G-PROTEIN SIGNALLING PATHWAYS AND OESTROGEN : A ROLE OF BALANCED MAINTENANCE IN OSTEOBLASTS

Abstract Oestrogen (E 2 ) is an important regulator of bone cell function and alterations in oestrogen levels may cause abnormal bone metabolism in vivo. In this study we examined the long term effects of 17β-oestradiol (17β-E 2 ) on G-proteins and the secondary signalling pathways of phospholipase C (PLC), cyclic adenosine monophosphate (cAMP), and 1,4,5-inositol triphosphate (IP 3 ). Cells from neonatal mouse calvariae were cultured in phenol red-free RPMI 1640 medium supplemented with charcoal stripped foetal calf serum for 192 h with either oestrogen (10 −8 M), or oestrogen withdrawal after 48 h. Cultures were stimulated for the final 48 h with IL-6 (10 −10 M), or left unstimulated. Western blot analysis was undertaken on osteoblast membrane preparations obtained by 10 mM Tris-HCl, 0.1 mM EDTA pH 7.8 and centrifugation at 40 000× g for 2 h. For cAMP study, cells were stimulated with IL-6 for either 15 min or 30 min. Intracellular cAMP was extracted from cells and measured by ELISA methodology. For the IP 3 assay, cells were stimulated with IL-6 for 20 s and IP 3 levels measured using radioimmunoassay. The blots revealed increased levels of G i α-, and G q α-proteins with oestrogen withdrawal and IL-6 stimulation. This was in comparison to cells which were unstimulated, or stimulated with IL-6 with continuous 17β-E 2 , or IL-6 alone. G s α expression decreased with oestrogen withdrawal compared to the control. Limited amounts of G i α-, G s α-, and G q α-proteins were identified with continuous 17β-E 2 . The levels of PLC isoforms PLCβ 1-2 were not affected by the differing oestrogen conditions. The cAMP production induced by IL-6 stimulation for 30 min and withdrawal of 17β-E 2 was lower and significantly different compared to the control study ( P P 2 had no effect on the formation of intracellular IP 3 , although IL-6 significantly lowered IP 3 levels in all the groups compared to the control ( P