Expression of an estrogen receptor agonist in differentiating osteoblast cultures

Abstract

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-␤, but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100°C and separable from the bulk of the remaining alcohol-and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a Mr of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease.intracrine ͉ selective estrogen receptor modulator ͉ steroid S ex steroids cause direct genomic transcriptional effects through specific receptors that contain ligand binding, dimerization, DNA binding, and gene transactivation domains. They also cause indirect stimulatory or inhibitory effects through complexes composed of activated hormone receptor and other transcription factors or through variations in the activation potential of other transcriptional components (1-6) The skeleton is a well recognized target for sex steroids. Bone fragility is notable when sex steroid levels fall in women after menopause, in elderly males, or after sex organ ablation (7-11). In such cases, bone loss follows a release from native constraints on bone resorption that largely result from changes in growth regulators expressed by osteoblasts and from opposing effects on osteoblast and osteoclast activation and apoptosis, leading to an overall increase in bone remodeling (12-16). Importantly, imbalances in bone remodeling are restored by sex hormone replacement therapy. Regardless of a clear benefit for bone, use of native sex steroids risks inappropriate gene activation in other tissues. The possibility of breast, cardiovascular, and prostate disease that can occur in this context (17-19) has driven the search for sex hormone receptor modulators with tissue-or function-restricted effects. However, the value of agents like these may be complicated by steroid and precursor metabolizing enzymes expressed by osteoblasts (3,20,21).Many cells u...

Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual

“…In full support of the contention that the unliganded ERα has a diametrically opposite effect to that of estrogens on periosteal bone expansion, it is documented that estrogens suppress periosteal bone formation and that estrogen deficiency in female rodents and women increases periosteal apposition (2,24). Be that as it may, we cannot completely exclude the possibility that some residual estrogens in the charcoal-stripped serum or estrogens produced by osteoblasts themselves or perhaps estrogen-like alternative ligands could have activated the ERα (25,26).…”

MG63 and MC3T3-E1 Osteoblastic Cell Lines Response to Raloxifene

“…After 7 days of contact with raloxifene, MC3T3-El cells, used at the 60th passage of culture, showed a significant reduction of both cell proliferation and their alkaline phosphatase activity which may, at least partly, be due to a different capacity that these "aged" cells have to respond to estrogens (22) compared to younger cells. Present findings stress the validity of the in vitro cell model used to mimic in vivo elderly bone.…”

The Gene for Aromatase, a Rate-Limiting Enzyme for Local Estrogen Biosynthesis, Is a Downstream Target Gene of Runx2 in Skeletal Tissues

“…While these studies were in progress, it was revealed that osteoblasts produce endogenous estrogen receptor agonists (22). Moreover, Teplyuk and colleagues showed that Runx2 controls enzymes (e.g., Cyp11a1) that support the production of pregnenolone (46) as well as the expression of the nongenomic receptor for estrogen (Gpr30/Gper), which is necessary for osteoblast proliferation in cell culture (47).…”