-GHRELIN IS INVOLVED IN OSTEOBLAST-OSTEOCLAST INTERACTION THROUGH WNT/BETA CATENIN SIGNALLING PATHWAY

Aims: Considerable attention has been focussed on the effect of energy regulating peptides such as leptin and ghrelin on bone metabolism. Ghrelin, the natural ligand of the growth hormone secretagogue receptor (GHS-R), is considered a positive regulator of bone mass. Ghrelin, in fact, acts directly on osteoblasts and stimulates osteoblast proliferation and differentiation. However, the detailed mechanisms of ghrelin’s modulation of osteoblast activity remain unclear and require further studies. The Wnt/beta-catenin signalling pathway plays a crucial role in osteoblastic cell differentiation and bone formation. When Wnt receptor binding interactions are absent, beta-catenin is phosphorilated by glycogen synthase kinase 3b (GSK-3b) leading to beta-catenin degradation in the proteasome. Following Wnt receptor activation GSK-3b is phosphorylated and inactivated, leading to the stabilization of beta-catenin and its accumulation in the cytoplasm. beta-catenin then migrates to the nucleus where it regulates transcription, and modulates the expression of target genes responsible for osteoblast proliferation and differentiation. The aim of the present study was to investigate the involvement of the Wnt/beta-catenin signalling in the anabolic effect of ghrelin and the receptors involved in such activity. Methods: We used primary cultures of osteoblast like cells derived from rat calvariae exposed to increasing concentrations of ghrelin (10-11-10-7M) for 60 min. beta-catenin and GSK-3b protein levels were measured by Western Blot in the whole cell lysates. Results: Ghrelin significantly increases the phosphorylation and degradation of GSK-3b and therefore it increases beta-catenin levels and beta-catenin nuclear migration. These effects are due to a specific interaction of ghrelin with its GHSR1a receptor since pretreatment with the GHS-R1a receptor antagonist D-LYS-GHRP6 (10-7M for 30min), prevents ghrelin-induced beta-catenin stabilization and nuclear migration. Ghrelin-dependent induction of beta-catenin increases osteoblast proliferation as shown by MTT assay. Considering that osteoprotegerin (OPG), a target gene of the Wnt/ beta-catenin signaling, plays a key role in the regulation of cross-talk between osteoblasts and osteoclasts, we examined the effect of increasing ghrelin concentrations (10-11 - 10-8 M) on the expression of OPG mRNA measured by Real Time PCR in osteoblasts. Ghrelin (10-10 M) significantly increases OPG mRNA levels 24 h after treatment. Thus, it is conceivable that ghrelin by increasing the Wnt/beta-catenin signaling pathway not only exerts an anabolic action but it is also involved in osteoblast-osteoclast interaction. Conclusions: In conclusion, we have shown that ghrelin stimulates osteoblast proliferation and increases OPG expression at the same time through Wnt/beta-catenin signalling pathway. Even if further in vivo studies will be required to confirm this assumption, this study suggests ghrelin as a key candidate to maintain bone homeostasis when there is imbalance between resorption and formation as occurs in osteoporosis.