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

Martin Pellicelli¹, Julie A Miller, Alice Arabian

¹Research Unit, Shriners Hospitals for Children-Canada, Montreal, Quebec, Canada.

Molecular and Cellular Biology

|February 20, 2014

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.

Insights

Parathyroid hormone (PTH) signaling activates protein kinase A (PKA), which phosphorylates the transcriptional coregulator alpha-NAC (αNAC). This phosphorylation drives αNAC nuclear translocation, impacting gene transcription and bone mass regulation.

Area of Science:

Bone Biology
Molecular Endocrinology
Signal Transduction

Background:

Parathyroid hormone (PTH) receptor activation initiates cyclic AMP (cAMP) production, activating protein kinase A (PKA).
A deficiency in Gα(s) in osteoblasts mirrors a mutation causing cytoplasmic retention of the transcriptional coregulator alpha-NAC (αNAC), suggesting a shared pathway.
Understanding the molecular mechanisms linking PTH signaling to transcriptional regulation in bone is crucial for bone mass control.

Purpose of the Study:

To investigate the role of the transcriptional coregulator αNAC in PTH-mediated signaling pathways within osteoblasts.
To determine if PKA directly phosphorylates αNAC and if this modification affects its cellular localization and function.
To elucidate the impact of the Gα(s)-PKA-αNAC axis on bone mass regulation and gene expression.

Main Methods:

Treatment of osteoblasts with PTH(1-34) or a PKA-specific activator (6Bnz-cAMP).
In vitro kinase assays to assess αNAC phosphorylation by PKA.
Analysis of αNAC localization using cell-based assays and mutated variants (S99A, S99D).
Chromatin immunoprecipitation (ChIP) to determine αNAC binding to target gene promoters.
Assessment of bone mass, osteocyte number, and gene expression (Sost) in compound heterozygous mice.

Main Results:

PTH and 6Bnz-cAMP treatment induced nuclear translocation of αNAC in osteoblasts.
PKA directly phosphorylated αNAC at serine 99 in vitro.
Phosphorylation at S99 was essential for PTH-induced nuclear accumulation of αNAC.
αNAC accumulated at the Osteocalcin (Ocn) promoter following PTH or 6Bnz-cAMP stimulation.
Compound heterozygous mice for Gα(s) and αNAC exhibited reduced bone mass, increased osteocytes, and elevated Sost expression.

Conclusions:

αNAC serves as a direct substrate for PKA downstream of PTH receptor signaling.
PKA-mediated phosphorylation of αNAC at S99 promotes its nuclear translocation.
Nuclear accumulation of αNAC at target promoters regulates gene transcription involved in bone mass.
The Gα(s)-PKA-αNAC pathway is a critical regulator of bone homeostasis.

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