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1.
Science ; 241(4861): 81-4, 1988 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-3164526

RESUMO

High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.


Assuntos
Estradiol/metabolismo , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Ligação Competitiva , Núcleo Celular/metabolismo , DNA/genética , Estradiol/farmacologia , Humanos , Radioisótopos do Iodo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Peptídeos/genética , Pró-Colágeno/genética , Ratos , Receptores de Estrogênio/genética , Transcrição Gênica , Fatores de Crescimento Transformadores , Células Tumorais Cultivadas
2.
J Bone Miner Res ; 6(6): 531-41, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1887816

RESUMO

A finite human cell line was established from trabecular bone explants obtained from a 48-year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using the following independent criteria: (1) the presence of histochemically detectable alkaline phosphatase (AP) activity; (2) response to the calciotropic hormone 1,25-(OH)2D3 as assessed by increased AP activity; (3) synthesis and secretion of the osteoblast-specific marker bone gla protein; and (4) expression of alpha 1(I)-procollagen and alpha 1(III)-procollagen mRNAs in a pattern similar to that of other osteoblast-like cell lines. In addition to these classic osteoblast markers, BG688 cells also possess approximately 2400 high-affinity (Kd = 0.45 nM) 17 beta-estradiol (E2) binding sites per cell. The binding of E2 to these sites is specific, and of the steroid hormone agonists tested, E2 and diethylstilbestrol elicited the greatest amount of competition with radiolabeled E2. BG688 cells were also shown to respond to a physiologic concentration (10 nM) of E2. In vitro translation products of poly(A)+ RNA obtained from control and hormone-treated cells revealed a pleiotropic influence of E2 on the relative abundance of several mRNAs as assessed by two-dimensional gel electrophoretic analysis of their corresponding peptides. E2 also elicits a twofold increase in the steady-state concentration of alpha 1(I)-procollagen mRNA as demonstrated by northern blot hybridization. Thus, we here extend our previous data obtained in osteoblast-like osteosarcoma cells to indicate that a normal osteoblastic cell line is a target for the action of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Estradiol/farmacologia , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Northern Blotting , Calcitriol/farmacologia , Linhagem Celular , Dietilestilbestrol/metabolismo , Dietilestilbestrol/farmacologia , Estradiol/metabolismo , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Cinética , Osteoblastos/metabolismo , Osteocalcina/análise , Pró-Colágeno/genética , Radioimunoensaio , Transcrição Gênica/efeitos dos fármacos
3.
Endocrinology ; 128(6): 2723-30, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2036957

RESUMO

Clinical observations have demonstrated a positive effect of estrogens and androgens on the maintenance of structural bone integrity. This study examines the direct effects of androgenic hormones on the osteoblast-like human osteosarcoma cell line, HOS TE85. Employing radiolabeled dihydrotestosterone (DHT), 2800 saturable, high-affinity (dissociation constant = 0.66 nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific in that DHT and testosterone (T) displayed significantly greater competition than the progestins, progesterone and medroxyprogesterone. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. A human androgen receptor complementary DNA probe revealed a 9.5 kilobase transcript which corresponds to the predominant human androgen receptor transcript detected in human male reproductive tissues. Androgens were also found to elicit biological responses in HOS TE85 cells. Physiological concentrations of DHT and T decreased HOS TE85 cell proliferation as assessed by cell count. This finding suggests that DHT may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with T (24 h, 10 nM) enhanced the abundance of both alpha 1(I)-procollagen messenger RNA (mRNA) (5-fold) and transforming growth factor-beta mRNA (2.2 fold). The nonaromatizable androgen DHT (24 h, 10 nM) elicited an increase in the steady state concentration of alpha 1(I)-procollagen mRNA similar to the increase observed with T treatment. Thus, in addition to the recent discovery of estradiol receptors and estrogenic regulation of HOS TE85 cells, it is now evident that these osteoblast-like osteosarcoma cells also express high affinity androgen binding sites and can respond biologically to androgens.


Assuntos
Androgênios/metabolismo , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Pró-Colágeno/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Androgênios/farmacologia , Androgênios/fisiologia , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Homeostase , Humanos , Osteossarcoma/patologia , Receptores Androgênicos/genética , Células Tumorais Cultivadas
4.
J Biol Chem ; 270(10): 5191-7, 1995 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-7890629

RESUMO

There is strong experimental evidence that oxidized low density lipoprotein (Ox-LDL) plays an important role in atherosclerosis. However, the mechanisms by which Ox-LDL is formed in vivo are unknown. To test whether 15-lipoxygenase (15-LO) could play a role in oxidation of LDL by cells, we expressed 15-LO activity in murine fibroblasts, which do not normally have 15-LO activity, and tested their ability to modify LDL. Using a retroviral vector, we prepared fibroblasts that expressed 2- to 20-fold more 15-LO activity than control fibroblasts infected with a vector containing beta-galactosidase (lacZ). Compared with LDL incubated with lacZ cells, LDL incubated with 15-LO-containing cells were enriched with lipid hydroperoxides. When these LDL samples were subsequently subjected to oxidative stress, they were more susceptible to further oxidative modification, as judged by increased conjugated diene formation and by increased ability to compete with 125I-Ox-LDL for uptake by macrophages. These findings establish that cellular 15-LO can contribute to oxidative modification of LDL, but the quantitative significance of these findings to the in vivo oxidation of LDL remains to be established.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/metabolismo , Animais , Araquidonato 15-Lipoxigenase/biossíntese , Northern Blotting , Linhagem Celular , Células Cultivadas , DNA Complementar , Fibroblastos/metabolismo , Expressão Gênica , Vetores Genéticos , Humanos , Cinética , Macrófagos Peritoneais/metabolismo , Camundongos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Retroviridae , Transfecção , beta-Galactosidase/biossíntese , beta-Galactosidase/metabolismo
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