Inhibition of osteoclastogenesis by mechanically stimulated osteoblasts is attenuated during estrogen deficiency

Abstract

Osteoporotic bone loss and fracture have long been regarded to arise upon depletion of circulating estrogen, which increases osteoclastogenesis and bone resorption. Osteoblasts from human osteoporotic patients also display deficient osteogenic responses to mechanical loading. However, while osteoblasts play an important role in regulating osteoclast differentiation, how this relationship is affected by estrogen deficiency is unknown. This study seeks to determine how mechanically stimulated osteoblasts regulate osteoclast differentiation and matrix degradation under estrogen deficiency. Here, we report that osteoblast-induced osteoclast differentiation (indicated by tartrate-resistant acid phosphatase, cathepsin K, and nuclear factor of activated T cells, cytoplasmic 1) and matrix degradation were inhibited by estrogen treatment and mechanical loading. However, estrogen-deficient osteoblasts exacerbated osteoclast formation and matrix degradation in conditioned medium and coculture experiments. This was accompanied by higher expression of cyclooxygenase-2 and macrophage colony-stimulating factor, but not osteoprotegerin, by osteoblasts under estrogen deficiency. Interestingly, this response was exacerbated under conditions that block the Rho-Rho-associated protein kinase signaling pathway. This study provides an important, but previously unrecognized, insight into bone loss in postmenopausal osteoporosis, whereby estrogen-deficient osteoblasts fail to produce inhibitory osteoprotegerin after mechanical stimulation but upregulate macrophage colony-stimulating factor and cyclooxygenase-2 expression and, thus, leave osteoclast activity unconstrained.

Scl-Ab reverts pro-osteoclastogenic signaling and resorption in estrogen deficient osteocytes

“…Although membrane-bound RANKL has been shown to be more potent than soluble RANKL [51], the results presented here suggests that under estrogen de cient conditions this may not be the case. In vitro osteoblast-osteoclast studies previously reported an increase in matrix degradation in estrogen de cient groups compared to estrogen treated group [52]. Here we report a signi cant increase in bone resorption through use of bovine bone resorption assays, in estrogen de cient groups compared to estrogen treated groups.…”

“…Following 15 days of expansion in α-MEM culture media at 37 °C, OCY454 cells were treated with estrogen (E: 10 nM 17βestradiol) for 6 days. To simulate postmenopausal conditions (ED), we implemented a model that rst accustomed osteocytes to estrogen before a subsequent period where estrogen supplementation was discontinued, known as estrogen withdrawal, based on our previous established postmenopausal model using MC3T3-E1 and MLO-Y4 cells [23,52]. OCY454 cells were treated with 17β-estradiol (10 nM) for 3 days before it was withdrawn from the media for a further 3 days.…”

“…Mechanically stimulated OCY454 cells from each group (E, E + Scl-Ab, ED and ED + Scl-Ab) were seeded at 25,000 cells per well in a 48 well plate. After 24 hours RAW264.7 cells were seeded on top of OCY454 cells at 10,000 per well based on a previous study [52] and BMM cells were seeded at 34,000 cells per well. Cells were co-cultured in the presence of 15 ng/mL of RANKL for a further 6 days.…”

Scl-Ab reverts pro-osteoclastogenic signalling and resorption in estrogen deficient osteocytes 

“…Although membrane-bound RANKL has been shown to be more potent than soluble RANKL [59], the results presented here suggests that under estrogen de cient conditions this may not be the case. In vitro osteoblast-osteoclast studies previously reported an increase in matrix degradation in estrogen de cient groups compared to estrogen treated group [60]. Here we report a signi cant increase in bone resorption through use of bovine bone resorption assays, in estrogen de cient groups compared to estrogen treated groups.…”

“…Following 15 days of expansion in α-MEM culture media at 37°C, OCY454 cells were treated with estrogen (E: 10 nM 17βestradiol) for 6 days. To simulate postmenopausal conditions (ED), we implemented a model that rst accustomed osteocytes to estrogen before a subsequent period where estrogen supplementation was discontinued, known as estrogen withdrawal, based on our previous established postmenopausal model using MC3T3-E1 and MLO-Y4 cells [27,60]. OCY454 cells were treated with 17β-estradiol (10 nM) for 3 days before it was withdrawn from the media for a further 3 days.…”