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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:186.)
© 2005 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Protein Kinase C Pathway Is Involved in Transcriptional Regulation of C-Reactive Protein Synthesis in Human Hepatocytes

Yuri Ivashchenko; Frank Kramer; Stefan Schäfer; Andrea Bucher; Kerstin Veit; Vinzenz Hombach; Andreas Busch; Olaf Ritzeler; Jürgen Dedio; Jan Torzewski

From the Aventis Pharma Deutschland GmbH (Y.I., F.K., S.S., K.V., A. Busch, O.R., J.D.), DG Cardiovascular Research, Frankfurt am Main; and the Department of Internal Medicine II–Cardiology (A. Bucher, V.H., J.T.), University of Ulm, Germany.

Correspondence to Jan Torzewski, MD, MPhil, University of Ulm, Department of Internal Medicine II–Cardiology, Robert Koch Str.8, 89081 Ulm, Germany. E-mail jan.torzewski{at}medizin.uni-ulm.de

Objective— C-Reactive protein (CRP) is the prototype acute phase protein and a cardiovascular risk factor. Interleukin-1ß (IL-1ß) and IL-6 stimulate CRP synthesis in hepatocytes. We searched for additional pathways regulating CRP expression.

Methods and Results— Primary human hepatocytes (PHHs) were treated with IL-1ß, IL-6, and protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). CRP was analyzed by quantitative RT-PCR and ELISA. PDBu significantly induced CRP transcription by 21.0±9.24-fold and protein release by 2.9±0.5-fold. Transcriptional regulation was studied in detail in hepatoma G2 (HepG2) cells stably transfected with the 1-kb CRP promoter (HepG2–ABEK14 cells). In these cells, PDBu significantly induced CRP transcription by 5.39±0.66-fold. Competetive inhibition with bisindolylmaleimide derivative LY333531 abolished PDBu-mediated promoter activation. Competetive inhibition with IB kinase inhibitor I229 also inhibited PDBu effects. Importantly, IL-8 significantly induced CRP release in PHHs by 58.675±19.1-fold, which was blockable by LY333531.

Conclusions— This study describes a novel PKC-dependent transcriptional regulation of CRP gene expression, which, in analogy to the classical IL-1ß and IL-6 pathways, is operational in hepatocytes only. It also identifies IL-8 as a potential physiological PKC activator. HepG2–ABEK14 cells may be useful for high throughput screening to identify inhibitors of CRP synthesis for the prevention of cardiovascular disease.

We identify the PKC pathway as a novel pathway in the regulation of CRP synthesis in primary human hepatocytes. Stable transfection of HepG2 cells with the 1kb CRP promoter results in a cell line strongly inducible by IL-1ß/IL-6. Using this cell line, we show that PKC-mediated CRP transcription is NF-B dependent. Moreover, this cell line may be useful for high throughput screening to identify inhibitors of CRP synthesis.

Key Words: atherosclerosis • C-reactive protein • drug development • gene expression • protein kinase C

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