G-protein signalling pathways and oestrogen: a role of balanced maintenance in osteoblasts

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 17 beta-oestradiol (17 beta-E-2) on G-proteins and the secondary signalling pathways of phospholipase C (PLC), cyclic adenosine monophosphate (cAMP), and 1,4,5-inositol triphosphate (IP3). 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 x 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 IP3 assay, cells were stimulated with IL-6 for 20 s and IP3 levels measured using radioimmunoassay. The blots revealed increased levels of G(i)alpha-, and G(q)alpha-proteins with oestrogen withdrawal and IL-6 stimulation. This was in comparison to cells which were unstimulated, or stimulated with IL-6 with continuous 17 beta-E-2, or IL-6 alone. G(s)alpha expression decreased with oestrogen withdrawal compared to the control. Limited amounts of Gia-, G(s)alpha-, and G(q)alpha-proteins were identified with continuous 17 beta-E-2. The levels of PLC isoforms PLC beta(1-2) were not affected by the differing oestrogen conditions. The cAMP production induced by IL-6 stimulation for 30 min and withdrawal of 17 beta-E-2 was lower and significantly different compared to the control study (P < 0.05). Also IL-6 activation with continuous oestradiol increased cAMP levels and was significantly different from the control cells (P < 0.01). However, 17 beta-E-2 had no effect on the formation of intracellular IP3, although IL-6 significantly lowered IP3 levels in all the groups compared to the control (P < 0.01). These results suggest that oestrogen modulates the signal transduction pathways of G-protein molecules, and the secondary pathways of cAMP in mouse osteoblast-like cells. (C) 1999 Elsevier Science B.V. Al rights reserved.