The cathepsin L gene is a direct target of FOXO1 in skeletal muscle

2010 ◽  
Vol 427 (1) ◽  
pp. 171-178 ◽  
Author(s):  
Yoshihiro Yamazaki ◽  
Yasutomi Kamei ◽  
Satoshi Sugita ◽  
Fumiko Akaike ◽  
Sayaka Kanai ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Cathepsin L ◽  
Skeletal Muscle Atrophy ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Direct Target

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

scholarly journals Mechanical loading of tissue engineered skeletal muscle prevents dexamethasone induced myotube atrophy

2020 ◽  
Author(s):  
Kathryn W. Aguilar-Agon ◽  
Andrew J. Capel ◽  
Jacob W. Fleming ◽  
Darren J. Player ◽  
Neil R. W. Martin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mechanical Loading ◽  
Muscle Atrophy ◽  
Mechanical Load ◽  
3D Models ◽  
Skeletal Muscle Atrophy ◽  
Treatment Modalities ◽  
Murine Cell Line

Abstract Skeletal muscle atrophy as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and aging, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. In vitro 3D-models of skeletal muscle provide a controllable, high throughput environment and mitigating many of the ethical and methodological constraints present during in vivo experimentation. This work aimed to determine if mechanical loading would offset dexamethasone (DEX) induced skeletal muscle atrophy, in muscle engineered using the C2C12 murine cell line. Mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX regardless of whether the loading occurred before or after 24 h of DEX treatment. Furthermore, mechanical load prevented increases in MuRF-1 and MAFbx mRNA expression, critical regulators of muscle atrophy. Overall, we demonstrate the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy.

Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 711-711
Author(s):  
Srimoyee Ghosh ◽  
Sergei B Koralov ◽  
Irena Stevanovic ◽  
Mark S Sundrud ◽  
Yoshiteru Sasaki ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Autoimmune Encephalitis ◽  
Cell Types ◽  
Wild Type ◽  
Ifn Γ

Abstract Abstract 711 Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells. NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases. We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls. To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells. To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease. In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures: No relevant conflicts of interest to declare.

Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration in vivo

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2835-2844 ◽  
Author(s):  
Mònica Suelves ◽  
Roser López-Alemany ◽  
Frederic Lluı́s ◽  
Gloria Aniorte ◽  
Erika Serrano ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Myoblast Fusion ◽  
Skeletal Muscle Regeneration ◽  
Wild Type ◽  
Plasmin Activity ◽  
Type Plasminogen Activator ◽  
Deficient Mice

Abstract Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and is implicated in biologic processes dependent upon proteolytic activity, such as tissue remodeling and cell migration. Active plasmin is generated from proteolytic cleavage of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Here, we have investigated the role of plasmin in C2C12 myoblast fusion and differentiation in vitro, as well as in skeletal muscle regeneration in vivo, in wild-type and Plg-deficient mice. Wild-type mice completely repaired experimentally damaged skeletal muscle. In contrast, Plg−/− mice presented a severe regeneration defect with decreased recruitment of blood-derived monocytes and lymphocytes to the site of injury and persistent myotube degeneration. In addition, Plg-deficient mice accumulated fibrin in the degenerating muscle fibers; however, fibrinogen depletion of Plg-deficient mice resulted in a correction of the muscular regeneration defect. Because we found that uPA, but not tPA, was induced in skeletal muscle regeneration, and persistent fibrin deposition was also reproducible in uPA-deficient mice following injury, we propose that fibrinolysis by uPA-dependent plasmin activity plays a fundamental role in skeletal muscle regeneration. In summary, we identify plasmin as a critical component of the mammalian skeletal muscle regeneration process, possibly by preventing intramuscular fibrin accumulation and by contributing to the adequate inflammatory response after injury. Finally, we found that inhibition of plasmin activity with α2-antiplasmin resulted in decreased myoblast fusion and differentiation in vitro. Altogether, these studies demonstrate the requirement of plasmin during myogenesis in vitro and muscle regeneration in vivo.

scholarly journals Protective Signatures of Roselle (Hibiscus sabdariffa L.) Calyx Fractions against Staphylococcus aureus in Drosophila Infection Model

2020 ◽  
Vol 27 (4) ◽  
pp. 306
Author(s):  
Firzan Nainu ◽  
M. Natsir Djide ◽  
Subehan Subehan ◽  
Sartini Sartini ◽  
Tri Puspita Roska ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Early Death ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Hibiscus Sabdariffa ◽  
Immune Stimulation ◽  
Wild Type ◽  
Toll Pathway

The rise of antibiotic-resistant Staphylococcus aureus-related clinical cases is an alarming chronicle for global communities. This research was conducted to examine the antistaphylococcal effect of roselle (Hibiscus sabdariffa L.) calyx fractions in the Drosophila model. In the infection experiment, wild-type and immunodeficient Drosophila were pricked with S. aureus and subsequently subjected to fly survivorship and colony-forming assays, in the presence or absence of roselle calyx fractions. The Involvement of immune stimulation in the host antibacterial protection was assessed in vitro using cell-based luciferase reporter assay and in vivo using RT-qPCR analysis on adult flies. A declining rate of fly survivorship and augmentation of bacterial growth were observable in S. aureus-infected wild-type flies but subject to improvement in the presence of roselle calyx fractions. Cell-based analysis revealed the absence of host immune stimulation via Drosophila Toll pathway and roselle calyx fractions-treated immune-deficient flies lacking for components in the Toll pathway were protected from infection-induced early death phenotype and harbored reduced number of S. aureus colonies. Overall, our data confirmed the in vivo anti-staphylococcal activity of roselle calyx fractions in Drosophila infection model and such protective signature was devoid of host immune stimulation.

scholarly journals Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 627-635 ◽  
Author(s):  
D.L. Song ◽  
G. Chalepakis ◽  
P. Gruss ◽  
A.L. Joyner
Keyword(s):  
Dna Binding ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Developing Brain ◽  
Molecular Evidence ◽  
Regulatory Sequences ◽  
Pax Genes

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

scholarly journals Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis

2019 ◽  
Vol 2 (3) ◽  
pp. e201900437 ◽  
Author(s):  
Milica Marinkovic ◽  
Claudia Fuoco ◽  
Francesca Sacco ◽  
Andrea Cerquone Perpetuini ◽  
Giulio Giuliani ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Control Mechanism ◽  
Notch Pathway ◽  
Skeletal Muscle Regeneration ◽  
Wild Type ◽  
Adult Skeletal Muscle ◽  
Pathological Conditions ◽  
Dystrophic Mice

Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. An unbiased mass spectrometry–based proteomic analysis of FAPs from muscles of wild-type and mdx mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in mdx FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.

scholarly journals Corepression of the P1 Addiction Operon by Phd and Doc

1998 ◽  
Vol 180 (23) ◽  
pp. 6342-6351 ◽  
Author(s):  
Roy Magnuson ◽  
Michael B. Yarmolinsky
Keyword(s):  
Fusion Protein ◽  
Binding Sites ◽  
Wild Type ◽  
Dna Complexes ◽  
Present Evidence ◽  
Cooperative Interactions ◽  
Regulatory Properties

ABSTRACT The P1 plasmid addiction operon encodes Doc, a toxin that kills plasmid-free segregants, and Phd, an unstable antidote that neutralizes the toxin. Additionally, these products repress transcription of the operon. The antidote binds to two adjacent sites in the promoter. Here we present evidence concerning the regulatory role of the toxin, which we studied with the aid of a mutation,docH66Y. The DocH66Y protein retained the regulatory properties of the wild-type protein, but not its toxicity. In vivo, DocH66Y enhanced repression by Phd but failed to affect repression in the absence of Phd, suggesting that DocH66Y contacts Phd. In vitro, a MalE-DocH66Y fusion protein was found to bind Phd. Binding of toxin to antidote may be the physical basis for the neutralization of toxin. DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. Although DocH66Y enhanced the binding of Phd to two adjacent Phd-binding sites, DocH66Y had relatively little effect on the binding of Phd to a single Phd-binding site, indicating that DocH66Y mediates cooperative interactions between adjacent Phd-binding sites. Several electrophoretically distinct protein-DNA complexes were observed with different amounts of DocH66Y relative to Phd. Maximal repression and specificity of DNA binding were observed with subsaturating amounts of DocH66Y relative to Phd. Analogous antidote-toxin pairs appear to have similar autoregulatory circuits. Autoregulation, by dampening fluctuations in the levels of toxin and antidote, may prevent the inappropriate activation of the toxin.

scholarly journals Trimetazidine Attenuates Dexamethasone-Induced Muscle Atrophy via Inhibiting NLRP3/GSDMD Pathway-Mediated Pyroptosis

2020 ◽  
Author(s):  
Li Wang ◽  
Ming-Qing He ◽  
Xi-Yu Shen ◽  
Kang-Zhen Zhang ◽  
Can Zhao ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Ring Finger ◽  
Muscle Performance ◽  
Skeletal Muscle Atrophy ◽  
C2c12 Myotubes ◽  
High Dose ◽  
Therapeutic Compound

Skeletal muscle atrophy is one of the major side effects of high dose or sustained usage of glucocorticoids. Pyroptosis is a novel form of pro-inflammatory programmed cell death that may contribute to skeletal muscle injury. Trimetazidine, a well-known anti-anginal agent, can also improve skeletal muscle performance both in human and mice. We here showed that dexamethasone induced atrophy, evidenced by the increase of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) expression , and the decrease of myotube diameter in C2C12 myotubes. Dexamethasone also induced pyroptosis, indicated by upregulated pyroptosis-related protein NLRP3, Caspase-1 and GSDMD. Knockdown of NLRP3 or GSDMD attenuated dexamethasone-induced myotube pyroptosis and atrophy. Trimetazidine administration ameliorated dexamethasone-induced muscle atrophy both in vivo and in vitro. Moreover, trimetazidine improved exercise tolerance, as evidenced by increased running distance and running time, as well as increased skeletal muscle mass in dexamethasone-treated mice. Mechanically, trimetazidine could reverse dexamethasone-induced activation of pyroptosis both in C2C12 myotubes and in mice. Taken together, our present study demonstrated that NLRP3/GSDMD pathway-mediated pyroptosis was involved in dexamethasone-induced skeletal muscle atrophy. Trimetazidine could partially alleviate dexamethasone-induced skeletal muscle atrophy, and increase the diameter of C2C12 myotubes via inhibiting pyroptosis. Thus, trimetazidine might be a potential therapeutic compound for the prevention of muscle atrophy in glucocorticoid-treated patients.

scholarly journals Use of a Two-Color Genetic Screen To Identify a Domain of the Global Regulator Lrp That Is Specifically Required forpap Phase Variation

1998 ◽  
Vol 180 (5) ◽  
pp. 1224-1231 ◽  
Author(s):  
Linda Kaltenbach ◽  
Bruce Braaten ◽  
Julie Tucker ◽  
Margareta Krabbe ◽  
David Low
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Phase Variation ◽  
Genetic Screen ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Global Regulator ◽  
Transcription In Vitro

ABSTRACT The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as ilvIH, activation ofpapBA transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp’s were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity forpap Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-lockedpap derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp’s are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.

beta3-tubulin is directly repressed by the engrailed protein in Drosophila

Development ◽  
1997 ◽  
Vol 124 (13) ◽  
pp. 2527-2536 ◽  
Author(s):  
N. Serrano ◽  
H.W. Brock ◽  
F. Maschat
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transgenic Line ◽  
Target Genes ◽  
Regulatory Protein ◽  
Polytene Chromosomes ◽  
First Intron ◽  
Direct Target

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

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