Caveolae and caveolin-1 are implicated in 1α,25(OH)2-vitamin D3-dependent modulation of Src, MAPK cascades and VDR localization in skeletal muscle cells

We previously reported that 1α,25(OH)2D3 induces non-transcriptional rapid responses through activation of MAPKs in C2C12 skeletal muscle cells. However, there is little information on the molecular mechanism underlying the initiation of 1α,25(OH)2D3 signaling through this pathway. Plasma membrane components have been involved in some non-genomic effects. In this work, we investigated the role of caveolae and caveolin-1 (cav-1) in 1α,25(OH)2D3-stimulation of c-Src and MAPKs. When proliferating cells were pretreated with methyl beta cyclodextrin (MβCD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1α,25(OH)2D3-dependent activation of ERK1/2, p38 MAPK and c-Src was suppressed. Similar results were obtained by siRNA technology whereby silencing of cav-1 expression abolished activation of c-Src and MAPKs induced by the hormone. By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment disrupted the colocalization of these proteins and redistributed them into cytoplasm and nucleus. Co-immunoprecipitation assays corroborated these observations. Changes in VDR localization after 1α,25(OH)2D3 exposure were also investigated. Confocal microscopy images showed that the hormone induces VDR translocation to the plasma membrane, and this effect is abolished by MβCD. Altogether, these data suggest that caveolae is involved upstream in c-Src-MAPKs activation by 1α,25(OH)2D3 and that VDR and cav-1 participate in the rapid signaling elicited by the hormone.Fil: Buitrago, Claudia Graciela. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentin

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 (H2O2)-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H2O2 cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H2O2 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, H2O2, 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.Fil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; ArgentinaFil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentin

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

17beta-Estradiol (E2) protects several non-reproductive tissues from apoptosis, including skeletal muscle. We have shown that E2 at physiological concentrations prevented apoptosis induced by H2O2 in C2C12 skeletal myoblasts. As we also demonstrated the presence of estrogen receptors in mitochondria, the present work was focused on the effects of E2 on this organelle. Specifically, we evaluated the actions of E2 on the mitochondrial permeability transition pore (MPTP) by the calcein-acetoxymethylester/cobalt method using fluorescence microscopy and flow cytometry. Pretreatment with E2 prevented MPTP opening induced by H2O2, which preceded loss of mitochondrial membrane potential. In addition, it was observed that H2O2 induced translocation of Bax to mitochondria; however, in the presence of the steroid this effect was abrogated suggesting that members of the Bcl-2 family may be regulated by E2 to exert an antiapoptotic effect. Moreover, E2 increased mitochondrial manganese superoxide dismutase protein expression and activity, as part of a mechanism activated by E2 that improved mitochondrial performance. Our results suggest a role of E2 in the regulation of apoptosis with a clear action at the mitochondrial level in C2C12 skeletal myoblast cells.Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Vitamin D analogue TX 527 down-regulates the NF-κB pathway and controls the proliferation of endothelial cells transformed by Kaposi sarcoma herpesvirus

Background and Purpose: The Kaposi sarcoma (KS)-associated herpesvirus GPCR (vGPCR) is a key molecule in the pathogenesis of KS, where it increases NF-κB gene expression and activates the NF-κB pathway. We investigated whether the less calcemic vitamin D analogue TX 527 inhibited the proliferation of endothelial cells transformed by vGPCR by modulation of the NF-κB pathway. Experimental Approach: Endothelial cells transformed by vGPCR (SVEC-vGPCR) were treated with TX 527. Proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium, inner salt (MTS) and cell cycle by flow cytometry. mRNA and protein levels were measured by real-time quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot analysis respectively. Key Results: TX 527, similar to bortezomib (0.5 nM), a proteasome inhibitor that inhibits the activation of NF-κB, reduced proliferation and induced G0/G1 cell cycle arrest in SVEC-vGPCR. TX 527 like 1α,25(OH)2D3, biological active form of vitamin D, decreased the activity of NF-κB comparable with the effect of bortezomib. Time-response studies showed that TX 527 significantly decreased NF-κB and increased IκBα mRNA and protein levels. The increase of IκBα was accompanied by a reduction in p65/NF-κB translocation to the nucleus. These responses were abolished when vitamin D receptor (VDR) expression was suppressed by stable transfection of shRNA against VDR. In parallel with NF-κB inhibition, there was a down-regulation of inflammatory genes such as IL-6, CCL2/MCP and CCL20/MIP3α. Conclusions and Implications: These results suggest that the anti-proliferative effects of the vitamin D analogue TX 527 in SVEC-vGPCR occur by modulation of the NF-κB pathway and are VDR dependent.Fil: González Pardo, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Verstuyf, A.. Katholikie Universiteit Leuven; BélgicaFil: Boland, Ricardo Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Russo, Ana Josefa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca2+-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca 2+ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca 2+-containing but not in a Ca2+-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y2 receptors and PI3K/Akt pathway activation involving Ca2+, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca2+ influx/CaM pathway.Fil: Katz, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Ayala Peña, Victoria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Santillán, Graciela Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Boland, Ricardo Leopoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentin

Vitamin D: beyond bone.

In recent years, vitamin D has been received increased attention due to the resurgence of vitamin D deficiency and rickets in developed countries and the identification of extraskeletal effects of vitamin D, suggesting unexpected benefits of vitamin D in health and disease, beyond bone health. The possibility of extraskeletal effects of vitamin D was first noted with the discovery of the vitamin D receptor (VDR) in tissues and cells that are not involved in maintaining mineral homeostasis and bone health, including skin, placenta, pancreas, breast, prostate and colon cancer cells, and activated T cells. However, the biological significance of the expression of the VDR in different tissues is not fully understood, and the role of vitamin D in extraskeletal health has been a matter of debate. This report summarizes recent research on the roles for vitamin D in cancer, immunity and autoimmune diseases, cardiovascular and respiratory health, pregnancy, obesity, erythropoiesis, diabetes, muscle function, and aging

High passage numbers induce resistance to apoptosis in C2C12 muscle cells

Cell lines with high passage numbers exhibit alterations in cell Morphology and functions. In the present work, C2C12 skeletal muscle cells with either low (60) passage numbers (identified as l-C2C12 or h-C2C12, respectively) were used to investigate the apoptotic response to H2O2 as a function of culture age h-C2C12. We found that older cultures (h-C2C12 group) were depleted of mitochondrial DNA (mtDNA). When we analyzed the behavior of Bad, Bax, caspase-3 and mitochondrial transmembrane potential, we observed that cells in the h-C2C12 group were resistant to H2O2 induction of apoptosis. We propose serially cultured C2C12 cells as a refractory model to H2O2-induced apoptosis. In addition, the data obtained in this work suggest that mtDNA is required for apoptotic cell death in skeletal muscle C2C12 cells.Fil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Ronda, Ana Carolina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Cátedra de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina;Fil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Química Biológica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Bahia Blanca; Argentina

El 17β-Estradiol y la Testosterona protegen a las mitocondrias contra el estrés oxidativo en Células del Músculo Esquelético

En trabajos previos demostramos que la testosterona (T) y el 17β-estradiol (E2) protegen a las células musculares C2C12 de la apoptosis inducida por peróxido de hidrógeno (H2O2). Conjuntamente evidenciamos la existencia de receptores de estrógenos y andrógenos en las mitocondrias. El presente trabajo se ha centrado en caracterizar los efectos de ambos esteroides en esta organela, que conducen a la supervivencia celular. Específicamente, se evaluaron las acciones de T y E2 sobre el potencial de membrana mitocondrial con el colorante JC-1 y sobre el poro de permeabilidad transitoria mitocondrial (MPTP) mediante el método de calcein-acetoxymethylester/cobalt, utilizando microscopía de fluorescencia y citometría de flujo. Demostramos que T y E2 previenen la apertura del MPTP y la pérdida de potencial de membrana mitocondrial inducidas por H2O2. Además, observamos que el H2O2 aumenta los niveles de expresión proteica del canal aniónico dependiente de voltaje (VDAC) e induce la translocación de Bax a mitocondria. Sin embargo, en presencia de las hormonas la translocación de Bax fue inhibida lo cual sugiere que los miembros de la familia Bcl -2 pueden ser regulados por E2 y T. Los eventos moleculares desencadenados por E2 y T a nivel mitocondrial se reflejaron en la morfología de las organelas. El análisis microscópico de las células C2C12 y cultivos primarios de músculo esquelético de ratón, mediante tinciones con verde de Jano y Mitotracker reveló un efecto protector de los esteroides contra el daño por estrés oxidativo inhibiendo la redistribución y picnosis mitocondrial.We have previously shown that testosterone (T) and 17β-estradiol (E2) protect C2C12 muscle cells against apoptosis induced by hydrogen peroxide (H2O2). Since we also showed the presence of estrogen and androgen Receptors in mitochondria, this work was focused on the effects of both steroids on this organelle, which result in cellular survival. Specifically, we evaluated the actions of T and E2 on the mitochondrial membrane potential with JC-1 dye and on the mitochondrial permeability transition pore (MPTP) by the calceinacetoxymethylester (AM)/cobalt method, using fluorescence microscopy and flow cytometry. We demonstrated that T and E2 prevent MPTP opening and the loss of mitochondrial membrane potential induced by H2O2. In addition, it was observed that H2O2 increase voltage-dependent anion channel (VDAC) protein expression levels and induce translocation of Bax to mitochondria. However, in the presence of the steroids Bax translocation was abrogated suggesting that members of the Bcl-2 family may be regulated by E2 and T. The observed effects triggered by E2 and T were reflected on mitochondrial morphology. Microscopic analysis of C2C12 cells and primary cultures of mouse skeletal muscle, with Janus Green and Mitotracker staining revealed a protective effect of the steroids against oxidative stress damage which included mitochondrial redistribution and pyknosis of the organelle.Fil: la Colla, Anabela Belén. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Pronsato, Lucía. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Ronda, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto Argentino de Oceanografía (i); Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Milanesi, Lorena Magdalena. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Vasconsuelo, Andrea Anahi. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; ArgentinaFil: Boland, Ricardo Leopoldo. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico Bahia Blanca. Instituto de Ciencias Biologicas y Biomedicas del Sur; Argentin

A duo of Potassium-responsive Histidine Kinases govern the multicellular destiny of Bacillus subtilis

Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host.Fil: Grau, Roberto Ricardo. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Oña, Paula. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kunert, Maritta. Instituto Max Planck Institut Fur Chemische Okologie; AlemaniaFil: Leñini, Cecilia Andrea. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gallegos Monterrosa, Ramses. Universitat Jena; AlemaniaFil: Mhatre, Eisha. Universitat Jena; AlemaniaFil: Vileta, Darío. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Donato, Veronica. Universidad Nacional de Rosario. Facultad de Cs.bioquimicas y Farmaceuticas. Departamento de Microbiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hölscher, Theresa. Universitat Jena; AlemaniaFil: Boland, Wilhem. Instituto Max Planck Institut Fur Chemische Okologie; AlemaniaFil: Kuipers, Oscar P.. University of Groningen; Países BajosFil: Kovács, Ákos T.. Universitat Jena; Alemani