Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy

2001 ◽  
Vol 281 (6) ◽  
pp. R2048-R2058 ◽  
Author(s):  
Abram M. Madiehe ◽  
Ling Lin ◽  
Christy White ◽  
H. Doug Braymer ◽  
George A. Bray ◽  
...  
Keyword(s):  
Dna Binding ◽  
Signaling Pathway ◽  
Leptin Receptor ◽  
Binding Activity ◽  
Janus Kinase ◽  
Adrenal Steroids ◽  
Western Blots ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Stat Signaling

Removal of adrenal steroids by adrenalectomy (ADX) slows or reverses the development of many forms of obesity in rodents, including those that are leptin or leptin receptor deficient. Obesity is associated with hyperleptinemia and leptin resistance. We hypothesized that glucocorticoids impair leptin receptor signaling and that removal thereof would activate the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway. The inhibitory effect of leptin (2.5 μg icv) on food intake was enhanced in ADX rats. A combination of ribonuclease protection assays, RT-PCR, Western blots, and mobility shift assays was used to evaluate the leptin signaling pathway in whole hypothalami from sham-operated, ADX and corticosterone-replaced ADX (ADX-R) Sprague-Dawley rats that were treated acutely with either saline vehicle or leptin intracerebroventricularly. ADX increased the expression of leptin receptor mRNA, increased STAT-3 mRNA and protein levels, induced constitutive STAT-3 phosphorylation and DNA binding activity, and also reduced suppressor of cytokine signaling-3 (SOCS-3) mRNA and protein levels. ADX and leptin treatment increased STAT-3 phosphorylation, but with no concomitant increase in DNA binding activity. Leptin and ADX decreased NPY mRNA expression, but their combination did not further decrease NPY mRNA. Corticosterone supplementation of ADX rats partially reversed many of these effects. In conclusion, ADX through activation of STAT-3 and inhibition of SOCS-3 activates the JAK-STAT signaling pathway. These effects most probably explain the ability to prevent the development of obesity by removal of adrenal steroids.

scholarly journals Biphasic regulation of extracellular-signal-regulated protein kinase by leptin in macrophages: role in regulating STAT3 Ser727 phosphorylation and DNA binding

2002 ◽  
Vol 364 (3) ◽  
pp. 875-879 ◽  
Author(s):  
Lisa O'ROURKE ◽  
Peter R. SHEPHERD
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Leptin Receptor ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Time Points ◽  
Dna Binding Activity ◽  
Erk Phosphorylation ◽  
Extracellular Signal ◽  
Stat3 Phosphorylation

Activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) requires dimerization that is induced by phosphorylation of Tyr705, but its activity can be further modulated by phosphorylation at Ser727 in a manner that is dependent on cell context and the stimulus used. The role of STAT3 Ser727 phosphorylation in leptin signalling is currently not known. While cells transfected with the signalling-competent long form of the leptin receptor (ObRb) have been used to study leptin signalling, these are likely to be of limited use in studying STAT3 Ser727 phosphorylation due to the importance of cell background in determining the nature of the response. However, we have recently found that J774.2 macrophages endogenously express high levels of ObRb, and using these cells we find that leptin stimulates STAT3 phosphorylation on both Tyr705 and Ser727. The phosphorylation of Ser727 was not affected by rapamycin or the protein kinase C inhibitor H7 [1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride]. While the MEK-1 [mitogen-activated protein kinase (MAP kinase)/extracellular-signal-related kinase (ERK) kinase-1] inhibitor PD98059 [(2-amino-3′-methoxyphenyl)oxanaphthalen-4-one] had no effect on leptin-stimulated phosphorylation of STAT3 Tyr705, it greatly attenuated leptin's effects on STAT3 Ser727 phosphorylation. Further, Ob's effect on the DNA binding activity of STAT3 was also greatly reduced at all time points by PD98059. Leptin-induced ERK activation in J774.2 cells shows a biphasic pattern, with an initial reduction in ERK phosphorylation for up to 10min following leptin stimulation, while at later time points phosphorylation of ERK was increased above basal levels. The increase in ERK activity corresponded with an increase in both phosphorylation of Ser727 and STAT3 DNA binding activity. These data provide the first evidence that ERK-mediated phosphorylation of Ser727 is required for full stimulation of STAT3 by leptin.

Interdiction of Src Family Kinase Activity as a Therapeutic Option for Aberrant JAK-STAT Signaling: Potential Targeted Therapy of JAK2 V617F in Myeloproliferative Disorders with Currently Available Kinase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2686-2686
Author(s):  
A.E. Schade ◽  
H. Szpurka ◽  
A. Jankowska ◽  
J. Bauer ◽  
E.D. Hsi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Binding Activity ◽  
Cytokine Receptors ◽  
Type I ◽  
Gm Csf ◽  
Dna Binding Activity ◽  
Stat Signaling ◽  
Erk Activity

Abstract Cytokine receptors preferentially associate with particular JAK-STAT combinations to transduce specific signals. For example, erythropoietin receptor (Epo-R) preferentially interacts with JAK2 to initiate signaling pathways via STAT5. The JAK2 V617F mutation (mJAK2) found in some myeloproliferative disorders (MPD) still requires binding to type I cytokine receptors to initiate signaling. Consequently, aberrant JAK-STAT signaling in MPD may require physiologic interactions with other pathways. Src family kinases (SFK) interact with various cytokine receptors resulting in close association between SFK and JAK-STAT pathways. We hypothesized that SFK activity plays a role in the activation of STAT5 and, given the importance of STAT5 in the pathogenesis of MPD, targeted inhibition of SFK could provide a novel therapeutic approach. First, we examined the effect the SFK inhibitors PP2 and SU6656 on the proliferation of the HEL cell line harboring mJAK2 and the Epo-dependent AML line UT7/Epo; SFK inhibition significantly diminished proliferation in both cell lines. These results imply that despite of the constitutive activity of mJAK2 or in the presence of Epo stimulated JAK2-STAT5 induction, SFK activation is required for proliferation. Since mJAK2 requires a functionally intact type I cytokine receptor, we examined Epo-R signaling in greater detail via phospho-specific immunoblotting. SFK inhibition resulted in diminished levels of phospho-SFK, coinciding with a similar degree of diminished phospho-STAT5. Simultaneously decreased induction of AKT and ERK pathways after SFK inhibition suggested SFK activity is also regulating a more global signaling network through the Epo-R. Inhibition of JAK2 activity potently suppressed phospho-STAT5, as well as ERK and AKT, without affecting SFK phosphorylation. Thus, SFK lies upstream of JAK2, or SFK and JAK2 may be regulating the second messenger pathways in parallel. In electrophoretic mobility shift assays to examine the effect of SFK inhibition on STAT5 DNA binding, SFK inhibition resulted in decreased STAT5 DNA binding despite constitutive activity of mJAK2. Analogous results were obtained after Epo stimulation in UT7/Epo cells. As expected, inhibition of JAK2 resulted in almost complete loss of STAT5 DNA binding. To confirm these results in primary cells, we examined the effects of SFK inhibition on primary monocytes from a patient with mJAK2. Stimulation with GM-CSF resulted in increased STAT5A DNA binding, but not STAT5B. In the presence of the SFK inhibitor PP2, GM-CSF induction of STAT5A DNA binding activity was completely inhibited. It is interesting to note that a key difference between STAT5A and STAT5B is the potential for ERK regulation of STAT5A DNA binding activity. Thus, showing here that SFK regulates ERK activity, and knowing that ERK activity can positively regulate STAT5A DNA binding, we propose a model in which SFK activity may modulate the JAK2-STAT5 signaling axis via the ERK pathway. In summary, our results demonstrate that while JAK2 is essential for this process, SFK activity appears to be necessary for full activation by positively modulating the JAK2-STAT5 axis. SFK inhibitors recently approved and in clinical trials may demonstrate efficacy in hematologic diseases characterized by aberrant JAK-STAT signaling, such as MPD.

Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation

1992 ◽  
Vol 12 (10) ◽  
pp. 4742-4750
Author(s):  
J Trejo ◽  
J C Chambard ◽  
M Karin ◽  
J H Brown
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Time Course ◽  
Firefly Luciferase ◽  
Receptor Activation ◽  
Binding Activity ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Firefly Luciferase Gene ◽  
The Difference

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

scholarly journals A novel STAT-like factor mediates lipopolysaccharide, interleukin 1 (IL-1), and IL-6 signaling and recognizes a gamma interferon activation site-like element in the IL1B gene.

1996 ◽  
Vol 16 (5) ◽  
pp. 2183-2194 ◽  
Author(s):  
J Tsukada ◽  
W R Waterman ◽  
Y Koyama ◽  
A C Webb ◽  
P E Auron
Keyword(s):  
Dna Binding ◽  
Signaling Pathway ◽  
Tyrosine Kinases ◽  
Interleukin 1 ◽  
Binding Specificity ◽  
Binding Activity ◽  
Gamma Interferon ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Activation Site

Binding of many cytokines to their cognate receptors immediately activates Jak tyrosine kinases and their substrates, STAT (signal transducers and activators of transcription) DNA-binding proteins. The DNA binding targets of STATs are sequence elements related to the archetypal gamma interferon activation site, GAS. However, association of interleukin 1 (IL-1) with Jak-STAT signaling has remained unresolved. We now report an element termed LILRE (lipopolysaccharide [LPS] and IL-1-responsive element) in the human prointerleukin 1beta gene (IL1B) which can be immediately induced by either lipopolysaccharide (LPS) or IL-1 protein to bind a tyrosine-phosphorylated protein. This LPS- and IL-1-induced factor (LIL factor) is recognized by an antibody raised against the N terminus of Stat1, but not by those specific for either the C terminus of Stat1 or any other GAS-binding STAT. Phosphotyrosine (P-Tyr) specifically inhibits formation of the LIL factor-DNA complex, suggesting the importance of P-Tyr for the DNA-binding activity, as has been found for all STAT dimers. Analysis of DNA-binding specificity demonstrates that the LIL factor possesses a novel GAS-like binding activity that contrasts with those of other STATs in a requirement for a G residue at position 8 (TTCCTGAGA). Further investigation has revealed that IL-6, but neither IL-4 nor gamma interferon, activates the LIL factor. Thus, the existence of such a STAT-like factor (LIL-Stat) relates the LPS and IL-1 signaling pathway to other cytokine receptor signaling pathways via the activation of STATs. Moreover, the unique DNA-binding specificity and antigenicity of this factor suggest that LPS, IL-1, and IL-6 may use a common signaling pathway.

Estradiol upregulates mesangial cell MMP-2 activity via the transcription factor AP-2

2002 ◽  
Vol 282 (1) ◽  
pp. F164-F169 ◽  
Author(s):  
Michael Guccione ◽  
Sharon Silbiger ◽  
Jun Lei ◽  
Joel Neugarten
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Protein Expression ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Binding Activity ◽  
Dependent Manner ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Metalloproteinase Activity

The accumulation of extracellular matrix in the glomerular mesangium reflects the net balance between the synthesis and degradation of matrix components. We have shown that estradiol suppresses the synthesis of types I and IV collagen by cultured mesangial cells (Kwan G, Neugarten J, Sherman M, Ding Q, Fotadar U, Lei J, and Silbiger S. Kidney Int 50: 1173–1179, 1996; Neugarten J, Acharya A, Lei J, and Silbiger S. Am J Physiol Renal Physiol 279: F309–F318, 2000; Neugarten J, Medve I, Lei J, and Silbiger SR. Am J Physiol Renal Physiol 277: F1–F8, 1999; Neugarten J and Silbiger S. Am J Kidney Dis 26: 147–151, 1995; Silbiger S, Lei J, and Neugarten J. Kidney Int 55: 1268–1276, 1998; Silbiger S, Lei J, Ziyadeh FN, and Neugarten J. Am J Physiol Renal Physiol 274: F1113–F1118, 1998). In the present study, we evaluated the effects of sex hormones on the activity of matrix metalloproteinase-2 (MMP-2) in murine mesangial cells, the synthesis of which is regulated by the transcription factor activator protein-2 (AP-2). Estradiol stimulated MMP-2 activity by increasing MMP-2 protein levels in a dose-dependent manner. These effects occurred at physiological concentrations of estradiol and were receptor mediated. Estradiol also increased AP-2 protein levels and increased binding of mesangial cell nuclear extracts to an AP-2 consensus binding sequence oligonucleotide. The ability of estradiol to increase AP-2 protein expression, AP-2/DNA binding activity, MMP-2 protein expression, and metalloproteinase activity was reversed by PD-98059, a selective inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade. We conclude that estradiol upregulates the MAPK cascade, which in turn stimulates the synthesis of AP-2 protein. The resultant increased AP-2/DNA binding activity leads to increased synthesis of MMP-2 and increased metalloproteinase activity. Stimulation of metalloproteinase activity by estradiol may contribute to the protective effect of female gender on renal disease progression.

Creatine feeding increases GLUT4 expression in rat skeletal muscle

2005 ◽  
Vol 288 (2) ◽  
pp. E347-E352 ◽  
Author(s):  
Jeong-Sun Ju ◽  
Jill L. Smith ◽  
Peter J. Oppelt ◽  
Jonathan S. Fisher
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Dna Binding ◽  
Binding Activity ◽  
Rat Skeletal Muscle ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Glut4 Expression ◽  
Edl Muscle ◽  
Glut4 Gene Expression

The purpose of this study was to investigate the potential role of creatine in GLUT4 gene expression in rat skeletal muscle. Female Wistar rats were fed normal rat chow (controls) or chow containing 2% creatine monohydrate ad libitum for 3 wk. GLUT4 protein levels of creatine-fed rats were significantly increased in extensor digitorum longus (EDL), triceps, and epitrochlearis muscles compared with muscles from controls ( P < 0.05), and triceps GLUT4 mRNA levels were ∼100% greater in triceps muscles from creatine-fed rats than in muscles from controls ( P < 0.05). In epitrochlearis muscles from creatine-fed animals, glycogen content was ∼40% greater ( P < 0.05), and insulin-stimulated glucose transport rates were higher ( P < 0.05) than in epitrochlearis muscles from controls. Despite no changes in [ATP], [creatine], [phosphocreatine], or [AMP], creatine feeding increased AMP-activated protein kinase (AMPK) phosphorylation by 50% in rat EDL muscle ( P < 0.05). Creatinine content of EDL muscle was almost twofold higher for creatine-fed animals than for controls ( P < 0.05). Creatine feeding increased protein levels of myocyte enhancer factor 2 (MEF2) isoforms MEF2A (∼70%, P < 0.05), MEF2C (∼60%, P < 0.05), and MEF2D (∼90%, P < 0.05), which are transcription factors that regulate GLUT4 expression, in creatine-fed rat EDL muscle nuclear extracts. Electrophoretic mobility shift assay showed that DNA binding activity of MEF2 was increased by ∼40% ( P < 0.05) in creatine-fed rat EDL compared with controls. Our data suggest that creatine feeding enhances the nuclear content and DNA binding activity of MEF2 isoforms, which is concomitant with an increase in GLUT4 gene expression.

scholarly journals Effect of dietary protein restriction on liver transcription factors

1996 ◽  
Vol 317 (2) ◽  
pp. 361-370 ◽  
Author(s):  
Norman W. MARTEN ◽  
Frances M. SLADEK ◽  
Daniel S. STRAUS
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dietary Protein ◽  
Function Analysis ◽  
Binding Activity ◽  
Protein Restriction ◽  
Scatchard Analysis ◽  
Western Blots ◽  
Dietary Protein Restriction ◽  
Dna Binding Activity

The transcription of several genes that are preferentially expressed in the liver, including the serum albumin, transthyretin and carbamyl phosphate synthetase-I genes, is specifically decreased in animals consuming inadequate amounts of dietary protein. The high level of transcription of these genes in the liver is directed in part by a number of liver-enriched transcription factors, including hepatocyte nuclear factors (HNF)-1, -3, and -4, and proteins of the CCAAT/enhancer-binding protein (C/EBP) family. In the present study, we investigated the possibility that the co-ordinate decrease in transcription of the nutritionally sensitive genes in protein-deprived rats results from altered activity of one or more of the liver-enriched transcription factors. For HNF-4, Western blots indicated no change in the level of nuclear HNF-4 protein in liver of protein-deprived animals, whereas we observed a 40% reduction in the DNA binding activity of HNF-4 as measured by electrophoretic mobility shift assay (EMSA). Furthermore, the binding affinity of HNF-4 for DNA was unaltered by dietary protein deprivation, while the number of HNF-4 molecules able to bind to DNA (Bmax) was reduced, as determined by Scatchard analysis. This indicates that in the protein-restricted rats a portion of the pool of HNF-4 protein is inactivated or otherwise prevented from binding to DNA. The overall DNA binding activity of C/EBPα and β was increased in protein-restricted animals. This change occurred in the absence of a change in the amount of the full-length forms of these two proteins, quantified by Western blotting. Interestingly, dietary protein restriction specifically increased the level of a truncated form of C/EBPβ (liver-enriched transcriptional inhibitory protein, LIP), which is a potent dominant negative inhibitor of C/EBP function. Analysis of HNF-3 DNA-binding activity by EMSA revealed that HNF-3α and β DNA binding was increased and that HNF-3γ DNA-binding activity was unchanged in protein-restricted animals. We also detected two apparently novel shift complexes with the HNF-3 probe by EMSA, both of which were decreased in protein-restricted animals. HNF-1 DNA-binding activity was increased by dietary protein restriction. We also examined the effect of protein restriction on the DNA-binding activity of two ubiquitous transcription factors, NF1 and Sp1. The DNA binding activity of the major NF1 isoforms was unchanged whereas the binding activity of Sp1 was increased in the protein-restricted animals. In summary, restriction of dietary protein resulted in a number of specific changes in the DNA-binding activity of various transcription factors. Because transcriptional activation typically involves the synergistic action of more than one transcription factor, small changes in the amount/activity of several factors could have a strong net effect on the transcription of many genes.

scholarly journals Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation.

1992 ◽  
Vol 12 (10) ◽  
pp. 4742-4750 ◽  
Author(s):  
J Trejo ◽  
J C Chambard ◽  
M Karin ◽  
J H Brown
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Time Course ◽  
Firefly Luciferase ◽  
Receptor Activation ◽  
Binding Activity ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Firefly Luciferase Gene ◽  
The Difference

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

C/EBP DNA-binding activity is upregulated by a glucocorticoid-dependent mechanism in septic muscle

2002 ◽  
Vol 282 (2) ◽  
pp. R439-R444 ◽  
Author(s):  
Gail Penner ◽  
Gyu Gang ◽  
Xiaoyan Sun ◽  
Curtis Wray ◽  
Per-Olof Hasselgren
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factors ◽  
Dna Binding ◽  
Cecal Ligation ◽  
Binding Activity ◽  
Nuclear Fraction ◽  
Mobility Shift ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Mobility Shift Assay

Sepsis-induced muscle cachexia is associated with increased expression of several genes in the ubiquitin-proteasome proteolytic pathway, but little is known about the activation of transcription factors in skeletal muscle during sepsis. We tested the hypothesis that sepsis upregulates the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)-β and -δ in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture, and control rats were sham operated. C/EBP-β and -δ DNA-binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In addition, C/EBP-β and -δ nuclear protein levels were determined by Western blot analysis. Sepsis resulted in increased DNA-binding activity of C/EBP, and supershift analysis suggested that this reflected activation of the β- and δ-isoforms of C/EBP. Concomitantly, C/EBP-β and -δ protein levels were increased in the nuclear fraction of skeletal muscle. In additional experiments, we tested the role of glucocorticoids in sepsis-induced activation of C/EBP-β and -δ by treating rats with the glucocorticoid receptor antagonist RU-38486. This treatment inhibited the sepsis-induced activation of C/EBP-β and -δ, suggesting that glucocorticoids participate in the upregulation of C/EBP in skeletal muscle during sepsis. The present results suggest that C/EBP-β and -δ are activated in skeletal muscle during sepsis and that this response is, at least in part, regulated by glucocorticoids.

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