Estrogen Receptor-Beta Regulates Mechanical Signaling in Primary Osteoblasts

Abstract

Mice lacking estrogen receptor-alpha (ERα) show reduced periosteal bone formation in response to mechanical loading suggesting that ERa plays an important role in bone mechanotransduction. Much less is known about the role of estrogen receptor-beta (ERβ) in mechanotransduction. Recent studies in female mice lacking ERβ show they have an enhanced response to mechanical loading suggesting that ERβ may inhibit load-induced periosteal bone formation. The enhanced response to loading is not observed in male mice lacking ERβ. PURPOSE: To determine the role of ERβ in osteoblast mechanotransduction in the presence and absence of estradiol. METHODS: Primary calvarial osteoblasts were harvested from 3–5 day-old wild-type (WT) and estrogen receptor-beta knockout (ERβ-−/−) mice and cultured in Dulbecco's minimum essential media (DMEM) + 10% fetal calf serum (FCS) and 1 % penicillin/ streptomycin (P/S). Early passage WT and ERβ−/− cells were plated onto collagen-coated glass slides. At 80% confluency, cells were loaded into parallel plate flow chambers and subjected to oscillatory fluid flow (OFF) which produced a fluid shear stress of 15 dyn/cm2 at 1 Hz. Cells were subjected to OFF for varying periods in the presence and absence of estradiol, and the activation of mechanical signaling pathways known to be important in bone physiology was observed. RESULTS: OFF significantly enhanced prostaglandin E2 (PGE2) release in WT and ERβ−/− cells in both the presence and absence of estradiol. PGE2 levels were significantly higher in ERβ−/− cells subjected to OFF and treated with estradiol compared to all other groups. Cyclooxygenase 2 (COX-2) expression was significantly greater in WT, but not ERβ−/− cells, in the absence of estradiol. Estradiol treatment rescued OFF-induced COX-2 expression in ERβ−/− cells. Estrogen receptor transcriptional activity, as measured by estrogen responsive element (ERE)-luciferase reporter activity, was significantly greater in ERβ-−/− cells regardless of flow conditions. OFF significantly enhanced transcriptional activity in ERβ−/− cells treated with estradiol. CONCLUSIONS: ERβ may normally suppress load-induced periosteal bone formation by regulating PGE2 release and ERE transcriptional activity in osteoblasts.