Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line
Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line.
The classical model of 17beta-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 mitochondrial-microsomal localization of ERalpha in the C2C12 skeletal muscle cell line. We first established [(3)H]17betaestradiol binding characteristics in whole cells in culture. Specific and saturable [(3)H]17betaestradiol binding sites of high affinity were then detected in mitochondrial fractions (K(d) = 0.43 nM; B(max) = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17betaestradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17beta-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ERalpha proteins were confirmed in C2C12 cells transfected with ERalpha siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ERalpha in skeletal muscle cells suggests an alternative mode of ER localization/function. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.
Milanesi L, de Boland AR, Boland R.
Departamento de BiologÃa, BioquÃmica y Farmacia, Universidad Nacional del Sur, 8000 BahÃa Blanca, Argentina.