Inducible deletion of Raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation
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PROVIDER: PRJNA889746 | ENA |
REPOSITORIES: ENA
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Project description:Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mTOR signaling. Here we wanted to better elucidate the functional role of mTOR on muscle contractility. Most loss of function studies for mTOR signaling have used the drug rapamycin to inhibit some of the signaling downstream of mTOR. However, as rapamycin does not completely inhibit all mTOR signaling, we generated a double k.o. for mTOR and for the scaffold protein of mTORC1, Raptor, in skeletal muscle. We found that dk.o. mice results in a more severe phenotype compared to Raptor or mTOR deletion alone. Indeed, they display muscle weakness, increased fiber denervation, and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibers and changes in the expression levels of calcium-related genes, like Serca1 and Casq1. Indeed, dk.o. mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes are linked to sarcomere organization, like Tcap and Fhod3. These results suggest a key role for mTOR signaling in maintaining a proper fiber relaxation in skeletal muscle.
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Project description:Recent studies have shown that mTOR signalling have a promotive effect on cancer. In our clinical work, we found that mTOR signalling is commonly activated in laryngeal cancer tissues and influence the metabolism of cancer cells. To investigate how mTORC1 exerts these functions and its downstream regulatory genes, we knocked down Raptor in the laryngeal cancer cell line LIU-LSC-1 and sequenced the transcriptome of wild-type and Raptor knockdown cells.
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Project description:Uterine contractile dysfunction leads to pregnancy complications such as preterm birth and labor dystocia. Progesterone is necessary to suppress uterine contractions to prevent premature labor. As humans maintain high levels of progesterone throughout parturition, a functional progesterone withdrawal hypothesis suggests that relative levels of myometrial progesterone receptor isoforms PGR-A and PGR-B switch at parturition, where PGR-B promotes a relaxed state and PGR-A result in increased uterine contractility. Our objective is to determine the effects of altered levels of PGR-B and PGR-A in the mouse myometrium on pregnancy and parturition using transgenic mouse models in which the relative levels of these isoforms are altered specifically in the myometrium. Overexpression of PGR-B is associated with a markedly increased gestational length compared to control mice. In both ex vivo and in vivo experiments, myometrium of PGR-B overexpressing mice have prolonged labor, a significant decrease in uterine contractility, and a high incidence of labor dystocia. Conversely, overexpression of PGR-A is associated with an increase in uterine contractility without a change in gestational length. Uterine RNAseq at mid-pregnancy identified isoform-specific downstream targets and genes that were commonly regulated by both PGR isoforms. Gene signature analyses further revealed that PGR-B promotes muscle relaxation and that PGR-A is pro-inflammation. High levels of PGR-B manifest a genetic profile of a blunted phospholipase C pathway that mediates oxytocin and angiotensin II induced muscle contraction. These findings provided in vivo support that PGR isoform levels determine distinct transcriptomic landscapes and pathways in myometrial function and labor.
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Project description:The myometrium is an important reproductive tissue composed primarily of smooth muscle cells. Contractility of the myometrial smooth muscle cells during the estrous cycle and pregnancy is modulated by estrogen. Despite much research, little is known about the molecular mechanism by which estrogen regulates myometrial contractility. This study investigates global gene expression profile of cultured human uterine smooth muscle cells (hUtSMCs) following 17M-NM-2-estradiol (E2) treatment using cDNA microarray technology. In response to E2 treatment 540 genes were identified as significantly differentially expressed. Gene ontology analysis identified several significant biological processes for these genes including muscle contraction, gland development, cell migration, cell adhesion, apoptosis, response to external stimulus and phosphorylation. In this study evidence is presented that in human smooth muscle cells 17M-NM-2-estradiol, contrary to expectations, altered expression of several genes that may favor myometrial relaxation. Genes related to focal adhesion function were downregulated suggesting a loosening of SMC connections to the ECM while upregulation of MMP9 is also associated with weakened ECM interactions. The hUtSMC system is an additional useful model to investigate steroid effects on smooth muscle cells in isolation from other myometrial cell types. In this study human myometrial smooth muscle cells (hUtSMC) purchased from Lonza were cultured and treated with 17beta estradiol. RNA was isolated after 6h, 24h and 72h from both treated and untreated control cells. The experiment was repeated at least 3 times.
2014-06-02 | E-GEOD-51470 | biostudies-arrayexpress
Project description:Little is known about the role of mTOR signaling in plasma cell differentiation and function. Furthermore, for reasons not understood, mTOR inhibition reverses antibody-associated disease in a murine model of systemic lupus erythematosus. Here, we have demonstrated that induced B-lineage specific deletion of the gene encoding RAPTOR, an essential signaling adaptor for the rapamycin-sensitive mTOR complex 1 (mTORC1) abrogated the generation of antibody-secreting plasma cells in mice. Acute treatment with rapamycin recapitulated the effects of RAPTOR deficiency, while both strategies led to the ablation of newly formed plasma cells in the spleen and bone marrow while also obliterating pre-existing germinal centers. Surprisingly, although perturbing mTOR activity caused a profound decline in serum antibodies that were specific for exogenous antigen or DNA, frequencies of long-lived bone marrow plasma cells were unaffected. Instead, mTORC1 inhibition led to decreased expression of immunoglobulin binding protein (BiP) and other factors needed for robust protein synthesis. Consequently, blockade of antibody synthesis was rapidly reversed after termination of rapamycin treatment. We conclude that mTOR signaling plays critical but diverse roles in early and late phases of antibody responses and plasma cell differentiation.
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Project description:Analysis of proteomic changes upon deletion of Raptor or inhibition by rapamycin in mouse muscles over expressing a constitutively active Akt.
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Project description:Rats with adenine-induced chronic renal failure (A-CRF) develop a reduction in the rate of relaxation of the thoracic aorta. The primary aim of this study was to elucidate the mechanisms underlying this abnormality. Male Sprague-Dawley rats received either chow containing adenine or were pair-fed with normal chow (controls). After 8-14 weeks arterial functions were analyzed ex vivo using wire myography and the thoracic aorta was analyzed by DNA microarray. Plasma creatinine levels were elevated ~8-fold in A-CRF rats. The rate of vascular relaxation following wash-out of KCl was reduced in A-CRF rats vs. controls in the thoracic aorta (P<0.01), abdominal aorta (P<0.05), and common carotid artery (P<0.05), but not in the common femoral artery. Endothelial denudation exaggerated the impairment in relaxation of thoracic aortas. Relaxation rates of thoracic aortas increased (P<0.01), but were not normalized, in response to wash-out of KCl with Ca2+-free buffer. Microarray and qRT-PCR analyses revealed altered gene expression levels for a number of genes involved in vascular smooth muscle cell excitation-contraction coupling in aortas of A-CRF rats. In conclusion, rats with A-CRF show a marked reduction in the rate of relaxation of larger conduit arteries localized proximal to the common femoral artery. This abnormality may be caused by reduced cytosolic Ca2+ clearance in vascular smooth muscle cells secondary to dysregulation of genes involved in excitation-contraction coupling.
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