Ghrelin Increases Beta-Catenin Level through Protein Kinase A Activation and Regulates OPG Expression in Rat Primary Osteoblasts.

Emanuela Mrak,Alessandro Rubinacci,Valeria Sibilia,Lavinia Casati,Francesca Pagani,Guido Zarattini

doi:10.1155/2015/547473

Emanuela Mrak, Alessandro Rubinacci + Show 4 more

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https://doi.org/10.1155/2015/547473

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Abstract

Ghrelin, by binding growth hormone secretagogue receptor (GHS-R), promotes osteoblast proliferation but the signaling mechanism of GHS-R on these cells remains unclear. Since canonical Wnt/β-catenin pathway is critically associated with bone homeostasis, we investigated its involvement in mediating ghrelin effects in osteoblasts and in osteoblast-osteoclast cross talk. Ghrelin (10−10M) significantly increased β-catenin levels in rat osteoblasts (rOB). This stimulatory action on β-catenin involves a specific interaction with GHS-R1a, as it is prevented by the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10−7M). The effect of ghrelin on β-catenin involves the phosphorylation and inactivation of GSK-3β via protein kinase A (PKA). Inhibition of PKA activity reduces the facilitatory action of ghrelin on β-catenin stabilization. Ghrelin treatment of rOB significantly increases the expression of osteoprotegerin (OPG), which plays an important role in the regulation of osteoclastogenesis, and this effect is blocked by D-Lys3-GHRP-6. Furthermore, ghrelin reduced RANKL/OPG ratio thus contrasting osteoclastogenesis. Accordingly, conditioned media from rOB treated with ghrelin decreased the number of multinucleated TRAcP+ cells as compared with the conditioned media from untreated-control rOB. Our data suggest new roles for ghrelin in modulating bone homeostasis via a specific interaction with GHSR-1a in osteoblasts with subsequent enhancement of both β-catenin levels and OPG expression.

Highlights

Accumulating evidence, including in vitro and in vivo studies in animal models as well as observational studies in humans, indicate a role for ghrelin in the control of bone cell activities [1,2,3,4].Ghrelin, an endogenous ligand of the GH secretagogue receptor (GHS-R), and its receptor GHS-R1a are expressed in several osteoblasts and osteoblastic cell lines
Since canonical Wnt/β-catenin pathway is critically associated with bone homeostasis, we investigated its involvement in mediating ghrelin effects in osteoblasts and in osteoblast-osteoclast cross talk
In order to study whether the observed β-catenin stabilization induced by ghrelin involves a specific interaction with its GHS-R1a, primary rat osteoblast-like cells were treated for 30 min with the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10−7 M), and challenged with ghrelin (10−10 M). β-catenin levels were measured in both the cytosolic and nuclear compartments after fractioning of the whole cell lysate

Summary

Introduction

Accumulating evidence, including in vitro and in vivo studies in animal models as well as observational studies in humans, indicate a role for ghrelin in the control of bone cell activities [1,2,3,4]. Since the canonical Wnt/β-catenin pathway is critically associated with the entire osteoblast life cycle, that is, commitment, proliferation, apoptosis, and function, we hypothesized that ghrelin exerts its anabolic action in bone through canonical Wnt signaling and β-catenin stabilization. This hypothesis is supported by the observation that in rat cortical neuron cells ghrelin exerts specific, receptor mediated antiapoptotic effects by activating ERK1/2 pathway and by stabilizing β-catenin via PI3K/Aktmediated inactivation of glycogen synthase kinase (GSK-3β) [20]. We evaluated whether or not ghrelin, by modulating the OPG/RANKL/RANK system, could influence osteoclastogenesis in cultured rat osteoclast precursors cells

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