IL-1β stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-κB

2003 ◽  
Vol 284 (5) ◽  
pp. R1249-R1254 ◽  
Author(s):  
Guangjo Luo ◽  
Dan D. Hershko ◽  
Bruce W. Robb ◽  
Curtis J. Wray ◽  
Per-Olof Hasselgren
Keyword(s):  
Skeletal Muscle ◽  
Dna Binding ◽  
Map Kinase ◽  
Muscle Cells ◽  
Binding Activity ◽  
C2c12 Cells ◽  
Luciferase Reporter ◽  
Kinase Signaling ◽  
Dna Binding Activity ◽  
Map Kinase Signaling

Recent studies suggest that the skeletal muscle may be a significant site of IL-6 production in various conditions, including exercise, inflammation, hypoperfusion, denervation, and local muscle injury. The mediators and molecular mechanisms regulating muscle IL-6 production are poorly understood. We tested the hypothesis that IL-6 production in muscle cells is regulated by IL-1β and that mitogen-activated protein (MAP) kinase signaling and NF-κB activation are involved in IL-1β-induced IL-6 production. Cultured C2C12 cells, a mouse skeletal muscle cell line, were treated with different concentrations (0.1–2 ng/ml) of IL-1β in the absence or presence of the p38 MAP kinase inhibitor SB-208350 or the p42/44 inhibitor PD-98059. Protein and gene expression of IL-6 were determined by ELISA and real-time PCR, respectively. NF-κB DNA binding activity was determined by electrophoretic mobility shift assay and by transfecting myocytes with a luciferase reporter plasmid containing a promoter construct with multiple repeats of NF-κB binding site. Treatment of myotubes with IL-1β resulted in a dose- and time-dependent increase of IL-6 production accompanied by an ∼25-fold increase in IL-6 mRNA levels. IL-1β stimulated NF-κB DNA binding activity and gene activation. SB-208350 and PD-98059 inhibited the increase in IL-6 production induced by IL-1β. The present results support the concept that skeletal muscle is an important site of IL-6 production. In addition, the results suggest the IL-1β regulates muscle IL-6 production at least in part by activating the MAP kinase pathway and NF-κB.

scholarly journals Dual-specificity phosphatase 4 is upregulated during skeletal muscle atrophy and modulates extracellular signal-regulated kinase activity

2019 ◽  
Vol 316 (4) ◽  
pp. C567-C581 ◽  
Author(s):  
Ashley N. Haddock ◽  
Sydney A. Labuzan ◽  
Amy E. Haynes ◽  
Caleb S. Hayes ◽  
Karina M. Kakareka ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Differentiation ◽  
Map Kinase ◽  
Reporter Gene ◽  
Muscle Cell ◽  
Muscle Cells ◽  
Skeletal Muscle Atrophy ◽  
Kinase Signaling ◽  
Muscle Cell Differentiation ◽  
Map Kinase Signaling

Skeletal muscle atrophy results from disparate physiological conditions, including denervation, corticosteroid treatment, and aging. The purpose of this study was to describe and characterize the function of dual-specificity phosphatase 4 (Dusp4) in skeletal muscle after it was found to be induced in response to neurogenic atrophy. Quantitative PCR and Western blot analysis revealed that Dusp4 is expressed during myoblast proliferation but rapidly disappears as muscle cells differentiate. The Dusp4 regulatory region was cloned and found to contain a conserved E-box element that negatively regulates Dusp4 reporter gene activity in response to myogenic regulatory factor expression. In addition, the proximal 3′-untranslated region of Dusp4 acts in an inhibitory manner to repress reporter gene activity as muscle cells progress through the differentiation process. To determine potential function, Dusp4 was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize to the nucleus of proliferating myoblasts. Furthermore, Dusp4 overexpression delayed C2C12 muscle cell differentiation and resulted in repression of a MAP kinase signaling pathway reporter gene. Ectopic expression of a Dusp4 dominant negative mutant blocked muscle cell differentiation and attenuated MAP kinase signaling by preferentially targeting the ERK1/2 branch, but not the p38 branch, of the MAP kinase signaling cascade in skeletal muscle cells. The findings presented in this study provide the first description of Dusp4 in skeletal muscle and suggest that Dusp4 may play an important role in the regulation of muscle cell differentiation by regulating MAP kinase signaling.

scholarly journals Overexpression of mitogen-activated protein kinase (ERK1) enhances T- cell cytokine gene expression: role of AP1, NF-AT, and NF-KB

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2470-2477 ◽  
Author(s):  
JH Park ◽  
L Levitt
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Dna Binding ◽  
Map Kinase ◽  
Binding Activity ◽  
Cytokine Gene Expression ◽  
Cytokine Gene ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

Abstract Transfected Jurkat cells overexpressing extracellular signal-regulated kinase (ERK1), also referred to as mitogen-activated protein (MAP) kinase, were selected by Western blotting assay using anti-ERK1 and antiphosphotyrosine antibodies in combination with a functional MAP kinase assay. We then asked whether enhanced ERK1 expression had any effect on induction of T-cell cytokine genes. The results show that overexpression of ERK1 enhances expression of T-cell interleukin-2 (IL- 2), IL-3, and granulocyte-macrophage colony-stimulating factor mRNA; no change was seen in expression of the alpha-actin gene. DNA-binding activities of the transcription factors AP1, NF-AT, and NF-kB were specifically increased twofold to fourfold in ERK1-overexpressing clones relative to nontransformed or vector-transformed cells, whereas no enhancement of CK1-CK2 protein DNA binding activity was detected after ERK1 overexpression. Additionally, increased NF-AT DNA binding activity was associated with functional enhancement of NF-AT transactivating activity in ERK1-overexpressing cells. These results provide direct evidence for the role of MAP kinase in the regulation of cytokine gene expression and indicate that such regulation is likely mediated through the enhanced DNA binding activity of specific nuclear transcription factors.

Treatment of cultured myotubes with the proteasome inhibitor β-lactone increases the expression of the transcription factor C/EBPβ

2007 ◽  
Vol 292 (1) ◽  
pp. C216-C226 ◽  
Author(s):  
Wei Wei ◽  
Hongmei Yang ◽  
Michael Menconi ◽  
Peirang Cao ◽  
Chester E. Chamberlain ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Proteasome Inhibitor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dna Binding Activity ◽  
Cultured Myotubes ◽  
Nuclear Levels ◽  
Factor C

The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is poorly understood. We tested the hypothesis that C/EBPβ levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor β-lactone resulted in increased nuclear levels of C/EBPβ as determined by Western blotting and immunofluorescent detection. This effect of β-lactone reflected inhibited degradation of C/EBPβ. Surprisingly, the increased C/EBPβ levels in β-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with β-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPβ and Gadd153/CHOP interacted and colocalized in the nuclei of the β-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPβ DNA-binding activity was restored in β-lactone-treated myotubes. The results suggest that C/EBPβ is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPβ and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPβ in skeletal muscle.

scholarly journals Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells: evidence for Erk-1/2 activation

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 692-699 ◽  
Author(s):  
Toshiya Nishibe ◽  
Graham Parry ◽  
Atsushi Ishida ◽  
Salim Aziz ◽  
Jacqueline Murray ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Smooth Muscle Cells ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Muscle Cells ◽  
Binding Activity ◽  
Oncostatin M ◽  
Dna Binding Activity ◽  
Atherosclerotic Lesions

Abstract Tissue factor (TF), a transmembrane glycoprotein, initiates the extrinsic coagulation cascade. TF is known to play a major role in mediating thrombosis and thrombotic episodes associated with the progression of atherosclerosis. Macrophages at inflammatory sites, such as atherosclerotic lesions, release numerous cytokines that are capable of modulating TF expression. This study examined the role of oncostatin M (OSM), a macrophage/ T-lymphocyte–restricted cytokine, in the expression of TF in vascular smooth muscle cells (SMCs). It is reported here that OSM stimulated a biphasic and sustained pattern of TF messenger RNA (mRNA). The effect of OSM on TF mRNA expression was regulated at the transcriptional level as determined by nuclear run-offs and transient transfection of a TF promoter-reporter gene construct. OSM-induced TF expression was regulated primarily by the transcription factor NF-κB. Activation of NF-κB by OSM did not require IκB-α degradation. Inhibition of MEK activity by U0126 prevented OSM-induced TF expression by suppressing NF-κB DNA binding activity as determined by gel-shift analysis. Further, inhibition of Erk-1/2 protein by antisense treatment resulted in suppression of TF mRNA expression, indicating a role for Erk-1/2 in modulating NF-κB DNA binding activity. These studies suggest that the induced expression of TF by OSM is primarily through the activation of NF-κB and that activation of NF-κB is regulated in part by the MEK/Erk-1/2 signal transduction pathway. This study indicates that OSM may play a key role in promoting TF expression in SMCs within atherosclerotic lesions.

Single-Stranded DNA-Binding Proteins (SSBPs) Promote the Oligomerization of LIM-Domain Binding Protein Ldb1

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2437-2437
Author(s):  
Ying Cai ◽  
Lalitha Nagarajan ◽  
Stephen J. Brandt
Keyword(s):  
Dna Binding ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Lim Domain ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
E Box

Abstract The multifunctional LIM domain-binding protein Ldb1 is important in multiple developmental programs, including hematopoiesis. An evolutionarily conserved family of proteins with single-stranded DNA-binding activity, the SSBPs, has been shown to act as Ldb1 partners and augment its biological actions. We recently established that Ssbp2 and Ssbp3 were components of an E-box-GATA DNA-binding complex in murine erythroid progenitors containing the LIM-only protein Lmo2 and transcription factors Tal1, E2A, and Gata1 and showed these SSBPs stimulated E box-GATA DNA-binding activity and inhibited Ldb1 ubiquitination and subsequent proteasomal degradation (Genes & Dev.21:942–955, 2007). As its SSBP interaction domain (Ldb1/Chip conserved domain or LCCD) is adjacent to Ldb1’s N-terminal dimerization domain (DD), we sought to determine whether SSBP binding affected Ldb1 dimerization. To investigate, the Ldb1 coding region was fused to the DNA-binding domain of the yeast transcription factor GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD). These fusion proteins were then expressed in mammalian cells with a luciferase reporter linked to a promoter with iterated GAL4 binding sites. Luciferase activity became detectable with coexpression of the VP16AD-Ldb1 and GAL4DBD-Ldb1 fusions, presumably from Ldb1 dimerization, which increased markedly with simultaneous expression of SSBP2. In contrast, SSBP2 (ΔLUFS) and Ldb1 (ΔLCCD) mutants incapable of interacting with Ldb1 and SSBPs, respectively, were inactive, suggesting that SSBP2 augmentation of Ldb1 dimerization involved direct protein-protein interactions. To exclude an effect of SSBP2 on turnover of Ldb1 fusion proteins, radiolabeled full-length Ldb1 and SSBP3 were prepared by in vitro transcription/translation, mixed, and subjected to chemical crosslinking. Addition of the crosslinker bis(sulfosuccinimidyl)-suberate (BS3) to Ldb1, but not SSBP3, led to the appearance of a radiolabeled protein with mobility in denaturing polyacrylamide gels approximately twice that of Ldb1, consistent with an Ldb1 homodimer. When SSBP3 and Ldb1 were mixed together and crosslinked, a dose-related increase was noted in a more retarded species predicted to contain two molecules each of Ldb1 and SSBP3, together with a decrease in monomeric Ldb1. Finally, two well-characterized dimerization-defective Ldb1 mutants, Ldb1(200–375) and Ldb1(50–375), failed to support the formation of the higher molecular weight species or to homodimerize. Thus, the SSBPs promoted assembly of ternary complexes incorporating both SSBP and Ldb1 in a manner dependent on Ldb1 dimerization. The failure to observe Ldb1-SSBP heterodimers in cross-linking experiments suggests, further, that the SSBPs interacted with preformed Ldb1 dimers. In summary, either through an allosteric effect on Ldb1’s DD or by altering the equilibrium between monomeric and dimeric species, the SSBPs promote Ldb1 oligomerization. Together with inhibition of Ldb1 ubiquitination and turnover, this would serve to augment Ldb1 function.

Analysis Of E2A Point Mutations In B Cell Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4850-4850
Author(s):  
Eroica Soans ◽  
John K Choi
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Cell Leukemia ◽  
Wild Type ◽  
Dna Binding Activity ◽  
E Box ◽  
B Cell Leukemia

Abstract Introduction TCF3 encodes for E2A protein, which belongs to the helix loop helix transcription factor family. E2A activates transcription of downstream genes by binding to E-box motifs as a homo or hetero dimer. E2A plays an important role in B lymphocyte development. Therefore deletion or mutations in TCF3 or even lowered activity of E2A are causes of B cell leukemia and lymphomas. Recently, three mutations V557E, D561E and N551K in E2A were isolated in Burkitt’s lymphoma (Schmitz, Young et al. 2012). The first two mutations are present in the homo dimerization region of E2A while N551K is present in the DNA binding region. Though the paper enumerated role of TCF3 in Burkitt’s lymphoma but the significance of these TCF3 mutations or mechanism needed further characterization. We hypothesized that these TCF3 mutations have an alternate mechanism as compared to wild type TCF3 and therefore may affect B cell development. Methods We characterized three TCF3 mutants by cloning them into in MIGR1 backbone using TOPO cloning. E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. DNA binding activity was measured using a DNA protein binding colorimetric assay. Results V557E and D561E mutants have lower activity as compared to wild type E2A as studied using E2A-specific luciferase reporter assay; while N551K showed no activity in the same assay as compared to wild type E2A activity. Similarly V557E and D561 form weaker bonds with the E box motifs while N551K showed no DNA binding activity as studied using colorimetric DNA-protein binding assay. The plasmid expressions were verified using western blot analysis. Conclusion Our findings suggest mutations V557E and D561E may follow a similar pathway as wild-type E2A but have lower activity. The N551K mutation has an alternate pathway to wild type TCF3 that may impact B cell proliferation, survival and development. Disclosures: No relevant conflicts of interest to declare.

Mechanical strain regulates syndecan-4 expression and shedding in smooth muscle cells through differential activation of MAP kinase signaling pathways

2007 ◽  
Vol 292 (1) ◽  
pp. C517-C525 ◽  
Author(s):  
Matheau A. Julien ◽  
Peiyi Wang ◽  
Carolyn A. Haller ◽  
Jing Wen ◽  
Elliot L. Chaikof
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Map Kinases ◽  
Muscle Cells ◽  
Kinase Signaling ◽  
Fold Reduction ◽  
Map Kinase Signaling

Syndecan-4 (S4) belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding as well as cell-matrix and cell-cell interactions, and is induced in neointimal smooth muscle cells (SMCs) after balloon catheter injury. We investigated S4 expression in SMCs in response to several force profiles and the role of MAP kinase signaling pathways in regulating these responses. S4 mRNA expression increased in response to 5% and 10% cyclic strain (4 h: 200 ± 34% and 182 ± 17%, respectively; P < 0.05) before returning to basal levels by 24 h. Notably, the SMC mechanosensor mechanism was reset after an initial 24-h “preconditioning” period, as evident by an increase in S4 gene expression following a change in cyclic stress from 10% to 20% (28 h: 181 ± 1%; P < 0.05). Mechanical stress induced a late decrease in cell-associated S4 protein levels (24 h: 70 ± 6%; P < 0.05), with an associated increase in S4 shedding (24 h: 537 ± 109%; P < 0.05). To examine the role of MAP kinases, cells were treated with U-0126 (ERK1/2 inhibitor), SB-203580 (p38 inhibitor), or JNKI I (JNK/SAPK inhibitor). Late reduction in cell-associated S4 levels was attributed to ERK1/2 and p38 signaling. In contrast, accelerated S4 shedding required both ERK1/2 (5-fold reduction in accelerated shedding; P < 0.05) and JNK/SAPK (4-fold reduction; P < 0.05) signaling. Given the varied functions of S4, stress-induced effects on SMC S4 expression and shedding may represent an additional component of the proinflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall.

scholarly journals Acute resistance exercise activates MAP kinase signaling in skeletal muscle of overweight women

2007 ◽  
Vol 21 (6) ◽  
Author(s):  
Justin Crane ◽  
Matthew Douglass ◽  
Todd Trappe ◽  
Scott Trappe ◽  
Matthew Harber
Keyword(s):  
Skeletal Muscle ◽  
Resistance Exercise ◽  
Map Kinase ◽  
Kinase Signaling ◽  
Overweight Women ◽  
Map Kinase Signaling

Extracellular-regulated protein kinase cascades are activated in response to injury in human skeletal muscle

1998 ◽  
Vol 275 (2) ◽  
pp. C555-C561 ◽  
Author(s):  
Doron Aronson ◽  
Jørgen F. P. Wojtaszewski ◽  
Anders Thorell ◽  
Jonas Nygren ◽  
David Zangen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Map Kinase ◽  
Muscle Injury ◽  
Human Skeletal Muscle ◽  
Signaling Cascades ◽  
Vastus Lateralis Muscle ◽  
Kinase Signaling ◽  
Transcriptional Responses ◽  
Map Kinase Signaling ◽  
Stimulation Of

The mitogen-activated protein (MAP) kinase signaling pathways are believed to act as critical signal transducers between stress stimuli and transcriptional responses in mammalian cells. However, it is not known whether these signaling cascades also participate in the response to injury in human tissues. To determine whether injury to the vastus lateralis muscle activates MAP kinase signaling in human subjects, two needle biopsies or open muscle biopsies were taken from the same incision site 30–60 min apart. The muscle biopsy procedures resulted in striking increases in dual phosphorylation of the extracellular-regulated kinases (ERK1 and ERK2) and in activity of the downstream substrate, the p90 ribosomal S6 kinase. Raf-1 kinase and MAP kinase kinase, upstream activators of ERK, were also markedly stimulated in all subjects. In addition, c-Jun NH2-terminal kinase and p38 kinase, components of two parallel MAP kinase pathways, were activated following muscle injury. The stimulation of the three MAP kinase cascades was present only in the immediate vicinity of the injury, a finding consistent with a local rather than systemic activation of these signaling cascades in response to injury. These data demonstrate that muscle injury induces the stimulation of the three MAP kinase cascades in human skeletal muscle, suggesting a physiological relevance of these protein kinases in the immediate response to tissue injury and possibly in the initiation of wound healing.

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