Elsevier

Cellular Signalling

Volume 16, Issue 7, July 2004, Pages 837-846
Cellular Signalling

JNK signaling involved in the effects of cyclic AMP on IL-1β plus IFNγ-induced inducible nitric oxide synthase expression in hepatocytes

https://doi.org/10.1016/j.cellsig.2004.01.001Get rights and content

Abstract

cAMP significantly inhibits IL-1β+IFNγ-induced iNOS gene expression in hepatocytes, but the signaling pathways responsible for the effect are not known. PKA inhibitors, H89, PKI, and KT5720, had no effect on the recovery of the inhibitory effects of cAMP on cytokine-induced hepatocyte iNOS expression and activity. The JNK inhibitor, SP 600125, effectively reversed the inhibitory effects of cAMP on iNOS expression and significantly increased iNOS promoter activity. A cAMP analogue, dbcAMP, significantly induced JNK signaling and increased AP-1 binding activity in hepatocytes. The JNK activator, anisomycin, inhibited iNOS expression and transcription in hepatocytes as well as AP-1 binding activity; and SP600125 reversed this effect of anisomycin. Overexpression of c-Jun in hepatocytes inhibited IL-1β+IFNγ-induced nitrite accumulation and iNOS promoter activity while dominant negative c-Jun partially reversed the inhibitory effects of cAMP on nitrite accumulation. We conclude that JNK signaling plays an important role in the inhibitory effects of cAMP on IL-1β+IFNγ-induced iNOS gene expression in cultured hepatocytes.

Introduction

Depending on the cell type, increased cAMP can exert either stimulatory or inhibitory effects on cytokine-induced iNOS expression. For example, the cAMP analogues, dbcAMP, glucagon, 8-brcAMP, and PGE2, inhibit cytokine-induced iNOS expression in hepatocytes, kupffer cells, islet cells, fibroblasts, and astrocytes but cAMP increases iNOS expression in adipocytes, smooth muscle cells, skeletal muscle cells, and renal mesangial cells; details was well reviewed in Ref. [1]. To date, understanding of the molecular mechanisms responsible for the bi-directional effects of cAMP is still limited. The effects of cAMP on the regulation of iNOS gene expression and iNOS activity may depend on cell-specific regulation of the transcription factors important in iNOS expression such as NF-κB [2], [3], [4], C/EBP [5], [6], and AP-1 [5], [7]. This hypothesis is based on the finding that some transcription factors are rich in certain tissues and that the relative difference in transcription factor abundance may mediate tissue-specific gene expression. In addition, the activation status of essential transcription factors may be regulated by cAMP through the alteration of cell signaling pathways.

cAMP typically exerts its effects through PKA-mediated phosphorylation of the CREB that interacts with the cis-acting cyclic AMP response element (CRE) [8]. Recent studies have identified that direct coupling of cAMP to Rap1 activation by cAMP-GEFs and Ras/MAPK signaling pathway as an important alternative to the cAMP messenger system beside PKA [9], [10]. The effects of cAMP on the MAPK cascade and cell proliferation are cell type-specific and occur through both PKA- dependent and -independent pathways [11], [12]. As for JNK signaling pathway, cAMP analogues activated JNK signaling in DDT1 MF-2 cells but failed to activate JNK in human embryonic kidney (HEK) 293 cell line [13]. In a human helper T cell clone, dbcAMP even inhibited the activities of JNK1 [14]. In the present work, we tested the role of PKA and JNK pathway on the effects of cAMP on iNOS expression in hepatocytes. AP-1 is a known repressor of iNOS transcription [15], [16]. It is mostly involved in c-Jun N-terminal kinase (JNK) signaling in hepatocytes. We show that the JNK signaling plays an important role in the inhibitory effect of cAMP on IL-1β plus IFNγ-induced iNOS gene expression in primary rat hepatocytes.

Section snippets

Reagents and plasmids

Human recombinant IL-1β was purchased from DuPont (Boston, MA) and murine recombinant IFNγ was purchased from Life Technologies. Antibodies used in this study were purchased from Cell Signaling Technology (c-Jun and phosphorylated c-Jun, Beverly, MA), BD Transduction Laboratory (iNOS and CREB, San Diego, CA), and Santa Cruz Biotechnology (polyclonal antibodies against total JNK1 FL, phosphorylated JNK1 G-7, and c-Jun C-terminal, Santa Cruz, CA). Protein kinase A inhibitors 14–22 amide

Effects of PKA and JNK inhibitors on the effects of cAMP on IL-1β plus IFNγ-induced iNOS expression

Typically, cAMP mediates its effects through stimulating PKA to phosphorylate downstream protein targets, for example, CREB [8]. We first tested the role of PKA signaling on cAMP's inhibitory effects on IL-1β+IFNγ-induced iNOS gene expression in primary cultured rat hepatocytes. Primary hepatocytes were pre-incubated with a cell-permeable PKA pseudo-sequence peptide PKI (amide 14–22) to inhibit PKA activity for 2 h, and then cells were stimulated with of IL-1β plus IFNγ for 24 h with or without

Discussion

cAMP activates PKA and causes reversible phosphorylation of protein substrates that regulate a vast number of cellular processes [8]. This reversible protein phosphorylation and G protein coupling to target proteins is highly ubiquitous and represents a universal mechanism for cellular signaling in eukaryotic cells. Recently, unexpected and novel insights have continued to appear. One important finding is that cAMP exerts its different effects through phosphorylation of different PKA subunits

Acknowledgements

We thank Drs. K.F. Beck and J. Pfeilschifter for providing PGL3/2 plasmid, Dr. M.J. Birrer for providing pcDNA3.1-c-Jun and pcDNA3.1-TAM expression plasmids, and Dr. W.H. Walker for providing CREB and I-CREB expression plasmids. This work was supported by DK55664 (BGH) from the National Institute of Health.

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