Sepsis induces the transcription of the glucocorticoid receptor in skeletal muscle cells

2003 ◽  
Vol 105 (3) ◽  
pp. 383-391 ◽  
Author(s):  
Xiaoyan SUN ◽  
Joshua M. V. MAMMEN ◽  
Xintian TIAN
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
L6 Myotubes ◽  
Breakdown Rates ◽  
Dependent Protein ◽  
Vitro Model ◽  
Energy Dependent ◽  
L6 Myocytes

Evidence from a recent study indicates that glucocorticoids (GCs) mediate skeletal muscle proteolysis during sepsis via the GC receptor (GR) pathway. Attempts to identify the mechanisms regulating GR gene expression in skeletal muscle during sepsis have been hampered by the lack of an appropriate in vitro model system that can mimic in vivo septic conditions. In the present study, we report that GR gene transcription in L6 myocytes in vitro is up-regulated by treatment with sera from septic rats in a manner similar to that measured in septic rats in vivo. Sera from septic rats were collected from animals in which sepsis was induced by caecal ligation and puncture and from control rats that were sham-operated. Finally, by treating L6 myotubes with the GR antagonist RU 38486, thereby preventing sepsis-induced GR transcription, we confirmed that the possible septic effect on the GR was due to increased GCs. L6 myocytes treated with sera from septic rats might therefore be useful as an experimental model for identifying the molecular mechanisms by which the GR regulates muscle cachexia during sepsis. Furthermore, RU 38486 inhibited the sepsis-induced increase in total and myofibrillar energy-dependent protein breakdown rates in incubated extensor digitorum longus muscles from septic and sham-operated rats, as measured by release of tyrosine and 3-methylhistidine respectively. Our results demonstrate for the first time that sepsis induces GR transcription in skeletal muscle, and supports the hypothesis that the GC-induced proteolysis under sepsis is partially a consequence of GR activation.

scholarly journals Activation of protein kinase B β and γ isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B α

1998 ◽  
Vol 331 (1) ◽  
pp. 299-308 ◽  
Author(s):  
Kay S. WALKER ◽  
Maria DEAK ◽  
Andrew PATERSON ◽  
Kevin HUDSON ◽  
Philip COHEN ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Rat Hepatocytes ◽  
Dependent Protein Kinase ◽  
L6 Myotubes ◽  
Dependent Protein ◽  
Stimulation Of

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBα, PKBβ and PKBγ) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, as well as PDK1. The activation of PKBβ and PKBγ by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBα, namely Thr309 (PKBβ) and Thr305 (PKBγ). PKBγ which had been activated by PDK1 possessed a substrate specificity identical with that of PKBα and PKBβ towards a range of peptides. The activation of PKBγ and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBα and PKBβ with similar kinetics. After stimulation of adipocytes, the activity of PKBβ was twice that of PKBα, but in hepatocytes PKBα activity was four-fold higher than PKBβ. Insulin induced the activation of PKBα in rat skeletal muscle in vivo, with little activation of PKBβ. Insulin did not induce PKBγ activity in adipocytes, hepatocytes or skeletal muscle, but PKBγ was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

Identity of the renin cell is mediated by cAMP and chromatin remodeling: an in vitro model for studying cell recruitment and plasticity

2008 ◽  
Vol 294 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Ellen Steward Pentz ◽  
Maria Luisa S. Sequeira Lopez ◽  
Magali Cordaillat ◽  
R. Ariel Gomez
Keyword(s):  
Chromatin Remodeling ◽  
In Vitro Model ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Histone H4 Acetylation ◽  
Renin Gene ◽  
Vitro Model

The renin-angiotensin system (RAS) regulates blood pressure and fluid-electrolyte homeostasis. A key step in the RAS cascade is the regulation of renin synthesis and release by the kidney. We and others have shown that a major mechanism to control renin availability is the regulation of the number of cells capable of making renin. The kidney possesses a pool of cells, mainly in its vasculature but also in the glomeruli, capable of switching from smooth muscle to endocrine renin-producing cells when homeostasis is threatened. The molecular mechanisms governing the ability of these cells to turn the renin phenotype on and off have been very difficult to study in vivo. We, therefore, developed an in vitro model in which cells of the renin lineage are labeled with cyan fluorescent protein and cells actively making renin mRNA are labeled with yellow fluorescent protein. The model allowed us to determine that it is possible to culture cells of the renin lineage for numerous passages and that the memory to express the renin gene is maintained in culture and can be reenacted by cAMP and chromatin remodeling (histone H4 acetylation) at the cAMP-responsive element in the renin gene.

scholarly journals Regulation of myogenic progenitor proliferation in human fetal skeletal muscle by BMP4 and its antagonist Gremlin

2006 ◽  
Vol 175 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Natasha Y. Frank ◽  
Alvin T. Kho ◽  
Tobias Schatton ◽  
George F. Murphy ◽  
Michael J. Molloy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Side Population ◽  
Functional Studies ◽  
Morphogenetic Protein ◽  
Study Gene Expression ◽  
Main Population ◽  
Myogenic Progenitor

Skeletal muscle side population (SP) cells are thought to be “stem”-like cells. Despite reports confirming the ability of muscle SP cells to give rise to differentiated progeny in vitro and in vivo, the molecular mechanisms defining their phenotype remain unclear. In this study, gene expression analyses of human fetal skeletal muscle demonstrate that bone morphogenetic protein 4 (BMP4) is highly expressed in SP cells but not in main population (MP) mononuclear muscle-derived cells. Functional studies revealed that BMP4 specifically induces proliferation of BMP receptor 1a–positive MP cells but has no effect on SP cells, which are BMPR1a-negative. In contrast, the BMP4 antagonist Gremlin, specifically up-regulated in MP cells, counteracts the stimulatory effects of BMP4 and inhibits proliferation of BMPR1a-positive muscle cells. In vivo, BMP4-positive cells can be found in the proximity of BMPR1a-positive cells in the interstitial spaces between myofibers. Gremlin is expressed by mature myofibers and interstitial cells, which are separate from BMP4-expressing cells. Together, these studies propose that BMP4 and Gremlin, which are highly expressed by human fetal skeletal muscle SP and MP cells, respectively, are regulators of myogenic progenitor proliferation.

scholarly journals Endogenous Galectin-3 is Required for Skeletal Muscle Repair

2020 ◽  
Author(s):  
Daniel Giuliano Cerri ◽  
Lilian Cataldi Rodrigues ◽  
Vani Maria Alves ◽  
Juliano Machado ◽  
Víctor Alexandre Félix Bastos ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Culture ◽  
Molecular Mechanisms ◽  
Repair Process ◽  
Muscle Repair ◽  
Galectin 3 ◽  
Self Repair ◽  
Myoblast Cell

ABSTRACTSkeletal muscle has the intrinsic ability to self-repair through a multifactorial process, but many aspects of its cellular and molecular mechanisms are not fully understood. There is increasing evidence that some members of the mammalian β-galactoside-binding protein family (galectins) are involved in the muscular repair process (MRP), including galectin-3 (Gal-3). However, there are many questions about the role of this protein on muscle self-repair. Here, we demonstrate that endogenous Gal-3 is required for: i) muscle repair in vivo using a chloride-barium myolesion mouse model, and ii) mouse primary myoblasts myogenic programming. Injured muscle from Gal-3 knockout mice (GAL3KO) showed persistent inflammation associated with compromised muscle repair and the formation of fibrotic tissue on the lesion site. In GAL3KO mice, osteopontin expression remained high even after 7 and 14 days of the myolesion, while MyoD and myogenin had decreased their expression. In GAL3KO mouse primary myoblast cell culture, Pax7 detection seems to sustain even when cells are stimulated to differentiation and MyoD expression is drastically reduced. These findings suggest that the detection and temporal expression levels of these transcriptional factors appear to be altered in Gal-3-deficient myoblast cell culture compared to Wild Type (WT) cells. We observed Gal-3 expression in WT states, both in vivo and in vitro, in sarcoplasm/cytoplasm and myonuclei; as differentiation proceeds, Gal-3 expression is drastically reduced, and its location is confined to the sarcolemma/plasma cell membrane. We also observed a change in the temporal-spatial profile of Gal-3 expression and muscle transcription factors levels during the myolesion. Overall, these results demonstrate that endogenous Gal-3 is required for the skeletal muscle repair process.

scholarly journals Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy

2019 ◽  
Author(s):  
Takehito Sugasawa ◽  
Tome Yoshiya ◽  
Yoshinori Takeuchi ◽  
Naoya Yahagi ◽  
Rahul Sharma ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Binding Protein ◽  
Cold Stimulation ◽  
Vitro Assay ◽  
Mitochondrial Dna Copy Number ◽  
Local Cryotherapy

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injury. However, its molecular mechanisms are unknown. To clarify these mechanisms, in this study, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cAMP response element-binding protein 1 (CREB1) was markedly phosphorylated (as pCREB1) and CREB-binding protein (CBP) was recruited to pCREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes such as Pgc-1α involved in mitochondrial biogenesis. The foregoing results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes in a manner dependent on cold stimulation frequency and duration. This information may serve as an impetus for further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

scholarly journals Reconstitution of Calcium-Regulated Parathyroid Hormone Secretion from Streptolysin-O-Permeabilized Parathyroid Cells by Guanosine 5′-O-(Thio)Triphosphate*

Endocrinology ◽  
1997 ◽  
Vol 138 (3) ◽  
pp. 1170-1179 ◽  
Author(s):  
Lisa M. Matovcik ◽  
Steven S. Rhee ◽  
Jean F. Schaefer ◽  
Barbara K. Kinder
Keyword(s):  
Hormone Secretion ◽  
Dependent Manner ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Calcium Calmodulin Dependent ◽  
Streptolysin O ◽  
Dependent Protein ◽  
Vitro Model

Abstract Intracellular Ca2+ levels determine the amount of PTH secretion from parathyroid cells. Dissociated calf parathyroid cells were permeabilized with streptolysin-O (SLO) to provide an in vitro model system to examine Ca2+-dependent regulation of hormone secretion. PTH release from these cells was energy dependent and increased by cytosolic cofactors. Guanosine 5′-O-(thio)triphosphate (GTPγS) increased PTH secretion from SLO-permeabilized cells in a dose-dependent manner from 0.1–100 μm. In the absence of GTPγS there was no relationship between the ambient Ca2+ concentration and the rate of PTH secretion. However, in the presence of GTPγS, intracellular Ca2+ inhibited PTH secretion with an EC50 of approximately 0.1 μm, corresponding to physiological intracellular Ca2+ levels. Thus, the addition of GTPγS to SLO-permeabilized parathyroid cells reconstituted the inverse relationship between extracellular Ca2+ and PTH secretion that is observed in vivo and in intact cells. The data indicate that this effect is mediated at least in part by heterotrimeric guanosine triphosphatases. In addition, calcium/calmodulin-dependent protein kinase II appears to mediate low Ca2+-dependent PTH secretion from these cells.

Regulation of GLUT4 biogenesis in muscle: evidence for involvement of AMPK and Ca2+

2002 ◽  
Vol 282 (5) ◽  
pp. E1008-E1013 ◽  
Author(s):  
Edward O. Ojuka ◽  
Terry E. Jones ◽  
Lorraine A. Nolte ◽  
May Chen ◽  
Brian R. Wamhoff ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Culture Medium ◽  
Mitochondrial Enzymes ◽  
L6 Myotubes ◽  
Glut4 Expression ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
L6 Myocytes ◽  
Enhancer Factor

There is evidence suggesting that adaptive increases in GLUT4 and mitochondria in skeletal muscle occur in parallel. It has been reported that raising cytosolic Ca2+ in myocytes induces increases in mitochondrial enzymes. In this study, we tested the hypothesis that an increase in cytosolic Ca2+ induces an increase in GLUT4. We found that raising cytosolic Ca2+ by exposing L6 myotubes to 5 mM caffeine for 3 h/day for 5 days induced increases in GLUT4 protein and in myocyte enhancer factor (MEF)2A and MEF2D, which are transcription factors involved in regulating GLUT4 expression. The caffeine-induced increases in GLUT4 and MEF2A and MEF2D were partially blocked by dantrolene, an inhibitor of sarcoplasmic reticulum Ca2+ release, and completely blocked by KN93, an inhibitor of Ca2+-calmodulin-dependent protein kinase (CAMK). Caffeine also induced increases in MEF2A, MEF2D, and GLUT4 in rat epitrochlearis muscles incubated with caffeine in culture medium. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), which activates AMP-activated protein kinase (AMPK), also induced approximately twofold increases in GLUT4, MEF2A, and MEF2D in L6 myocytes. Our results provide evidence that increases in cytosolic Ca2+and activation of AMPK, both of which occur in exercising muscle, increase GLUT4 protein in myocytes and skeletal muscle. The data suggest that this effect of Ca2+ is mediated by activation of CAMK and indicate that MEF2A and MEF2D are involved in this adaptive response.

scholarly journals SOX2 and BRN2 as Key Transcription Factors in Neural Rosette Formation In Vitro

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Zuzana Hudáčová ◽  
Keyword(s):  
Transcription Factors ◽  
Neural Development ◽  
Molecular Mechanisms ◽  
Rosette Formation ◽  
In Vivo Studies ◽  
Confounding Variables ◽  
Vitro Model ◽  
Underlying Processes

Although neurogenesis has been well studied, its molecular mechanisms remain largely unknown due to the challenges posed by the complexity of the underlying processes. Whilst in vivo studies can be used to study neurogenesis, the inability to control confounding variables complicate findings. Therefore, the purpose of this study was to identify the markers of in vitro neural rosette formation and describe the formation of neural rosettes from pluripotent stem cells using immunofluorescence analysis. The protocol of stem cell cultivation and induction of neural rosette formation was tested. Following, two transcription factors, BRN2 and SOX2, were fluorescently labelled and cells were imaged over a period of eight days. It was identified that SOX2 and BRN2 are expressed during in vitro neural rosette formation. These results are concurrent with in vivo neurogenesis, which suggests that neural rosettes could be a suitable in vitro model for researching neural development. Given that mistakes can arise during neurogenesis, such as neural tube defects, developing robust models to understand the formation of the nervous system is important. Moving forward, a detailed molecular understanding of neural rosette formation has the potential to be used for targeting specific transcription factors to treat or prevent problematic neurogenesis.

scholarly journals Cytotoxicity of atropine to human corneal endothelial cells by inducing mitochondrion-dependent apoptosis

2016 ◽  
Vol 241 (13) ◽  
pp. 1457-1465 ◽  
Author(s):  
Qian Wen ◽  
Ting-Jun Fan ◽  
Cheng-Lei Tian
Keyword(s):  
Corneal Endothelium ◽  
Molecular Mechanisms ◽  
Anticholinergic Drug ◽  
Death Receptor ◽  
Time Dependent ◽  
Dependent Manner ◽  
Plasma Membrane Permeability ◽  
Vitro Model

Atropine, a widely used topical anticholinergic drug, might have adverse effects on human corneas in vivo. However, its cytotoxic effect on human corneal endothelium (HCE) and its possible mechanisms are unclear. Here, we investigated the cytotoxicity of atropine and its underlying cellular and molecular mechanisms using an in vitro model of HCE cells and verified the cytotoxicity using cat corneal endothelium (CCE) in vivo. Our results showed that atropine at concentrations above 0.3125 g/L could induce abnormal morphology and viability decline in a dose- and time-dependent manner in vitro. The cytotoxicity of atropine was proven by the induced density decrease and abnormality of morphology and ultrastructure of CCE cells in vivo. Meanwhile, atropine could also induce dose- and time-dependent elevation of plasma membrane permeability, G1 phase arrest, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation of HCE cells. Moreover, 2.5 g/L atropine could also induce caspase-2/-3/-9 activation, mitochondrial transmembrane potential disruption, downregulation of anti-apoptotic Bcl-2 and Bcl-xL, upregulation of pro-apoptotic Bax and Bad, and upregulation of cytoplasmic cytochrome c and apoptosis-inducing factor. In conclusion, atropine above 1/128 of its clinical therapeutic dosage has a dose- and time-dependent cytotoxicity to HCE cells in vitro which is confirmed by CCE cells in vivo, and its cytotoxicity is achieved by inducing HCE cell apoptosis via a death receptor-mediated mitochondrion-dependent signaling pathway. Our findings provide new insights into the cytotoxicity and apoptosis-inducing effect of atropine which should be used with great caution in eye clinic.

scholarly journals Tau accumulation induces synaptic impairment and memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling

2016 ◽  
Vol 113 (26) ◽  
pp. E3773-E3781 ◽  
Author(s):  
Yaling Yin ◽  
Di Gao ◽  
Yali Wang ◽  
Zhi-Hao Wang ◽  
Xin Wang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Camp Response Element Binding ◽  
Memory Deficit ◽  
Nuclear Fraction ◽  
Wild Type ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

Intracellular accumulation of wild-type tau is a hallmark of sporadic Alzheimer’s disease (AD), but the molecular mechanisms underlying tau-induced synapse impairment and memory deficit are poorly understood. Here we found that overexpression of human wild-type full-length tau (termed hTau) induced memory deficits with impairments of synaptic plasticity. Both in vivo and in vitro data demonstrated that hTau accumulation caused remarkable dephosphorylation of cAMP response element binding protein (CREB) in the nuclear fraction. Simultaneously, the calcium-dependent protein phosphatase calcineurin (CaN) was up-regulated, whereas the calcium/calmodulin-dependent protein kinase IV (CaMKIV) was suppressed. Further studies revealed that CaN activation could dephosphorylate CREB and CaMKIV, and the effect of CaN on CREB dephosphorylation was independent of CaMKIV inhibition. Finally, inhibition of CaN attenuated the hTau-induced CREB dephosphorylation with improved synapse and memory functions. Together, these data indicate that the hTau accumulation impairs synapse and memory by CaN-mediated suppression of nuclear CaMKIV/CREB signaling. Our findings not only reveal new mechanisms underlying the hTau-induced synaptic toxicity, but also provide potential targets for rescuing tauopathies.

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