Várias condições podem estar associadas com a atrofia muscular, tais como inatividade, envelhecimento, septicemia, diabetes, câncer e uso de glicocorticoides. Todas estas condições levam a atrofia muscular através de mecanismos que incluem aumento da degradação proteica e/ou redução na síntese proteica, envolvendo pelo menos cinco sistemas: lisossomal, da calpaína, das caspases, metaloproteinases e o sistema ubiquitina-proteasoma (SUP). Glicocorticoides, tais como a dexametasona, acarretam atrofia muscular atuando em quase todos esses sistemas, com significante ativação do SUP e lisossomal, afetando uma importante via de trofismo muscular, a via do IGF-1/PI-3K/Akt/mTOR. Ácidos graxos poli-insaturados, como o Ômega-3 (ômega-3), têm sido utilizados de forma benéfica na atenuação da atrofia muscular que ocorre na septicemia e na caquexia associada ao câncer, no entanto, sua atuação sobre a atrofia muscular induzida por glicocorticoides ainda não foi avaliada. Objetivo: Avaliar se a suplementação do ácido graxo ômega-3 influenciaria o desenvolvimento da atrofia muscular induzida pela dexametasona em ratos. Metodologia: Vinte e quatro ratos Wistar suplementados e não suplementados com ômega-3 (40 dias) foram submetidos à administração de dexametasona subcutânea (5mg/Kg/dia) nos últimos 10 dias, formando assim quatro grupos: Controle (CT), dexametasona (DX), ômega3 e dexametasona+ômega3 (DX+ômega3). Através de estudo de comportamento motor, histológico, PCR em tempo real e Western Blotting foram avaliados respectivamente, o número de grandes e pequenos movimentos em campo aberto; a área de secção transversa das fibras musculares (fibras I, IIA e IIB); a expressão dos genes MyoD, Miogenina, MuRF-1, Atrogina-1 e Miostatina; e a expressão de proteínas relacionadas com a via do IGF-1/PI-3K/Akt/mTOR: Akt, GSK3beta, FOXO3a e mTOR, totais e fosforiladas. Resultados: A dexametasona produziu diminuição na quantidade de pequenos movimentos, atrofia muscular em fibras do tipo IIB e diminuição na expressão de P-Akt, P-GSK3ômega e P-FOXO3a/FOXO3a total. A suplementação com Ômega-3 não se mostrou eficaz na atenuação de tais alterações. Por outro lado, o Ômega-3 associado à dexametasona (grupo DX+3) induziu a maior expressão de atrogenes (MuRF-1 e atrogina-1) causando, adicionalmente, maior atrofia muscular em fibras do tipo I e IIA, além de menor expressão gênica de Miogenina. O Ômega-3 de forma isolada conduziu de forma significativa a maior expressão de Miostatina e MyoD, e de forma não significante elevou a expressão proteica de mTOR total e induziu menor ganho de peso corporal dos animais ao fim do estudo. Conclusão: A suplementação de Ômega-3 não foi capaz de atenuar as alterações comportamentais, atrofia muscular e perda de peso corporal causadas pela administração de dexametasona, levando por outro lado a maior atrofia das fibras musculares e aumento na expressão de atrogenes. Desta forma, este estudo sugere que suplementos alimentares usualmente considerados benéficos para saúde, tal como o ácido graxo Ômega-3, podem agir em interação com alguns medicamentos, como os glicocorticoides, potencializando seus efeitos colaterais / Many conditions can be related to muscle atrophy, such as inactivity, aging, sepsis, diabetes, cancer, as well as, glucocorticoid treatment. All these conditions lead to muscle atrophy through mechanisms that include increase of protein degradation and/or decrease of protein synthesis involving at least five systems: lysossomal, calpain, caspases, metaloproteinases and ubiquitin proteasome system (UPS). Glucocorticoids, such as dexamethasone cause muscle atrophy acting in almost all of these systems, with a significant UPS activation and affecting an important pathway related to muscular trophism, IGF-1/PI-3k/Akt/mTOR pathway. Poly-unsaturated fatty acids, such as Omega-3 (omega-3), have been used beneficially to attenuation of muscle atrophy that occur in sepsis and cachexia related to cancer, however, its action in the glucocorticoid-induced muscle atrophy, has never been evaluated. Objective: Assess whether the omega-3 supplementation would influence the development of dexamethasone-induced muscle atrophy in rats. Methods: Twenty four Wistar rats supplemented and non-supplemented with omega-3 (40 days) were submitted to dexamethasone administration (5mg/kg/day) during the last 10 days, thus establishing 4 groups: control (CT), dexamethasone (DX), omega-3 and dexamethasone+omega-3 (DX+ omega-3). The amount of large and small movements in open field; muscle fiber cross sectional areas (I, IIA and IIB); MyoD, Myogenin, MuRF-1, Atrogin-1 and Myostatin gene expression; and protein expression of Akt, GSK3omega, FOXO3a and mTOR, total and phosphorylated forms were assessed, respectively, by: motor behavior testing, histological reactions, Real-time PCR and Western Blotting analysis. Results: Dexamethasone administration induced significant decrease of small motor movements, atrophy in type IIB muscle fibers and decrease of P-Akt, P-GSK3omega and P-FOXO3a/total FOXO3a expression. Omega-3 supplementation was not able to attenuate these changes. Instead, omega-3 associated to dexamethasone (DX+ omega-3 group) additionally induced higher muscle atrophy in type I, IIA muscle fibers, and reduced expression of Myogenin. The isolated use of Omega-3 led to a significant higher expression of Myostatin and MyoD, and a non-significant increase of total mTOR protein expression and less body weight gain at end of study. Conclusion: Supplementation of omega-3 was not able to attenuate motor behavioral changes, muscle atrophy and loss of body weight caused by dexamethasone administration, leading on the other hand to higher muscle fibers atrophy and increase in atrogenes expression. Therefore, this study suggests that food supplements, usually considered benefic to the health, such as Omega-3 fatty acid, may interact with some medications, such as glucocorticoids, potentiating its side effects

Várias condições podem estar associadas com a atrofia muscular, tais como inatividade, envelhecimento, septicemia, diabetes, câncer e uso de glicocorticoides. Todas estas condições levam a atrofia muscular através de mecanismos que incluem aumento da degradação proteica e/ou redução na síntese proteica, envolvendo pelo menos cinco sistemas: lisossomal, da calpaína, das caspases, metaloproteinases e o sistema ubiquitina-proteasoma (SUP). Glicocorticoides, tais como a dexametasona, acarretam atrofia muscular atuando em quase todos esses sistemas, com significante ativação do SUP e lisossomal, afetando uma importante via de trofismo muscular, a via do IGF-1/PI-3K/Akt/mTOR. Ácidos graxos poli-insaturados, como o Ômega-3 (ômega-3), têm sido utilizados de forma benéfica na atenuação da atrofia muscular que ocorre na septicemia e na caquexia associada ao câncer, no entanto, sua atuação sobre a atrofia muscular induzida por glicocorticoides ainda não foi avaliada. Objetivo: Avaliar se a suplementação do ácido graxo ômega-3 influenciaria o desenvolvimento da atrofia muscular induzida pela dexametasona em ratos. Metodologia: Vinte e quatro ratos Wistar suplementados e não suplementados com ômega-3 (40 dias) foram submetidos à administração de dexametasona subcutânea (5mg/Kg/dia) nos últimos 10 dias, formando assim quatro grupos: Controle (CT), dexametasona (DX), ômega3 e dexametasona+ômega3 (DX+ômega3). Através de estudo de comportamento motor, histológico, PCR em tempo real e Western Blotting foram avaliados respectivamente, o número de grandes e pequenos movimentos em campo aberto; a área de secção transversa das fibras musculares (fibras I, IIA e IIB); a expressão dos genes MyoD, Miogenina, MuRF-1, Atrogina-1 e Miostatina; e a expressão de proteínas relacionadas com a via do IGF-1/PI-3K/Akt/mTOR: Akt, GSK3beta, FOXO3a e mTOR, totais e fosforiladas. Resultados: A dexametasona produziu diminuição na quantidade de pequenos movimentos, atrofia muscular em fibras do tipo IIB e diminuição na expressão de P-Akt, P-GSK3ômega e P-FOXO3a/FOXO3a total. A suplementação com Ômega-3 não se mostrou eficaz na atenuação de tais alterações. Por outro lado, o Ômega-3 associado à dexametasona (grupo DX+3) induziu a maior expressão de atrogenes (MuRF-1 e atrogina-1) causando, adicionalmente, maior atrofia muscular em fibras do tipo I e IIA, além de menor expressão gênica de Miogenina. O Ômega-3 de forma isolada conduziu de forma significativa a maior expressão de Miostatina e MyoD, e de forma não significante elevou a expressão proteica de mTOR total e induziu menor ganho de peso corporal dos animais ao fim do estudo. Conclusão: A suplementação de Ômega-3 não foi capaz de atenuar as alterações comportamentais, atrofia muscular e perda de peso corporal causadas pela administração de dexametasona, levando por outro lado a maior atrofia das fibras musculares e aumento na expressão de atrogenes. Desta forma, este estudo sugere que suplementos alimentares usualmente considerados benéficos para saúde, tal como o ácido graxo Ômega-3, podem agir em interação com alguns medicamentos, como os glicocorticoides, potencializando seus efeitos colaterais / Many conditions can be related to muscle atrophy, such as inactivity, aging, sepsis, diabetes, cancer, as well as, glucocorticoid treatment. All these conditions lead to muscle atrophy through mechanisms that include increase of protein degradation and/or decrease of protein synthesis involving at least five systems: lysossomal, calpain, caspases, metaloproteinases and ubiquitin proteasome system (UPS). Glucocorticoids, such as dexamethasone cause muscle atrophy acting in almost all of these systems, with a significant UPS activation and affecting an important pathway related to muscular trophism, IGF-1/PI-3k/Akt/mTOR pathway. Poly-unsaturated fatty acids, such as Omega-3 (omega-3), have been used beneficially to attenuation of muscle atrophy that occur in sepsis and cachexia related to cancer, however, its action in the glucocorticoid-induced muscle atrophy, has never been evaluated. Objective: Assess whether the omega-3 supplementation would influence the development of dexamethasone-induced muscle atrophy in rats. Methods: Twenty four Wistar rats supplemented and non-supplemented with omega-3 (40 days) were submitted to dexamethasone administration (5mg/kg/day) during the last 10 days, thus establishing 4 groups: control (CT), dexamethasone (DX), omega-3 and dexamethasone+omega-3 (DX+ omega-3). The amount of large and small movements in open field; muscle fiber cross sectional areas (I, IIA and IIB); MyoD, Myogenin, MuRF-1, Atrogin-1 and Myostatin gene expression; and protein expression of Akt, GSK3omega, FOXO3a and mTOR, total and phosphorylated forms were assessed, respectively, by: motor behavior testing, histological reactions, Real-time PCR and Western Blotting analysis. Results: Dexamethasone administration induced significant decrease of small motor movements, atrophy in type IIB muscle fibers and decrease of P-Akt, P-GSK3omega and P-FOXO3a/total FOXO3a expression. Omega-3 supplementation was not able to attenuate these changes. Instead, omega-3 associated to dexamethasone (DX+ omega-3 group) additionally induced higher muscle atrophy in type I, IIA muscle fibers, and reduced expression of Myogenin. The isolated use of Omega-3 led to a significant higher expression of Myostatin and MyoD, and a non-significant increase of total mTOR protein expression and less body weight gain at end of study. Conclusion: Supplementation of omega-3 was not able to attenuate motor behavioral changes, muscle atrophy and loss of body weight caused by dexamethasone administration, leading on the other hand to higher muscle fibers atrophy and increase in atrogenes expression. Therefore, this study suggests that food supplements, usually considered benefic to the health, such as Omega-3 fatty acid, may interact with some medications, such as glucocorticoids, potentiating its side effects

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

Indiana University-Purdue University Indianapolis (IUPUI) / Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.