ISSN:
0730-2312
Keywords:
immortalized
;
clonal
;
alkaline phosphatase
;
osteocalcin
;
mineralization
;
vitamin D3
;
dexamethasone
;
parathyroid hormone
;
interleukin-6
;
bone
;
osteoporosis
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
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. © 1997 Wiley-Liss, Inc.
Additional Material:
11 Ill.
Type of Medium:
Electronic Resource
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