Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line?,

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

JOURNAL OF CELLULAR BIOCHEMISTRY

WILEY-LISS, DIV JOHN WILEY & SONS INC

Lugar: New York; Año: 2008 p. 1254 - 1273

0730-2312

Abstract The classical model of 17b-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrialmicrosomal localization of ERa in the C2C12 skeletal muscle cell line. We first established [3H]17bestradiol binding characteristics in whole cells in culture. Specific and saturable [3H]17bestradiol binding sites of high affinity were then detected in mitochondrial fractions (Kd¼0.43 nM; Bmax¼572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17bestradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17b-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ERa proteins were confirmed in C2C12 cells transfected with ERa siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ERa in skeletal muscle cells suggests an alternative mode of ER localization/function. J. Cell. Biochem. 104: 1254?1273, 2008. 2008 Wiley-Liss, Inc.