Ana Carolina Ronda

Role of 17β-estradiol and testosterone in apoptosis.

17β-Estradiol (E2) and Testosterone (T) exert actions in most animal tissues, in addition to the reproductive system. Thus, both sex steroid hormones affect growth and different cell functions in several organs. Accordingly, the nuclear estrogen (ER) and androgen (AR) receptors are ubiquitously expressed. Moreover, ER and AR may have non-classical intracellular localizations, e.g. plasma membrane, mitochondria and endoplasmic reticulum, raising additional complexity to the functional roles of E2 and T. In addition to the modulation of gene transcription by direct interaction with their cognate nuclear receptors, the steroids can rapidly activate signaling pathways by a non-genomic mechanism mediated by receptors identical to or different from known steroid receptors. Among various functions, E2 and T can regulate apoptosis through those pathways. In mitochondria, the presence of ER and AR and actions of estrogen and androgen have been shown, in keeping with the organelle being a control point of apoptosis. The most recurrent action for each steroid hormone is the protection of mitochondria against different insults, resulting in antiapoptosis. This review summarizes the molecular basis of the modulation of programmed cell death by E2 and T in several tissues.

MAP kinases p38 and JNK are activated by the steroid hormone 1α,25(OH)2‐vitamin D3 in the C2C12 muscle cell line

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1α,25(OH)2‐vitamin D3 [1α,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal‐regulated mitogen‐activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1α,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen‐activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1α,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c‐Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced‐p38 activation. Of relevance, 1α,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen‐activated protein kinase activating protein kinase 2 (MAPKAP‐kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation‐dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1α,25(OH)2D3 also promotes the phosphorylation of c‐jun N‐terminal protein kinases (JNK 1/2), the response is fast (0.5–1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1α,25(OH)2D3 (1 nM). The relative contribution of ERK‐1/2, p38, and JNK‐1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established. J. Cell. Biochem. 97: 698–708, 2006.

17β-Estradiol signaling in skeletal muscle cells and its relationship to apoptosis

17beta-estradiol exerts an antiapoptotic action in skeletal muscle cells through extranuclear ERalpha and beta. This protective action, mainly involves a non-genomic mechanism of ERK1/2 and PI3K/Akt activation and BAD phosphorylation. ERbeta plays a major role in the inhibition of apoptosis by 17beta-estradiol at the level of mitochondria, whereas ERalpha and ERbeta mediate the activation of Akt to the same extent, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway involved. The myopathies associated to estrogen deficit states may be related to the mechanisms by which estrogen regulates apoptosis.

Activation of MAPKs by 1α,25(OH)2-Vitamin D3 and 17β-estradiol in skeletal muscle cells leads to phosphorylation of Elk-1 and CREB transcription factors

The mitogen activated protein kinases (MAPKs) have been classified into at least six subfamilies, among which ERK1/2, JNK1/2 and p38 MAPK are the most extensively studied. The steroid hormones 1alpha,25-dihydroxy-Vitamin D(3) and 17beta-estradiol promote biological responses through activation of MAPK cascades in various cell types. We previously reported that 1alpha,25(OH)(2)D(3) rapidly (within 1 min) activates p38 MAPK in C2C12 skeletal muscle cells. In this work, using the same muscle cell line, we demonstrate that 1alpha,25(OH)(2)D(3) or 17beta-estradiol phosphorylate and activate ERK1/2 and p38 MAPK after longer treatment intervals, maximal effects seen at 90 and 30 min (ERK1/2) and at 60 and 15 min (p38 MAPK) for these hormones, respectively. Hormone-dependent ERK and p38 activation was abolished by MAPK specific inhibitors U0126 and SB203580. 1alpha,25(OH)(2)D(3) and 17beta-estradiol also induced the phosphorylation of CREB and Elk-1 transcription factors in an ERK1/2-dependent manner. Simultaneous addition of both hormones potentiated CREB phosphorylation. 1alpha,25(OH)(2)D(3)- and 17beta-estradiol-induced c-fos expression, which was mediated by p38 phosphorylation. The action of 17beta-estradiol on c-fos levels was also dependent on ERK1/2. These results suggest that MAPK signalling pathways play an important role in regulating early gene expression through CREB and Elk-1 activation in skeletal muscle cells.

Extracellular-regulated kinase and p38 mitogen-activated protein kinases are involved in the antiapoptotic action of 17β-estradiol in skeletal muscle cells

17beta-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 10(-8) 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(-8) 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.

Role of estrogen receptors, PKC and Src in ERK2 and p38 MAPK signaling triggered by 17β-estradiol in skeletal muscle cells.

We have previously reported in C2C12 murine skeletal muscle cells that 10(-8)M 17β-estradiol promotes MAPKs stimulation which in turn mediates the activation of CREB and Elk-1 transcription factors. In this work, we demonstrated that the hormone induces ERK2 phosphorylation (without affecting ERK1 activation) and also stimulates p38 MAPK, both in a dose-dependent manner. Moreover, estrogen receptors involvement in MAPKs activation by the estrogen was studied. The use of ICI182780 (1 μM), an antagonist of ERs, and specific siRNAs to block ERα and ERβ expression, demonstrated that ERα mediates ERK2 activation but not p38 MAPK phosphorylation by 17β-estradiol, and that ERβ isoform is not implicated in MAPKs activation by the hormone. Furthermore, Src and PKC contribution in estrogen stimulation of the MAPKs was investigated. Compounds PP2 and Ro318220, Src and PKC family inhibitors, respectively abrogated ERK2 and p38 MAPK phosphorylation by 17β-estradiol. Of interest, the hormone was able to induce Src and PKCδ activation. In addition, Ro318220 decreased estrogen-dependent Src modulation implicating PKC in hormone upregulation of Src. Accordingly, PP2 and Ro318220 suppressed CREB and Elk-1 phosphorylation as well as c-Fos and c-Jun oncoprotein levels induced by 17β-estradiol. Altogether, these data indicate that 17β-estradiol activates ERK2 through ERα and p38 MAPK in an ERα/β-independent manner and that PKC and Src proteins are key upstream components on MAPKs activation in C2C12 skeletal muscle cells.

17β-Estradiol Protects Mitochondrial Functions through Extracellular-Signal- Regulated Kinase in C2C12 Muscle Cells

Background/Aims: We have previously shown that exposure to 17β-estradiol (E2) prior to induction of apoptosis with H2O2 protects skeletal muscle cells against oxidative damage. However, the mechanism involved in the protective action of the hormone is poorly understood. In the present study, we focused on the mechanism by which ERK mediates this survival effect in connection with COXIV activity and mitochondrial membrane potential. Methods: Immunocytochemistry, Western blot, cytochrome c oxidase complex IV (COXIV) activity, coimmunoprecipitation and JC-1 dye by flow cytometry were carried out using C2C12 myoblasts as experimental model. Results: E2 is able to activate ERK and then induces its translocation to mitochondria. Using the pharmacological inhibitor of ERK activation U0126 we show that E2, through ERK activation, is able to enhance COXIV activity. Moreover, the hormone increases the interaction between COXIV and ERK. Also, we found that hydrogen peroxide decreases COXIV activity and that preincubation of the cells with E2 prior to induction of apoptosis prevents this effect. In addition, we observe that the estrogen inhibits the collapse of mitochondrial membrane potential induced by H2O2, involving ERK and COXIV. Conclusion: Our data demonstrate that E2 promotes ERK activation and translocation to mitochondria preventing the decline in COXIV activity and in turn, alteration of mitochondrial membrane potential by oxidative stress, in C2C12 myoblasts.

Intracellular Distribution and Involvement of GPR30 in the Actions of E2 on C2C12 Cells

G‐protein‐coupled receptor 30 (GPR30) is an estrogen receptor that initiates several rapid, non‐genomic signaling events triggered by E2. GPR30 has recently been identified in C2C12 cells; however, little is known about the intracelular distribution and its role in C2C12 myoblasts and myotubes. By western blotting and immunohistochemistry, we evidenced expression of GPR30. While in C2C12 myoblasts, the receptor was present in nucleus, mitochondria, and endoplasmic reticulum, in C2C12 myotubes, it was additionally found in cytoplasm. Using trypan blue uptake assay to determine cellular death and fluorescent microscopy to evaluate picnotic nuclei and mitochondrial distribution, we demonstated that treatment of C2C12 myoblasts with G1 (GPR30 agonist) did not protect the cells against apoptosis induced by H2O2 as E2. However, when G15 (GPR30 antagonist) was used, E2 could not prevent the damage caused by the oxidative stress. Further, some of the molecular mechanisms involved were investigated by wertern blot assays. Thus, E2 was able to induce AKT phosphorylation in apoptotic conditions and ERK phosphorylation in proliferating C2C12 cells but not when the cultures were incubated with G15. Additionally, using G15 antagonist we have found that GPR30 participates in the myogenin expression and creatine kinase activity stimulated by E2 in the first steps of C2C12 differentiation. Althogether these findings provide evidences showing that GPR30 is expressed in diverse intracellular compartments in undifferentiated and differentiated C2C12 cells and mediates E2 actions. J. Cell. Biochem. 117: 793–805, 2016.

In vitro effects of 1α,25(OH)2D3-glycosides from Solbone A (Solanum glaucophyllum leaves extract; Herbonis AG) compared to synthetic 1α,25(OH)2D3 on myogenesis

The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts. For these, differentiation markers and myogenic parameters were studied in C2C12 myoblasts. Results showed that Solbone A, likewise the synthetic hormone, increased creatine kinase activity at day 2 after differentiation induction (60%, p<0.05). Solbone A and synthetic 1α,25(OH)2D3 increased vitamin D3 receptor protein expression at 10nM (50% and 30%, respectively) and the transcription factor myogenin (80%, p<0.05). However, tropomyosin expression was not affected by both compounds. In addition, myosin heavy chain (MHC) protein expression was increased 30% at day 2 of differentiation. Solbone A or synthetic 1α,25(OH)2D3 had no effects on myogenin nor MHC cell localization. Cellular mass increased with myogenesis progression, being Solbone A more effective than synthetic 1α,25(OH)2D3. Finally, Solbone A, as well as synthetic 1α,25(OH)2D3, augmented the index fusion of cultured muscle fibers. In conclusion, these results demonstrated that Solbone A exhibit at least equal or greater effects on early myoblast differentiation as synthetic hormone, suggesting that plant glycosides could be an effective, accessible and cheaper substitute for synthetic 1α,25(OH)2D3 to promote muscle growth.

1α ,25(OH)2-Vitamin D3 and 17β-Estradiol: Two Steroid Partners Acting in Skeletal Muscle

Fil: Buitrago, Claudia Graciela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur. Universidad Nacional del Sur. Departamento de Biologia, Bioquimica y Farmacia. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentina