Two lines of evidence suggest that the calcium-sensing receptor (CaSR) in osteoblasts is a therapeutic target for the treatment of osteoporosis and reduction in fractures. Knockout of the CaSR in osteoblasts drastically reduced bone mass in mice (Chang,Sci Signal, 2008). Strontium, which has been shown to reduce fractures in randomised controlled trials, appears to act, at least in part, through the CaSR (Brennan, Brit J Pharm, 2009; Rybchyn, JBC, 2011). Divalent cations are not the only agents that activate the CaSR, which also responds to L-amino acids and small peptides (Broadhead, JBC, 2011). The current study assessed the effects of a tripeptide, S-methylglutathione (SMG), on signalling, proliferation, differentiation, and cell survival after H2O2-induced oxidative stress in primary human osteoblasts (HOBs) and HOBs immortalised by transfection with SV40 large T antigen. Bone cells were cultured in DMEM with 10% fetal calf serum and adapted to serum free media (1mM Ca2+) for 24 hours. Alkaline phosphatase activity increased in both bone cell types after 12 days treatment with 20 μM SMG compared with vehicle (p<0.001). Treatment with 20 μM SMG for 24 hours also prevented significant increases in apoptosis, measured by caspase-3 activity, in both bone cell types, in response to 2.5 h exposure to 50 μM H2O2 as an inducer of oxidative stress (p<0.05). Assessed via western blot, SMG increased phosphorylation of Akt at S473, mTOR at S2448, β-catenin at S552 (part of the canonical Wnt pathway) as well as Erk1/2 at Thr202/Y204 in HOBs. Knockdown of the CaSR by siRNA in HOBs, reduced SMG-stimulated phosphorylation of Akt (p<0.05), mTOR (p<0.01) and β-catenin (p<0.01). These results support the proposal that the new CaSR-acting modulator increases bone cell differentiation and protects against oxidative stress. The data indicate a role for this or a related analogue as a potential novel therapy for osteoporosis in humans.
Disclosure: The authors declared no competing interests. This work was funded by a project grant from the National Health and Medical Research Council of Australia.