The PTH-Gαs-Protein Kinase A Cascade Controls αNAC Localization To Regulate Bone Mass

The binding of PTH to its receptor induces Gαs -dependent cyclic AMP (cAMP) accumulation to turn on effector kinases, including protein kinase A (PKA). The phenotype of mice with osteoblasts specifically deficient for Gαs is mimicked by a mutation leading to cytoplasmic retention of the transcriptional coregulator αNAC, suggesting that Gαs and αNAC form part of a common genetic pathway. We show that treatment of osteoblasts with PTH(1–34) or the PKA-selective activatorN 6 -benzoyladenosine cAMP (6Bnz-cAMP) leads to translocation of αNAC to the nucleus. αNAC was phosphorylated by PKA at serine 99in vitro . Phospho-S99-αNAC 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 αNAC at theOsteocalcin (Ocn ) promoter. Altered gene dosages for Gαs and αNAC in compound heterozygous mice result in reduced bone mass, increased numbers of osteocytes, and enhanced expression ofSost . Our results show that αNAC is a substrate of PKA following PTH signaling. This enhances αNAC translocation to the nucleus and leads to its accumulation at target promoters to regulate transcription and affect bone mass.