RESUMO
In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.
Assuntos
Adipócitos/citologia , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Genisteína/farmacologia , Osteogênese/efeitos dos fármacos , Células Estromais/citologia , Fosfatase Alcalina/genética , Proteínas de Transporte/genética , Contagem de Células , Diferenciação Celular/genética , Fator D do Complemento , Estradiol/farmacologia , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicoproteínas/genética , Humanos , Cinética , Lipase Lipoproteica/genética , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Osteoblastos/citologia , Osteopontina , Osteoprotegerina , Ligante RANK , RNA Mensageiro/análise , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Estrogênio/fisiologia , Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Ácidos Siálicos/genética , Sialoglicoproteínas/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/análise , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta1RESUMO
Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17beta-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERalpha was used to validate the protocol. We expressed the full-length ERalpha protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERalpha. At physiological conditions (150 mM salt, 37 degrees C) we determined the 17beta-estradiol Kd for ERalpha to be 281 +/- 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37 degrees C. In homologous competition binding experiments using 17beta-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.
