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

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 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.

The Wnt antagonist secreted frizzled‐related protein‐1 controls osteoblast and osteocyte apoptosis

“…PGE 2 treatment of the HOB-03-C5 cells also suppressed ALP activity by 57% in a dose-dependent manner with an IC 50 of 0.7 nM (data not shown). This observation, together with induction of sFRP-1 expression, is consistent with promotion of cellular differentiation since the HOB-01-C1 pre-OCYs express high levels of sFRP-1 message and low amounts of ALP [Bodine et al, 1996a[Bodine et al, , 1997Bodine and Komm, 2002]. Treatment of mature-OB HOB-03-CE6 cells with PGE 2 also up-regulated sFRP-1 steady-state mRNA levels in a doseand time-dependent manner (Fig.…”

“…et al, 1996a, 1997Bodine and Komm, 2002], divided slowly in FBS-containing medium at the non-permissive temperature of 398C, and cell number increased by about 75% after 6 days in culture (Fig. 6B); this rate of cell division was similar to that of explant cultures of hOBs [Bodine et al, 1996a[Bodine et al, , 1997Bodine and Komm, 2002]. The mature-OB HOB-03-CE6 cells did not proliferate at the non-permissive temperature, and cell number remained constant over the 6-day incubation.…”

“…RT-PCR was performed using 1 mg of total RNA, primers that spanned the coding region of hFRP-1/SARP-2 (forward primer: 5 0 -GCTGGGGACTGCGCCTTTTGT-3 0 ; reverse primer: 5 0 -CCTGCCCCCGGGAGAATCACT-TA-3 0 ), 35 cycles of PCR, and the Advantage-GC PCR kit (BD Biosciences Clonetech). In order to detect expression of the mRNA, a Southern hybridization analysis was performed with the RT-PCR products using a 32 P-oligonucleotide probe, which specifically hybridized to bases 501-530 of the FRP-1/SARP-2 coding region [Bodine et al, 1997]. Likewise, RT-PCR of total RNA isolated from HOB-03-C5 cells treated with PGE 2 identified a 2.2 kb cDNA that spanned from the 5 0 -region of the FRP-1/SARP-2 cDNA to the 276 bp HOB sFRP-1 RADE fragment at the 3 0 -end.…”

“…Total cellular protein was extracted, and Western blot analysis for b-catenin was performed using a monoclonal antibody (Transduction Laboratories, Lexington, KY) as previously described [Bodine et al, 1996a[Bodine et al, , 1997Melkonyan et al, 1997;Bodine and Komm, 2002].…”

“…In order to characterize the molecular events associated with human OB differentiation and to identify new and important genes and pathways associated with the osteogenic process, we employed a collection of HOB cell lines [Bodine et al, 1996a[Bodine et al, , 1997Bodine and Komm, 2002] and a high-throughput robotic version of DD-PCR analysis known as RADE [Shiue, 1997;Chang et al, 2005]. For these experiments, we treated three of the HOB cell lines (HOB-03-C5, HOB-03-CE6, and HOB-01-C1), representing three stages of differentiation (pre-OBs, mature-Obs, and pre-OCYs, respectively) [Bodine and Komm, 2002], for 24 h with three osteogenic agents (PTH 1-34, PGE 2 , and TGFb1) [McCarthy et al, 2000;Vrotsos et al, 2003; sFRP-1 Controls Osteoblast and Osteocyte Apoptosis Qin et al, 2004;Quattrocchi and Kourlas, 2004;Rosen, 2004].…”