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