DNA Binding Activity of C/EBPbeta and C/EBPdelta for the Rat alpha₂-Macroglobulin Gene Promoter Is Regulated in an Acute-Phase Dependent Manner

T. S. Milosavljevic^*, M. V. Petrovic, I. D. Cvetkovic, and I. I. Grigorov

Department for Molecular Biology, Institute for Biological Research “Sinisa Stankovic”, 29th November st. 142, Belgrade 11060, Serbia, Federal Republic of Yugoslavia; fax: (381) 11-761-433; E-mail: pemi@ibiss.bg.ac.yu

^* To whom correspondence should be addressed.

Received August 3, 2001; Revision received November 7, 2001

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Turpentine-induced acute-phase (AP) response in rats is followed by transcriptional activation of the alpha₂-macroglobulin (MG) gene mediated by cytokine interleukin-6 (IL-6) and glucocorticoids. Based on nucleotide sequence analysis of the alpha₂-MG gene promoter regions responsive to IL-6, we postulated that binding of members of the liver-enriched CCAAT-enhancer-binding proteins (C/EBP) family of transcription factors to the type I IL-6 responsive element (IL-6RE) may participate in the transcriptional activation of this gene during AP response. Results of Western immunoblot and Northern-blot assays revealed coordinate changes in the pool levels of C/EBPalpha, -beta. and -delta protein isoforms and their genes expression in liver in response to turpentine. By means of an in vitro phosphorylation assay, South-Western blot, and selective proteolysis we have also found that only abilities of 35-kD C/EBPbeta and 27-kD C/EBPdelta to bind to the alpha₂-MG gene promoter were affected by phosphorylation. Based on these data we concluded that transcriptional induction of the rat alpha₂-MG gene during AP response correlates with both increased synthesis and phosphorylation-induced binding of 35-kD C/EBPbeta and 27-kD C/EBPdelta.

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KEY WORDS: acute-phase response, turpentine, alpha₂-macroglobulin gene, C/EBPalpha, C/EBPbeta, C/EBPdelta, DNA-binding activity, phosphorylation

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