pathway. We show that treatment of osteoblasts with PTH(1-34) or the PKA-selective activator N 6 -benzoyladenosine cAMP (6Bnz-cAMP) leads to translocation of ␣NAC to the nucleus. ␣NAC was phosphorylated by PKA at serine 99 in vitro. Phospho-S99-␣NAC accumulated in osteoblasts exposed to PTH(1-34) or 6Bnz-cAMP but not in treated cells expressing dominant-negative PKA. Nuclear accumulation was abrogated by an S99A mutation but enhanced by a phosphomimetic residue (S99D). Chromatin immunoprecipitation (ChIP) analysis showed that PTH(1-34) or 6Bnz-cAMP treatment leads to accumulation of ␣NAC at the Osteocalcin (Ocn) promoter. Altered gene dosages for G␣ s and ␣NAC in compound heterozygous mice result in reduced bone mass, increased numbers of osteocytes, and enhanced expression of Sost. Our results show that ␣NAC is a substrate of PKA following PTH signaling. This enhances ␣NAC translocation to the nucleus and leads to its accumulation at target promoters to regulate transcription and affect bone mass.

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We previously reported a loss-of-PITX1 expression in patients suffering of knee/hip osteoarthritis (OA). Search for the mechanism underlying this event led us to discover that PITX1 repression was triggered by the aberrant nuclear accumulation of Prohibitin (PHB1), an E2F1 co-repressor, in OA articular chondrocytes. In the current study, we assessed in details the involvement of E2F transcription factors in regulating PITX1 expression. We also analyzed other genes that are similarly regulated by E2F in regard to osteoarthritis. The transcriptional regulation of the PITX1 promoter by E2F1 was analyzed with the luciferase reporter assay, and chromatin immunoprecipitation assays, which confirmed direct E2F1-PITX1 interactions. The probable binding sites for E2F1 in the PITX1 promoter were identified by DNA pulldown experiments. In silico and in vitro analyses show that the PITX1 proximal promoter region contains 2 specific sequences that are bound by E2F1. Overexpression of E2F1 enhances PITX1 promoter activity and mRNA transcription. In primary control and osteoarthritis chondrocytes, real time RT-PCR was used to measure the mRNA expression levels of candidate genes under E2F1 transcriptional control. Transcription Factor Dp-1 (TFDP1) knockdown experiments confirmed that the E2F1-TFDP1 complex regulates PITX1. Knockdown of TFDP1, an E2F1 dimerization partner, inhibits the activating effect of E2F1 and reduces both PITX1 promoter activity and mRNA transcription. Real time RT-PCR results reveal reduced expression of TFDP1 and a similar downregulation of their targets PITX1, BRCA1, CDKN1A, and RAD51 in mid-stage OA chondrocytes. Collectively, our data define a previously uncharacterized role for E2F1 and TFDP1 in the transcriptional regulation of PITX1 in articular chondrocytes. Additional E2F1 targets may be affected in OA pathogenesis.