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Although stimulation of hepatic cells with interleukin-6 induces the expression of fibrinogen, the molecular basis for this regulation remains largely uncharacterized. A recent examination of the A alpha fibrinogen gene promoter identified a protein, termed the A alpha-core protein, that bound constitutively to the IL-6 response element [Liu, Z. & Fuller, G. M. (1995) J. Biol. Chem. 270, 7580-7586]. This current study provides further characterization of this regulatory protein. The data presented show the following: (i) The A alpha-core protein has a similar molecular weight and identical N-terminal sequence to that of the mitochondrial single-stranded DNA binding protein P16. (ii) The A alpha-core protein and P16 have similar characteristics in terms of DNA binding preference and antigenic properties. (iii) Overexpression of P16 gene in the hepatoma cell lines Hep G2 and Hep 3B enhances the IL-6-induced expression of A alpha fibrinogen. These results demonstrate that the A alpha-core protein is closely related to P16 and involved in the IL-6-regulated transcription of A alpha fibrinogen.

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Interleukin-6 (IL-6) and glucocorticoids are important mediators of inflammatory and immunological responses. Glucocorticoids are known to synergistically enhance IL-6-mediated cellular responses. We now show that IL-6 also has a synergistic effect upon glucocorticoid signaling. In particular, IL-6-activated STAT3 associates with ligand-bound glucocorticoid receptor to form a transactivating/signaling complex, which can function through either an IL-6-responsive element or a glucocorticoid-responsive element. These findings reveal a new level of interaction between these two crucial signaling cascades and indicate that activated STAT3 can also act as a transcriptional co-activator without direct association with its DNA binding motif.

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Fibrinogen, a hepatically derived class II acute phase protein, is the product of three separate genes, (A alpha, B beta, and gamma). The fibrinogen genes are expressed constitutively; however, their transcription can be significantly up-regulated by interleukin-6 (IL-6) and glucocorticoid. Inspection of the promoter region of the fibrinogen gamma gene revealed three hexanucleotide clusters of CTGGGA that are recognized as class II IL-6 responsive elements. Functional analyses of these regions (designated here as site I, site II, and site III according to their position in the promoter) were performed using luciferase reporter constructs and show a hierarchy of IL-6 response in which site II was the preferred functional site, site I was the next important site, and site III was the site least responsive to IL-6. Gel mobility shift assays using 25-base pair oligonucleotide probes derived from these three regions with the CTGGGA positioned in the middle and nuclear extracts from IL-6-treated primary hepatocytes reveal the presence of IL-6-induced high molecular weight complexes appearing 5 min after cytokine treatment. Supershift assays using anti-Stat3 antibody indicate that Stat3 is part of the IL-6-induced complex formed on the three gamma chain probes. The binding of Stat3 to the IL-6 responsive elements of the gamma probes is significantly weaker than to an alpha 2-macroglobulin probe. These findings show for the first time that Stat3 is involved in associating with the IL-6 responsive elements of fibrinogen gamma chain, a class II acute phase gene other than alpha 2-macroglobulin.

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Interleukin-6 (IL-6) not only regulates a variety of immune functions, but also is the most potent cytokine in inducing the hepatic acute phase proteins. We determined the effect of IL-6 on serum lipid levels and the mechanism of IL-6-induced hypertriglyceridemia in rats. Intravenous administration of IL-6 (0.1-10 micrograms/200 g BW) increased serum triglyceride levels in a dose-dependent manner. One hour after IL-6 administration, serum triglyceride levels were increased, with peak values at 2 h (2.2-fold increase). Serum cholesterol levels also increased, but the effect was delayed, first occurring at 4 h and peaking at 8 h (1.24-fold increase). IL-6 treatment increased hepatic triglyceride secretion without decreasing the clearance of triglyceride-rich lipoproteins, indicating that the hypertriglyceridemia was due to increased secretion by the liver. Furthermore, IL-6 stimulates lipolysis, and the increased delivery of FFA to the liver significantly contributed to the IL-6-induced hypertriglyceridemia. Neither alpha 1- nor beta-adrenergic receptor antagonists affected the hypertriglyceridemia induced by IL-6, whereas previous studies have shown that endotoxin-induced hypertriglyceridemia was blocked by alpha-adrenergic receptor antagonists. These results demonstrate that IL-6 induces hypertriglyceridemia by stimulating hepatic triglyceride secretion independent of endogenous catecholamines. Thus, changes in hepatic triglyceride metabolism are another acute phase response that can be induced by IL-6.

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Treatment of primary rat hepatocytes with ciliary neurotrophic factor (CNTF) resulted in increased fibrinogen mRNA levels and protein expression in a time- and dose- dependent manner. The stimulation was similar to but not as potent as the response observed with interleukin-6 (IL-6). Equilibrium binding studies using radioiodinated CNTF revealed approximately 1,900 binding sites per cell with a binding affinity of 1.6-2.0 nM, indicating the presence of specific CNTF receptors on the hepatocyte surface. Binding of CNTF to the hepatocyte surface could be competed by IL-6. Additionally, IL-6 binding was also competed by CNTF, although not entirely. These findings suggest that the CNTF-induced stimulation of fibrinogen gene expression occurs, at least in part, by this cytokine's interaction with the IL-6 receptor.

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Interleukin-6 (IL-6) is a multifunctional cytokine that exerts its effects on different target cells by interacting with a specific receptor. This interaction leads to the association and activation of a second membrane glycoprotein, gp130, which is the IL-6 signal transducing molecule. The nucleotide sequence of gp130 from a human B-cell line has been reported. We report here the cloning and sequence analysis of the gp130 molecule derived from rat liver. Comparison of gp130 molecules from the different species and cell types reveals 78% overall amino acid homology and 94% identity in the growth factor signaling domain. Two gp130 mRNA species, a moderately abundant species of 7.5 kb and a lesser one of 9.0 kb, were present in rat hepatocytes. Ribonuclease protection analyses demonstrated the presence of gp130 mRNA in four different nontransformed cell types: hepatocytes, astrocytes, fibroblasts, and endothelial cells. The sequences between both gp130s in the different cell types are quite similar, supporting the prediction that the different responses initiated by IL-6 on different target cells are modulated by cell-specific proteins distal to the activated gp130 molecule.

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