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β2-Adrenergic receptor regulates osteoblast differentiation and migration and C-terminal β-catenin phosphorylation
Leah E Worton1, Anna C Curtin2, Edith M Gardiner1
1Department of Orthopaedic Surgery and Sports Medicine, University of Washington, Seattle, WA, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA.
Abstract:
The sympathetic nervous system modulates bone mass in part by inhibiting bone formation through the osteoblastic β2-adrenergic receptor (β2AR), but understanding of the bone anabolic processes hindered by β2AR signaling is incomplete. Canonical Wnt signaling is anabolic in bone, promoting osteoblastic bone formation when the multifunctional β-catenin protein is unphosphorylated at N-terminal residues. In the present study, osteoblastic consequences of Wnt-independent β-catenin C-terminal phosphorylation downstream of β2AR activation were investigated. Treatment with the β-adrenergic agonist isoproterenol (ISO) decreased mineralized nodule formation in differentiating cultures of MC3T3 pre-osteoblasts and BMSCs from WT mice, but not BMSCs from β2AR deficient mice. After treatment with ISO or the β2AR agonist salbutamol there was increased phosphorylation of β-catenin at serine residues 552 and 675, whereas phosphorylation at canonical Wnt-dependent N-terminal sites was unaffected. These C-terminal serine phosphorylation events were inhibited by pre-treatment with β-adrenergic antagonist propranolol or PKA inhibitor H89, indicating that β-catenin phosphorylation downstream of β2AR occurs via G-protein coupled signaling. These phosphorylation events were also dependent on PAK4 kinase activation. Also, co-immunoprecipitation studies suggested enhanced interaction of β-catenin protein phosphorylated at Ser552 with cadherin 11. Finally, β-adrenergic stimulation increased osteoblast attachment and reduced migration, and transfection studies indicated a requirement for serine phosphorylated β-catenin in these effects. These data suggest that increased interaction between cadherin and the C-terminal serine-phosphorylated β-catenin species may contribute to β2AR delay of osteoblast migration. Thus, β-catenin is a novel downstream effector of β2AR signaling and may link β2AR activity to changes in osteoblast cell structure and function.
Insights
Sympathetic nervous system activation inhibits bone formation via osteoblastic beta2-adrenergic receptors (β2AR). This study reveals beta-catenin phosphorylation downstream of β2AR signaling affects osteoblast function and migration.
Area of Science:
- Bone Biology
- Cell Signaling
- Adrenergic Receptor Signaling
Background:
- The sympathetic nervous system regulates bone mass by inhibiting bone formation through osteoblastic beta2-adrenergic receptors (β2AR).
- Canonical Wnt signaling promotes osteoblastic bone formation via unphosphorylated beta-catenin.
- The precise mechanisms by which β2AR signaling hinders bone anabolism are not fully understood.
Purpose of the Study:
- To investigate the osteoblastic consequences of Wnt-independent beta-catenin C-terminal phosphorylation downstream of β2AR activation.
- To elucidate the role of beta-catenin as a downstream effector of β2AR signaling in osteoblasts.
Main Methods:
- Utilized MC3T3 pre-osteoblasts and bone marrow-derived mesenchymal stem cells (BMSCs) from wild-type (WT) and β2AR-deficient mice.
- Administered β-adrenergic agonists (isoproterenol, salbutamol) and antagonists (propranolol).
- Assessed mineralized nodule formation, beta-catenin phosphorylation (Western blot, co-immunoprecipitation), and osteoblast attachment/migration.
Main Results:
- β2AR activation decreased mineralized nodule formation in WT but not β2AR-deficient BMSCs.
- Isoproterenol and salbutamol increased beta-catenin phosphorylation at Ser552 and Ser675, independent of canonical Wnt sites.
- β-catenin phosphorylation was mediated by G-protein coupled signaling, PKA, and PAK4 kinase, and enhanced beta-catenin interaction with cadherin 11.
- β-adrenergic stimulation increased osteoblast attachment and reduced migration, dependent on serine-phosphorylated beta-catenin.
Conclusions:
- Beta-catenin is a novel downstream effector of β2AR signaling in osteoblasts.
- β2AR-mediated beta-catenin C-terminal phosphorylation influences osteoblast function, including attachment and migration.
- Enhanced interaction between cadherin and C-terminal phosphorylated beta-catenin may contribute to β2AR-induced delay in osteoblast migration, impacting bone formation.
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