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Estrogens can regulate apoptosis in various cellular systems. The present study shows that 17b-estradiol (E 2 ), at physiological concentrations, abrogates DNA damage, poly (ADP-ribose) polymerase cleavage, and mitochondrial cytochrome c release induced by H 2 O 2 or etoposide in mouse skeletal muscle C2C12 cells. This protective action, which involved PI3K/Akt activation and Bcl-2 associated death agonist (BAD) phosphorylation, was inhibited by antibodies against the estrogen receptor (ER) a or b isoforms, or transfecting siRNA specific for each isoform. The inhibition of the antiapoptotic action of E 2 at the mitochondrial level was more pronounced when ER-b was immunoneutralized or suppressed by mRNA silencing, whereas transfection of C2C12 cells with either ER-a siRNA or ER-b siRNA blocked the activation of Akt by E 2 , suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway evaluated. These results indicate that E 2 exerts antiapoptotic effects in skeletal muscle cells which are mediated by ER-b and ER-a and involve the PI3K/Akt pathway. Journal of Endocrinology (2008) 196, 385-397 IntroductionThe estrogen 17b-estradiol (E 2 ) is a steroid hormone whose actions involve genomic and non-genomic mechanisms (Bjornstrom & Sjoberg 2005). It is generally accepted that the majority of the effects of the hormone are mediated via two estrogen receptors (ERs), namely ER-a and ER-b, which are members of the nuclear receptor superfamily, by regulating nuclear estrogen responsive genes (Evans 1988, Tsai & O'Malley 1994, Beato et al. 1996, Pettersson et al. 2000, Hall et al. 2001, Hewitt & Korach 2002. Also, several investigators have pointed out the possibility that the ER could be non-classically associated with intracellular membranes (Parikh et al. 1980, Watson & Muldoon 1985, Muldoon et al. 1988, Monje & Boland 1999, Watson et al. 1999. Moreover, there is evidence showing that ER-a and ER-b may be located in the plasma membrane (Luconi et al. 1999, Norfleet et al. 2000, Ropero et al. 2002, Li et al. 2003 and mitochondrial compartments (Zheng & Ramirez 1999, Horvat et al. 2001, Monje & Boland 2002, Yang et al. 2004, Solakidia et al. 2005. In addition, the non-genomic events triggered by E 2 suggest the ability of the hormone to activate extranuclear receptors (Bjornstrom & Sjoberg 2005). Among the rapid nontranscriptional actions of E 2 , the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been shown in various cellular lines (Fernando & Wimalasena 2004, Guo et al. 2006. PI3K regulates phosphoinositide metabolism and is responsible for the generation of phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ;Vanhaesebroeck et al. 2001, Osaki et al. 2004. The activation of PI3K results in PIP 3 -mediated activation of the serine-threonine kinase Akt by phosphorylation. In turn, phospho-Akt modulates the function of numerous substrates involved in the regulation of cell functions as for example apoptosis (Coffer et al. 1998, Vanhaesebroeck et al....

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17b-Estradiol (E 2 ) stimulates the mitogen-activated protein kinases (MAPKs) in various cellular types. We have shown that the hormone activates extracellular-regulated kinase (ERK) and p38 MAPK in skeletal muscle cells. However, the functions of MAPK modulation by the estrogen in muscle cells have not been studied yet. We have recently reported antiapoptotic actions of E 2 in C2C12 cells. Here, the role of MAPKs mediating the hormone effect in muscle cells was investigated. The results showed that cells exposed to 0 . 5 mM hydrogen peroxide (H 2 O 2 ) presented cytoskeleton disorganization, mitochondrial redistribution, and picnotic/ fragmented nuclei. Pretreatment with 10K8 M E 2 prevented these morphological apoptotic characteristics, except in the presence of ERK or p38 MAPK inhibitors, U0126 and SB203580 respectively. Mitochondrial membrane integrity was also studied. Preincubation of cultures with 10 K8 M E 2 abrogated H 2 O 2 effects such as Janus Green oxidation, presence of cytochrome c oxidase activity in the cytoplasm, and SMAC/DIABLO release from mitochondria. When MAPKs were inhibited, the hormone could not prevent mitochondrial membrane damage exerted by oxidative stress. Blocking experiments with small interfering RNAs confirmed that both ERK and p38 MAPKs mediate the antiapoptotic effects of the hormone at the mitochondrial level. Further, some of the molecular mechanisms involved were also investigated. Thus, E 2 was able to induce AKT (Ser473) and BAD (Ser112) phosphorylation in C2C12 cells in the absence or in the presence of H 2 O 2 but not when the cultures were incubated with H 2 O 2 and MAPK inhibitors. Altogether, these results show that E 2 exerts a survival action in skeletal muscle cells involving ERK and p38 MAPK activation.

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