Non-classical localization of estrogen receptors in C2C12 murine skeletal muscle cell line.?.

LORENA MILANESI; ANA RUSSO DE BOLAND; RICARDO BOLAND

Rosario

Congreso; XXI Reunión Anual de la Asociación Argentina de Osteología y Metabolismo Mineral (AAOMM).; 2006

Asociación Argentina de Osteología y Metabolismo Mineral (AAOMM)

NON-CLASSICAL LOCALIZATION OF ESTROGEN RECEPTORS IN C2C12 MURINE SKELETAL MUSCLE CELL LINE. Milanesi Lorena M, Russo de Boland Ana J and  Boland Ricardo L. Laboratorio de Química Biológica. Universidad Nacional del Sur. 8000. Bahía Blanca.  milanesi@criba.edu.ar There is data indicating that the known estrogen receptor (ER) a and the novel subtype ER b may reside in the cell membrane, mitochondrial and perinuclear compartments. Thus a parallel reservoir of extra nuclear ERs may also exist. In this work we show biochemical and immunochemical evidence that the ER a and b isoforms are found localized at the mitochondrial and microsomal level in C2C12 murine skeletal muscle cell line. The presence of specific intracellular binding sites for ER a and b was confirmed by competition assays in whole cells using [3H]17b-estradiol and an excess of non-radioactive 17b-estradiol, 17a-estradiol, estradiol macromolecular derivatives, agonist and antagonists. By competitive binding assays in subcellular fractions we verified that the steroid binding sites were mostly localized in mitochondria and microsomes. Antibodies against the two ER isoforms blocked the specific binding in whole cells and in mitochondrial and microsomal fractions, indicating that the steroid binding sites were structurally related to the ER. By using different antibodies against the ER a and b isoforms and estradiol macromolecular derivatives in Western and Ligand blot analysis and immunocytochemical assays employing conventional and confocal microscopy, we confirmed the non-classical localization for ER a and b. Transient transfections of whole cells with specific short interfering RNAs (siRNAs) for ER a and b, could reduce the specific binding previously detected in non-transfected cells and also decreased the immunoreactivity observed by Western blots and immunocytochemistry. These results point out that ER a and b receptor entities are targeted into extra nuclear compartments in muscle C2C12 cells.