Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line.
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 ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-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 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 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-beta 1. 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 17 beta-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]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 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 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-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 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.
Bodine
P V
PV
Women's Health Research Institute, Wyeth-Ayerst, Radnor, Pennsylvania 19087, USA. bodinep@war.wyeth.com
Green
J
J
Harris
H A
HA
Bhat
R A
RA
Stein
G S
GS
Lian
J B
JB
Komm
B S
BS
D000368:Aged; D000469:Alkaline Phosphatase / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming; D002117:Calcitriol / Q000494:pharmacology; D002455:Cell Division; D002460:Cell Line; D002461:Cell Line, Transformed; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011959:Receptors, Estradiol / Q000235:genetics; D013696:Temperature
D000952:Antigens, Polyomavirus Transforming; D011959:Receptors, Estradiol; D015675:Osteocalcin; D004958:Estradiol; D000469:Alkaline Phosphatase; D002117:Calcitriol