. “Expresión and Subcellular Localization of 17-estradiol Binding proteins in C2C12 Cell Line”.

MILANESI, LORENA; ANA RUSSO DE BOLAND; BOLAND, RICARDO

JOURNAL OF BONE AND MINERAL RESEARCH

AMER SOC BONE & MINERAL RES

Año: 2005 vol. 20 p. 311 - 311

0884-0431

The classical cellular mechanism of 17b -estradiol actions is assumed to involve the steroid diffusion through the plasma membrane; it’s binding to a cytoplasmic/nuclear receptor (ER), and subsequent transcription and protein synthesis regulation. In addition to this mechanism, increasing evidence for non-genomic estrogen short-term effects in different cells types has been accumulated and led to hypothesize the existence of cell-surface resident receptor forms triggering membrane events. These cell- surface protein receptors have been detected in different cell lines (MCF-7, SHM, HT-29) and the expression of an estrogen binding protein and its effects on the development and function of human and bovine skeletal muscle was reported. The present study was focused to analyze the subcellular localization of ER binding proteins in C2C12 cells (a murine skeletal muscle cell line).  By competition assays we have detected binding sites for [3H]17b-estradiol in total homogenates. The subcellular localization of these binding sites was predominantly mithocondrial-microsomal. The sites were specific for the steroid hormone 17b-estradiol, as progesterone was no effective to compete with the radioactive ligand. The nonsteroidal estrogenic and antiestrogenic compounds diethylstilbestrol, tamoxifen and ICI 182, 780 were as effective as 17b-estradiol in competition studies.  The protein nature of the estrogen binding sites was clearly indicated by their sensitivity to trypsin degradation. Specific antibodies against different domains of the classical ER (ERa) could reduce the specific radioligand binding. Immunochemical studies employing the same monoclonal antibodies detected the 67 kDa immunoreactive band expected for the ERa in nuclear and cytosolic fractions and, additionally, low molecular weight bands present in the mitochondrial and microsomal fractions. These reactive bands were able to bind the steroid hormone in Ligand blot assays. Also these studies showed a clear surface labeling of the macromolecular complexes E2-BSA-FITC in non-permeabilized living cells. The characterization of these estrogen binding proteins and the signaling cascades activated thereby by the steroid hormone in skeletal muscle cells may provide information about molecular events implicated in alterations in muscle contractility and development which occur in menopause.