Estrogen deficiency attenuates fluid flow-induced [Ca²⁺]_i oscillations and mechanoresponsiveness of MLO-Y4 osteocytes

It has been proposed that estrogen regulates the mechanosensitivity of osteocytes; however, the effects of estrogen deficiency that arises during postmenopausal osteoporosis on mechanical stimulation-induced calcium signaling in osteocytes remain elusive. Here,we pretreated MLO-Y4 osteocyteswith 10 nM E₂ for 2, 3 and 5 d, then simulated postmenopausal conditions either by estrogen withdrawal (EW) from culture medium, or by inhibiting the estrogen receptor by using fulvestrant and estrogen (FE; ICI 182,780) in vitro.We investigated [Ca²⁺]_i oscillations and mechanobiologic responses of osteocytes (EW and FE) that were exposed to oscillatory fluid flow (OFF; 1 Pa, 0.5 Hz).We demonstrated that estrogen treatment enhanced OFF-induced [Ca²⁺]_i oscillations and that this effect was abrogated both by FE and EW. Moreover, osteocytes in both estrogen-depleted groups (EW and FE) had reduced levels ofNOand prostaglandin E₂ release, down-regulated dentin matrix protein-1, sclerostin, osteopontin, osteocalcin, and alkaline phosphatasemRNA expression, and reduced F-actin fiber formation after OFF stimulation comparedwith estrogen-treated cells.We propose a link between estrogen deficiency and alterations in [Ca²⁺]_i-mediated mechanosensitivity of osteocytes, which ultimately alter osteocyte function and differentiation.