17β-Estradiol abrogates apoptosis in murine skeletal muscle cells through estrogen receptors: role of the phosphatidylinositol 3-kinase/Akt pathway

Abstract

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....

Estradiol exerts antiapoptotic effects in skeletal myoblasts via mitochondrial PTP and MnSOD

“…In previous studies, we showed that E 2 abrogates H 2 O 2 -induced apoptosis in skeletal muscle cells involving both ERa and ERb, PI3K/Akt/Bad, HSP27, and MAPKs (Vasconsuelo et al 2008, Ronda et al 2010). Accordingly, a protective effect of the steroid on mitochondria integrity was evidenced, as cytochrome c release by H 2 O 2 was inhibited by E 2 through ERb (Vasconsuelo et al 2008). The data obtained in this work, using the C2C12 murine skeletal myoblasts line, deepen the knowledge of molecular actions triggered by E 2 , which result in mitochondrial protection.…”

“…Anti-SOD2 (1:1000) was from Santa Cruz Biotechnology, Inc. Anti-beta tubulin (1:10 000) and anti-Bax Cell culture and treatment C2C12 murine skeletal myoblasts obtained from American Type Culture Collection (Manassas, VA, USA) were cultured in growth medium (DMEM) supplemented with 10% heat-inactivated (30 min, 56 8C) fetal bovine serum, 1% nistatine, and 2% streptomycin. These highly myogenic cells have been widely used to study muscle functions (Burattini et al 2004, Vasconsuelo et al 2008, Pronsato et al 2012. Cells were incubated at 37 8C in a humid atmosphere of 5% CO 2 in air.…”

“…Then 10 K8 M E 2 or vehicle 0.001% isopropanol (control) was added w120 min before induction of apoptosis with hydrogen peroxide (H 2 O 2 ) at the times indicated (ranging from 30 min to 4 h). This time and concentration range of the oxidant has been previously used to study apoptosis in C2C12 cells (Jiang et al 2005, Vasconsuelo et al 2008, Siu et al 2009, Pronsato et al 2012. H 2 O 2 was diluted in culture medium without serum at a final concentration of 0.5 mM in each assay.…”

“…Typical changes of apoptosis such as nuclear fragmentation, cytoskeleton disorganization, mitochondrial reorganization/dysfunction, and cytochrome c release induced by H 2 O 2 , were abolished when cells were previously exposed to E 2 (Vasconsuelo et al 2008). Likewise, we demonstrated that E 2 exerts its antiapoptotic effect through ERs with non-classical localization involving MAPKs, HSP27, and the survival PI3K/Akt pathway, which phosphorylates and inactivates proapoptotic members of the Bcl-2 family (Vasconsuelo et al 2008, Ronda et al 2010. The mitochondrial protein cytochrome c plays a key role in apoptosis (reviewed by Jiang & Wang (2004)).…”

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

“…However, cell decision-making in the DDR is influenced by cell cycle status (5). Much less is known about the intrinsic properties of DDR and DNA repair in postmitotic, terminally differentiated cells such as neurons and multinucleated myofibers in skeletal muscle (6)(7)(8)(9)(10). Terminally differentiated skeletal muscle cells have prominent down-regulation of DNA repair mechanisms and a low capacity for DNA base excision repair (11), although these cells are resistant to the cell killing effects of DNA single-strand break (SSB) inducers (10).…”