Lorena Milanesi

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

Estrogens can regulate apoptosis in various cellular systems. The present study shows that 17beta-estradiol (E2), at physiological concentrations, abrogates DNA damage, poly (ADP-ribose) polymerase cleavage, and mitochondrial cytochrome c release induced by H2O2 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) alpha or beta isoforms, or transfecting siRNA specific for each isoform. The inhibition of the antiapoptotic action of E2 at the mitochondrial level was more pronounced when ER-beta was immunoneutralized or suppressed by mRNA silencing, whereas transfection of C2C12 cells with either ER-alpha siRNA or ER-beta siRNA blocked the activation of Akt by E2, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway evaluated. These results indicate that E2 exerts antiapoptotic effects in skeletal muscle cells which are mediated by ER-beta and ER-alpha and involve the PI3K/Akt pathway.

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.

Expression and localization of estrogen receptor α in the C2C12 murine skeletal muscle cell line

The classical model of 17β‐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 ERα in the C2C12 skeletal muscle cell line. We first established [3H]17βestradiol binding characteristics in whole cells in culture. Specific and saturable [3H]17βestradiol binding sites of high affinity were then detected in mitochondrial fractions (Kd = 0.43 nM; Bmax = 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 17βestradiol‐BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17β‐estradiol treatment. Western and Ligand blot approaches corroborated the non‐classical localization. Expression and subcellular distribution of ERα proteins were confirmed in C2C12 cells transfected with ERα siRNA and by RT‐PCR employing specific primers. The non‐classical distribution of native pools of ERα in skeletal muscle cells suggests an alternative mode of ER localization/function. J. Cell. Biochem. 104: 1254–1273, 2008.

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.

Expression and subcellular distribution of native estrogen receptor β in murine C2C12 cells and skeletal muscle tissue

We have recently described the expression and intracellular localization of ER alpha in murine C2C12 cells and skeletal muscle tissue. In separate studies, a protective role of 17beta-estradiol against apoptosis exerted mainly at the mitochondrial level was also shown in the C2C12 muscle cell line. However, this functional evidence was in accordance with the participation of ER beta. We have then here investigated the expression and subcellular distribution of native ER beta in similar skeletal muscle cultured cells and tissue developed in vivo. ER beta was detected by immunoblotting using specific antibodies and ligand blot analysis after subcellular fractionation. Immunolocalization was confirmed using conventional and confocal microscopy. ER beta was found to a great extent in mitochondria and in lower amounts in the cytosolic fraction, differently to ER alpha which localizes in microsomes, cytosol, mitochondria, and also in the nucleus of muscle tissue. ER beta expression was also demonstrated by RT-PCR. Finally, the mitochondrial localization of native ER beta in C2C12 muscle cells was corroborated after transient transfection with specific ER beta siRNAs. These data raise the possibility that the antiapoptotic action of 17beta-estradiol in muscle cells may be related in part to a direct action of the hormone on mitochondria through ER beta.

Vitamin D compounds in plants

Abstract An appreciable number (15) of flowering plants, specially within the Solanaceae family, have been shown to contain vitamin D 3 and its hydroxylated derivatives, including 1α,25(OH) 2 -vitamin D 3 [1α,25(OH)D 3 ], a pluripotent hormone in animals. These secosteroids have also been detected in members of the Cucurbitaceae, Fabaceae and Poaceae families. On the basis of recent cladistic analysis it is possible to predict that the synthesis of these compounds is a characteristic of the Angiosperms. Highly specific and sensitive bioassays and analytical procedures of high resolution are now available which may allow confirmation of this hypothesis. Solanum glaucophyllum is the species which accumulates 1α,25(OH)D 3 to the greatest extent. The metabolite is distributed among different tissues of the plant as a free steroid or glycoside derivatives. The presence of 7-dehydrocholesterol, vitamin D 3 , 25(OH)D 3 and 1α,25(OH) 2 D 3 in S. glaucophyllum and other species has been unequivocally demonstrated. Moreover, recent studies with radioactive precursors, protein immunoblot and RNA hybridization analysis suggest the presence of specific hydroxylases for vitamin D 3 and 25(OH)D 3 structurally related to the corresponding enzymes in vertebrates. It appears then that plants possess a similar synthetic route to 1α,25(OH) 2 D 3 as in animals. However, of unique biological importance, evidence obtained with S. glaucophyllum and Nicotiana glauca supports the operation of a non-photolytic reaction of vitamin D 3 synthesis. Both intermediates and enzymes of the vitamin D 3 pathway can be detected in cell and tissue cultures thus affording a convenient experimental model for studies on its molecular characterization and regulation. This is also endowed with biotechnological significance. Vitamin D 3 compounds may play a function in Angiosperms. Studies in vitro have shown that, like mitogenic plant hormones, they stimulate root growth and differentiation through activation of the Ca 2+ messenger system. There are vitamin D 3 and 1α,25(OH) 2 D 3 binding proteins in plants which exhibit characteristics suggesting a regulatory function. It remains to be established whether these binders act as true steroid hormone receptors.

Testosterone exerts antiapoptotic effects against H2O2 in C2C12 skeletal muscle cells through the apoptotic intrinsic pathway

Experimental data indicate that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H(2)O(2))-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H(2)O(2) cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H(2)O(2) induces BAD inactivation, inhibition of poly(ADP-ribose) polymerase cleavage, and a decrease in BAX levels, and impedes the loss of mitochondrial membrane potential, suggesting that the hormone participates in the regulation of the apoptotic intrinsic pathway. Simultaneous treatment with testosterone, H(2)O(2), and the androgen receptor (AR) antagonist, flutamide, reduces the effects of the hormone, pointing to a possible participation of the AR in the antiapoptotic effect. The data presented allow us to begin to elucidate the mechanism by which the hormone prevents apoptosis in skeletal muscle.

Nicotiana glauca: another plant species containing vitamin D3 metabolites

Vitamin D(3)-related compounds have been detected in various plant species, mostly belonging to the Solanaceae. In this work we show that Nicotiana glauca, a widespread member of this taxonomic family, contains 7-dehydrocholesterol, vitamin D(3) and hydroxylated derivatives bearing precursor-product metabolic relationships in vertebrates. Leaves collected in the field and callus cultures were used. By means of specific radioreceptor binding assays and mass spectrometry of purified fractions obtained from plant tissue lipid extracts by Sephadex LH-20 and Sep-Pak C18 chromatography followed by HPLC, we established the presence of 7-dehydrocholesterol, vitamin D(3), 25(OH)-vitamin D(3) and 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)), the latter being a hormonally relevant metabolite in animals. These results indicate that N. glauca may represent a useful species in which to characterize the biosynthetic pathway and physiological functions of vitamin D(3) compounds in plants. In addition, tissue culture of N. glauca could become a significant tool for biotechnological production of 1alpha,25(OH)(2)D(3).

Actions of 17β-estradiol and testosterone in the mitochondria and their implications in aging.

A decline in the mitochondrial functions and aging are two closely related processes. The presence of estrogen and androgen receptors and hormone-responsive elements in the mitochondria represents the starting point for the investigation of the effects of 17β-estradiol and testosterone on the mitochondrial functions and their relationships with aging. Both steroids trigger a complex molecular mechanism that involves crosstalk between the mitochondria, nucleus, and plasma membrane, and the cytoskeleton plays a key role in these interactions. The result of this signaling is mitochondrial protection. Therefore, the molecular components of the pathways activated by the sexual steroids could represent targets for anti-aging therapies. In this review, we discuss previous studies that describe the estrogen- and testosterone-dependent actions on the mitochondrial processes implicated in aging.

Effect of culture conditions on the synthesis of vitamin D3 metabolites in Solanum glaucophyllum grown in vitro

In cultured Solanum glaucophyllum we have recently described the operation of a light-independent pathway of 1alpha,25-dihydroxy-vitamin D(3) (1alpha,25(OH)(2)D(3)) biosynthesis which involves similar intermediates as in vertebrates. In this work we investigated factors influencing the formation of 1alpha,25(OH)(2)D(3) and related sterols in S. glaucophyllum grown in vitro in darkness. Callus tissue and cells cultured in Murashige and Skoog medium in the absence of light were employed. Lipids were extracted with chloroform-methanol. The remaining water soluble fraction was incubated with beta-glucosidase to release vitamin D(3) compounds from their glycoconjugated derivatives followed by organic solvent extraction. Vitamin D(3) derivatives were isolated by Sephadex LH-20 and high-performance liquid chromatography (HPLC). HPLC or competitive protein binding assays with intestine 1alpha,25(OH)(2)D(3) receptor and serum vitamin D binding protein were used to quantify the metabolites. The levels of 1alpha,25(OH)(2)D(3) in calli varied according to the tissue explant origin, e.g. stem>leaf>fruit. For all organs, the metabolite was mainly present as free sterol (>80% of total). There were larger amounts of 25(OH)D(3) than 1alpha,25(OH)(2)D(3). It was found that Ca(2+), auxin and kinetin are important factors upregulating 1alpha,25(OH)(2)D(3) synthesis in S. glaucophyllum tissue and cells. Irradiation with UV light increased vitamin D(3) but not 1alpha,25(OH)(2)D(3) levels. In agreement with these results, incubation of cells with [3H]25(OH)D(3) revealed a low conversion rate to [3H]1alpha,25(OH)(2)D(3). The operation of a light-dependent pathway formation of vitamin D(3) coupled to higher expression of 25(OH)D(3)-1alpha-hydroxylase may account for the large concentrations of 1alpha,25(OH)(2)D(3) normally found in differentiated field-grown plants.