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.