Probing glucocorticoid‐dependent osteogenesis in rat and chick cells in vitro by specific blockade of osteoblastic differentiation with progesterone and RU38486

Abstract

Glucocorticoids and sex-steroids can modulate osteogenesis in vivo and in vitro. Although the effects of glucocorticoids on bone cells in vitro have been described in detail, the role of sex-steroids is not as well defined. We examined whether sex-steroids influence bone metabolism indirectly by regulating glucocorticoid effects on bone. Interactions of the sex-steroid progesterone or its analog RU38486 with the glucocorticoid dexamethasone (dex) were studied in functional assays of osteogenesis. Three osteoblastic models were evaluated: (1) the rat bone marrow stromal cell (RBMC) nodule system; (2) the chick periosteal osteogenesis (CPO) model; and (3) ROS 17/2.8 cells. RU38486, progesterone, and unlabelled dex competitively inhibited 3H-dex uptake by ROS 17/2.8 cells as well as its (3H-dex) binding to cytosol preps. Both RU38486 and progesterone inhibited dex-induced increases in alkaline phosphatase in CPO cultures, in RBMC cultures, and in ROS 17/2.8 cells. Dex-induced decreases in cell proliferation in ROS 17/2.8 cells were reversed by RU38486 but dex-induced increases in proliferation in the CPO model were not affected. In CPO cultures, dex-induced increases in collagen synthesis were inhibited completely by RU38486 and progesterone. Dex-dependent nodule formation in the RBMC was blocked by RU38486. Both RU38486 and dex mediated reduction of calcium uptake in the CPO model but did not affect mineralized tissue area. The data indicate that RU38486 and progesterone competitively inhibit dex-mediated stimulation of osteogenesis in vitro; this inhibition is exerted on early but not late stage differentiation events of osteoprogenitor cells.

Inhibition of dioxin effects on bone formation in vitro by a newly described aryl hydrocarbon receptor antagonist, resveratrol

“…First, we wanted to investigate in more detail how the prototypical AhR ligand, TCDD, might affect bone cell differentiation and bone formation in vitro using previously characterized osteogenic model systems (Maniatopoulos et al 1988, Tenenbaum et al 1995, Sukhu et al 1997. Secondly, presuming that TCDD did inhibit osteodifferentiation and/or osteogenesis, we also wanted to determine whether these putative effects could be inhibited by resveratrol, thereby 'protecting' bone from the potentially harmful actions of TCDD.…”

“…CPO and SBMC cultures were assayed for AP activity using paranitrophenol-phosphate (pN-p) as the substrate (Tenenbaum et al 1995, Demetriou et al 1996. The intensity of the color change was evaluated using a Titertek Multiskan MCC/340 Spectrophotometer (Flow Laboratories, Mississauga, ON, Canada) and compared with a standard curve obtained from a serial dilution of 10 mM pN with bicarbonate buffer (pH 7·4) in flat bottomed Titertek 96-well plates as described previously (Demetriou et al 1996).…”

“…Calcium incorporation was assayed in the pellets produced from centrifugation of the homogenized CPO cultures following hydrolysis in 500 µl 0·5 M HCl and then measured using atomic absorption spectrophotometry (Sukhu et al 1997). In some cases, AP activity was also measured using direct staining in situ of SBMC cultures in 96-well plates as described previously (Tenenbaum et al 1995, Demetriou et al 1996. Mineralization in the SBMC cultures was determined using Alizarin Red staining also in situ, which was assessed with the Titertek spectrophotometer set at 525 nm.…”

“…Radiolabeled collagen was measured in CPO cultures incubated with [ 14 C]glycine (Amersham Pharmacia Biotech Inc., Quebec City, Canada, 59 mCi/mmol) at a concentration of 10 µCi/ml media in each group over days 4-6 as described previously (Tenenbaum et al 1995). Fluorography was used to analyze the amount of newly synthesized 14 C-labeled collagen in each sample.…”

Glucocorticosteroid-induced spinal osteoporosis: scientific update on pathophysiology and treatment

“…In addition, HRT has been shown to reduce significantly the risk of hip, vertebral and other osteoporotic fractures in post-menopausal women [118]. On the other hand, progesterone binds competitively to the GC receptor of the osteoblast and may act as a GC antagonist [99,135]. However, published randomized controlled trials of HRT in the prevention or treatment of GIOP are rare.…”