Signaling networks from Gbeta1 subunit to transcription factors and actin remodeling via a membrane-located ERbeta-related protein in the rapid action of daidzein in osteoblasts

Although estrogen replacement has been the main therapy to prevent and treat osteoporosis, there are concerns about its safety. Phytoestrogens have attracted attention to their potential impacts in osteoporosis prevention and treatment. Among phytoestrogens, the isoflavone daidzein (Dz) acts on transcription via the intracellular estrogen receptors (ER), mainly ERbeta, in osteoblasts, but mimics only part of the estrogen effects. Since estradiol also exerts rapid effects in osteoblasts, we investigated the multistep processes involved in the rapid actions of low (1-100 pM) doses of daidzein. Dz bound to a membrane moiety, related to ERbeta since the calcium response to Dz was blocked by an anti-ERbeta antibody directed against the C-terminus, but not by a double-stranded siRNA specific for ERbeta. This protein was coupled to a pertussis toxin (PTX)-sensitive Gbeta1 subunit whose transducer was PLC-beta2, which triggered a rapid (5 sec) mobilization of calcium from the endoplasmic reticulum. Dz phosphorylated within 15 sec ERK1/2 whose phosphorylation involved two routes: Gbeta1/PLC-beta2/PKC/c-Raf-1/MEK1/2 and Gbeta1/PI3K/cSrc/c-Raf-1/MEK1/2 as shown using several inhibitors. Dz induced rapid (1 min) changes in the actin cytoskeleton via the two routes. The rapid (20 sec) phosphorylation of Elk-1 and CREB by Dz involved Gbeta1 and ERK1/2. All the processes were insensitive to the estradiol antagonist ICI 182,780. In conclusion, the rapid effects of Dz seem to be biologically relevant for the function of osteoblast in bone since the isoflavone activates transcription factors linked to early genes controlling cellular proliferation and differentiation, and modulates actin cytoskeleton which controls cell adhesion, division, or secretion.