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

Abstract

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.

Regulation of Bone Remodeling by Parathyroid Hormone

“…Therefore, additional intracellular signaling nodes downstream from the PTH receptor must exist. Nascent polypeptide-associated complex and coregulator α (αNAC) is another PKA substrate that shuttles from the cytoplasm to the nucleus on phosphorylation (Pellicelli et al 2014). In the nucleus, αNAC associates with bZIP family transcription factors and enhances their activity (Akhouayri et al 2005).…”

Connexin43 and Runx2 Interact to Affect Cortical Bone Geometry, Skeletal Development, and Osteoblast and Osteoclast Function

“…Similar approaches have been used to detect interactions of genes controlling bone remodeling. (44,45) These compound Gja1 þ/-Runx2 þ/animals (referred to as Cmpd) are viable and thus provide a way to assess the effects of Cx43 and Runx2 deficiency on bone while avoiding the perinatal lethality caused by homozygous null mutations of either gene individually. (20,28) If Cx43 and Runx2 do functionally intersect, then we hypothesized that there would be a unique (or more than additive) skeletal phenotype in the skeletal properties of the Cmpd animals not seen in singly hemizygous (Gja1 þ/or Runx2 þ/-) or WT littermate controls.…”

Parathyroid Hormone Signaling in Osteocytes

“…Therefore, additional intracellular signaling nodes downstream of the PTH receptor must exist. Nascent polypeptide‐associated complex and co‐regulator alpha (αNAC) is another PKA substrate that shuttles from the cytoplasm to the nucleus upon phosphorylation . In the nucleus, αNAC associates with bZIP family transcription factors and enhances their activity .…”