The PTH-Gαs-protein kinase A cascade controls αNAC localization to regulate bone mass

Mol Cell Biol. 2014 May;34(9):1622-33. doi: 10.1128/MCB.01434-13. Epub 2014 Feb 18.

Abstract

The binding of PTH to its receptor induces Gα(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 Gα(s) 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 activator N(6)-benzoyladenosine cAMP (6Bnz-cAMP) leads to translocation of αNAC to the nucleus. αNAC was phosphorylated by PKA at serine 99 in 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 the Osteocalcin (Ocn) promoter. Altered gene dosages for Gα(s) and αNAC in compound heterozygous mice result in reduced bone mass, increased numbers of osteocytes, and enhanced expression of Sost. 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Active Transport, Cell Nucleus
  • Amino Acid Sequence
  • Animals
  • Bone Diseases, Metabolic / genetics
  • Bone Diseases, Metabolic / metabolism
  • Bone Diseases, Metabolic / pathology
  • Cell Nucleus / metabolism
  • Chromogranins
  • Cyclic AMP / analogs & derivatives
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • GTP-Binding Protein alpha Subunits, Gs / metabolism*
  • Male
  • Mice
  • Molecular Chaperones / analysis
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Osteoblasts / metabolism
  • Osteocalcin / genetics
  • Parathyroid Hormone / metabolism*
  • Phosphorylation
  • Promoter Regions, Genetic
  • Sequence Alignment
  • Signal Transduction

Substances

  • Chromogranins
  • Molecular Chaperones
  • Parathyroid Hormone
  • nascent-polypeptide-associated complex
  • Osteocalcin
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Gnas protein, mouse
  • GTP-Binding Protein alpha Subunits, Gs