Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line

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Abstract
Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis for ERα mRNA and transactivation of adenovirus‐estrogen response element (ERE)‐tk‐luciferase by 17β‐estradiol (17β‐E2) for functional ER protein. One of these cell lines, termed HOB‐03‐CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature‐sensitive SV40 large T antigen, proliferated at the permissive temperature (34°C) but stopped dividing at the nonpermissive temperature (&ge 39°C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1&agr 25‐dihydroxyvitamin D3 in a dose‐dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red‐S and von Kossa histochemical staining, and responded to dexamethasone, all‐trans retinoic acid, and transforming growth factor‐β1. This cell line expressed 42‐fold less ER message than MCF‐7 human breast cancer cells, as determined by quantitative RT‐PCR. However, adenovirus‐ERE‐tk‐luciferase activity was upregulated three‐ to fivefold in these cells by 17β‐E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co‐treatment with the anti‐estrogen ICI‐182, 780. Cytosolic extracts of these cells specifically bound [125I]‐17β‐E2 in a concentration‐dependent manner with a Bmax of 2.7 fmoles/mg protein (∼ 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel‐shift analysis using a [32P]‐ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA‐binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17β‐E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17β‐E2 suppressed parathyroid hormone‐induced cAMP production, as well as basal interleukin‐6 mRNA expression; conversely, the steroid upregulated the steady‐state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17β‐E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone. J. Cell. Biochem. 65:368–387.