Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Ikegami, Ayao

doi:10.1210/en.135.2.782

Cited by 20 publications

(17 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, this cell line clearly contains the basic transcriptional apparatus to up-regulate E 2 -stimulated gene activity, because transfected cells expressing control normal ER␣ increased EREtk81Luc activity up to 20-fold in response to 10 nM E 2 . These findings with normal ER␣ are in agreement with other studies that examine normal ER␣ activation of E 2 -responsive gene transcription in various OS cell lines (29)(30)(31)(32)(33). Our data also confirm previous studies that show that the ⌬5ER␣ isoform has minimal activity when expressed alone.…”

Section: Discussionsupporting

confidence: 93%

A Tumor-Specific Truncated Estrogen Receptor Splice Variant Enhances Estrogen-Stimulated Gene Expression

Chaidarun

Alexander

1998

Molecular Endocrinology

View full text Add to dashboard Cite

This study examines the cooperative effects of a human estrogen receptor-alpha (ERalpha) isoform on estrogen (E2)-mediated gene activation in U2-OS osteosarcoma cells. Delta5ERalpha, an alternatively spliced ERalpha variant lacking exon 5, is coexpressed with normal ERalpha in several E2-responsive neoplastic tissues. However, the potential interactions of delta5ERalpha with normal ERalpha have not been functionally characterized. Delta5ERalpha encodes the hormone-independent trans-activating function (AF-1), as well as the constitutive receptor dimerization and DNA-binding domains. It is generated by an alternate splice event that omits exon 5 and alters the reading frame of the resulting mRNA. The delta5ERalpha protein is prematurely truncated and lacks the majority of the hormone-binding and activating function-2 (AF-2) domains. When delta5ERalpha mammalian expression vector was transfected alone in human ERalpha/ERbeta-negative osteosarcoma U2-OS cells, it had no effect on either basal or E2-mediated EREtk81Luc reporter transcriptional activity, while transfected cells expressing control normal ERalpha increased EREtk81 Luc activity up to 20-fold in response to 10 nM E2. However, when delta5ERalpha was cotransfected with normal ERalpha, both basal and E2-stimulated EREtk81Luc reporter activation were increased approximately 500% over levels observed when cells were transfected with ERalpha alone. Similar effects of delta5ERalpha and normal ERa coexpression were observed using an E2-responsive human C3 promoter/luciferase reporter construct. The effects of delta5ERalpha on normal ERalpha were further assessed in U2-OS cells stably transfected with normal ERalpha. Transfection of increasing amounts of delta5ERalpha expression vector into [ERalpha+]OS cells resulted in potentiation of E2-stimulated ERELuc activity in a synergistic, dose-dependent manner. Moreover, coexpression of delta5ERalpha in [ERalpha+]OS cells improved E2 sensitivity 100-fold over cells expressing ERalpha alone. Proliferation rates of stable U2-OS cell lines expressing delta5ERalpha were significantly increased (P < 0.05), with cell doubling times reduced from 35 h in control parental U2-OS cells to 28 h in [delta5ERalpha]OS cells. However, growth rates were not affected by either E2 or tamoxifen treatment. Electromobility shift/supershift assays using nuclear extracts of U2-OS cells stably transfected with ERalpha and delta5ERalpha confirmed the constitutive binding of delta5ERalpha and ERalpha protein to estrogen-response element (ERE) sequence independent of E2 and also showed an increase in delta5ERalpha/ERalpha-ERE complexes with E2 treatment. These data are consistent with interactive effects of normal ERalpha and delta5ERalpha on transcription from classic ERE gene promoters. Delta5ERalpha appears to therefore act as a dominant positive receptor that increases both basal and E2-stimulated gene transactivation of normal ERalpha.

show abstract

Section: Discussionsupporting

confidence: 93%

A Tumor-Specific Truncated Estrogen Receptor Splice Variant Enhances Estrogen-Stimulated Gene Expression

Chaidarun

Alexander

1998

Molecular Endocrinology

View full text Add to dashboard Cite

show abstract

“…The HOB-03-CE6 cells expressed relatively high levels of ER mRNA by RT-PCR, although this level was considerably less than that expressed by the rat uterus. By contrast, the HOB-03-C2 cells expressed low levels of ER message, and we could not detect ER mRNA in a clone of HOS-TE85 human osteosarcoma cells, even though this cell line has been reported to express low levels of functional ERs [Komm et al, 1988;Etienne et al, 1990;Ikegami et al, 1993Ikegami et al, , 1994.…”

Section: Hob-03-ce6 Cells Express Functional Erscontrasting

confidence: 76%

“…Compared to cell lines like MCF-7 human breast cancer cells [Kasid et al, 1984], these osteoblast models contain low numbers (60-4,500/ cell) of high affinity (K d 5 0.05-1.1 nM) ERs [Komm et al, 1988;Eriksen et al, 1988;Kaplan et al, 1988;Etienne et al, 1990;Benz et al, 1991;Keeting et al, 1992;Masuyama et al, 1992;Migliaccio et al, 1992;Davis et al, 1994]. Estrogen has been reported to have numerous effects on osteoblasts in vitro: these include the regulation of DNA synthesis and cellular proliferation [Gray et al, 1987;Bankson et al, 1989;Ernst et al, 1989;Liel et al, 1992;Masuyama et al, 1992;Ikegami et al, 1994;Majeska et al, 1994;Verhaar et al, 1994]; the stimulation of alkaline phosphatase, creatine kinase, lactate dehydrogenase, g-glutamyl transferase, and aspartate aminotransferase activity [Gray et al, 1987;Bankson et al, 1989;Somjen et al, 1989;Majeska et al, 1994;Verhaar et al, 1994]; the upregulation of transferrin, transforming growth factor (TGF)-b1, insulin-like growth factor (IGF)-I, a1 type (I) procollagen, progesterone receptor, c-fos, heat-shock protein, and interleukin-1b (IL-1b) expression [Komm et al, 1988;Eriksen et al, 1988;Bankson et al, 1989;Gray et al, 1989a,b;Oursler et al, 1991;Cooper and Uoshima, 1994;Harris et al, 1992;Majeska et al, 1994;Pivirotto et al, 1995]; the suppression of cytokineinduced IL-6 and tumor necrosis factor-a (TNF-a) expression [Girasole et al, 1992;…”

mentioning

confidence: 99%

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

Bodine¹,

Green

Harris³

et al. 1997

J. Cell. Biochem.

View full text Add to dashboard Cite

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.

show abstract

“…The subcellular localization of rat ER and ER protein was examined as described (Ikegami et al 1994). Briefly, 293T cells, transfected with each expression vector, were reseeded and allowed to grow to subconfluence on chambered glass slides.…”

Section: Western Blot Analysis Immunocytochemistry and Immunohistochmentioning

confidence: 99%

Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus

Hiroi¹,

Inoue²,

Watanabe³

et al. 1999

Journal of Molecular Endocrinology

134

View full text Add to dashboard Cite

In order to investigate the localization of estrogen receptor (ER) and ER in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ER antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ER antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ER antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ER antibody. In rat ovary, positive signals were shown with anti-ER antibody in the nuclei of granulosa cells. No specific immunostaining was observed with anti-ER antibody. Although ER was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ER was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ER and ER in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs.

show abstract

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Cited by 20 publications

References 0 publications

A Tumor-Specific Truncated Estrogen Receptor Splice Variant Enhances Estrogen-Stimulated Gene Expression

A Tumor-Specific Truncated Estrogen Receptor Splice Variant Enhances Estrogen-Stimulated Gene Expression

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

Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus

Contact Info

Product

Resources

About