FUNCTIONAL-PROPERTIES OF A CONDITIONALLY PHENOTYPIC, ESTROGEN-RESPONSIVE, HUMAN OSTEOBLAST CELL-LINE

Osteoblasts are established targets of estrogen action in bone. We scr eened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase c hain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tkluciferase by 17 beta- estradiol (17 beta-E-2) for functional ER protein. One of these cell l ines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensit ive SV40 large T antigen, proliferated al the permissive temperature ( 34 degrees C) but stopped dividing al the nonpermissive temperature (g reater than or equal to 39 degrees C). Alkaline phosphatase activity a nd osteocalcin secretion were upregulated by 1 alpha,25-dihydroxyvitam in D-3 in a dose-dependent manner. The cells also expressed type I col lagen and other bone matrix proteins, secreted a variety of growth fac tors 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-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast ca ncer cells, as determined by quantitative RT-PCR. However, adenovirus- ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E-2 with an EC50 of 64 pM. Furthermore, this upregul ation was suppressed by co-treatment with the anti-estrogen ICI-182,78 0. Cytosolic extracts of these cells specifically bound [I-125]-17 bet a-E-2 in a concentration-dependent manner with a B-max of 2.7 fmoles/m g protein (similar to 1,200 ERs/cell) and a K-d of 0.2 nM. DNA gel-shi ft analysis using a [P-32]-ERE demonstrated the presence of ERs in nuc lear extracts of these cells. Moreover, binding of the extracts to thi s ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous res ponses to 17 beta-E-2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E-2 suppressed para thyroid hormone-induced cAMP production, as well as basal interleukin- 6 mRNA expression; conversely, the steroid upregulated the steady-stat e expression of alkaline phosphatase message in these cells. In summar y, we have identified a clonal, conditionally phenotypic, human osteob last cell line that expresses functional ERs and exhibits endogenous r esponses to 17 beta-E-2. This cell line will be a valuable in vitro mo del for exploring some of the molecular mechanisms of estrogen action in bone. (C) 1997 Wiley-Liss, Inc.