Vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide inhibit the MEKK1/MEK4/JNK signaling pathway in LPS-stimulated macrophages

Abstract

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, downregulate TNFα expression in LPS-stimulated peritoneal macrophages and Raw 264.7 cells. We showed previously that VIP/PACAP change the composition of the CRE-binding complex in the TNFα promoter from ^highc-Jun/^lowCREB, characteristic for LPS-stimulated macrophages, to ^lowc-Jun/^highCREB, characteristic for the unstimulated cells. In the present study we examined the effects of VIP/PACAP on the MEKK1/MEK4/JNK transduction pathway, and on the subsequent changes in Jun family members. Our studies indicate that VIP/PACAP inhibit MEKK1 activity, and the subsequent phosphorylation of MEK4, JNK, and c-Jun. Treatment with VIP or PACAP results in a decrease in AP-1 binding, and a marked change in the composition of the AP-1 complexes from c-Jun/c-Fos to JunB/c-Fos. Western blots confirm that VIP stimulates JunB production in LPS-stimulated macrophages. Both the inhibition of the MEKK1/MEK4/JNK pathway, leading to the reduction in phosphorylated c-Jun, and the stimulation of JunB, are mediated through the specific VPAC1 receptor and the cAMP/PKA pathway. The VIP/PACAP interference with the stress-induced SAPK/JNK pathway in stimulated macrophages may represent a significant element in the regulation of the inflammatory response by the endogenous neuropeptides.

Introduction

Macrophages, participants in both innate and specific immunity, have numerous functions, such as phagocytosis, antigen processing and presentation, secretion of both pro- and anti-inflammatory cytokines, production of reactive oxygen and nitrogen intermediates. Following stimulation with microbial products like LPS, macrophages secrete several pro-inflammatory products such as TNFα, IL-12, IL-1, IL-6 and nitric oxide (NO), followed later by the secretion of the anti-inflammatory cytokines IL-10 and TGFβ (Laskin and Pendino, 1995). Macrophage activation is controlled by a number of regulatory molecules. Among these, the ‘macrophage deactivating factors’ have received considerable interest lately (Kunkel et al., 1988, Tsunawaki et al., 1988, Fiorentino et al., 1991, Trepicchio et al., 1996, Muchamuel et al., 1997). We and others showed that neuropeptides such as the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP) function as macrophage deactivating factors, inhibiting the production of IL-6, IL-12, TNFα and NO, and enhancing IL-10 production in vivo and in vitro (Hernanz et al., 1996, Martinez et al., 1998, Dewit et al., 1998, Xin and Subramaniam, 1998, Delgado et al., 1999a, Delgado et al., 1999b, Delgado et al., 1999c, Delgado et al., 1999d, Delgado et al., 1999e). The inhibition of proinflammatory mediators is responsible, at least partially, for the protective effect of VIP and PACAP in vivo in a murine model for septic shock (Delgado et al., 1999f), and might contribute to their role as survival factors for lung and neuronal cells in injury models (Said, 1996a, Said, 1996b, Said et al., 1998). The effects of VIP and PACAP on the expression of macrophage-derived factors are exerted at a transcriptional level, and involve the regulation of several transcription factors, such as NFkB, CREB, c-Jun, and IRF-1 (Delgado et al., 1998, Delgado et al., 1999c, Delgado et al., 1999d, Delgado and Ganea, 1999). We have shown previously that the inhibition of NFkB translocation is cAMP-independent, subsequent to a stabilizing effect on IkB (Delgado and Ganea, 1999). In contrast, the VIP/PACAP effects on CREB, c-Jun and IRF-1 are cAMP-dependent (Delgado et al., 1998, Delgado et al., 1999c, Delgado et al., 1999d, Delgado and Ganea, 1999). Recently, we reported that the VIP/PACAP inhibition of IRF-1 synthesis in LPS-stimulated macrophages is mediated through the Jak1/STAT1 transduction pathway (Delgado and Ganea, in press). In this study we investigate the transduction pathways involved in the VIP/PACAP reduction of phosphorylated c-Jun in LPS-stimulated Raw 264.7 macrophages.

LPS treatment of macrophages leads to the activation of all three MAPK cascades, i.e. the mitogen-activated protein kinase (MAPK; ERK), the stress-activated protein kinase (JNK) and the p38 kinase (Weinstein et al., 1991, Weinstein et al., 1992, Beaty et al., 1994, Shapira et al., 1994, Chen and Wang, 1999). JNK regulates the activity of several transcription factors such as c-Jun, ATF2 and ELK-1. JNK itself is activated in response to stress signals such as osmotic and heat shock, UV light, protein synthesis inhibitors, DNA-damaging drugs, and proinflammatory cytokines (Derijard et al., 1994, Galcheva-Gargova et al., 1994, Kyriakis et al., 1994). The activation of JNK is mediated through phosphorylation of threonine and tyrosine residues by the upstream MAPK kinases MKK4/7 (MEK4/7) (reviewed in Tibbles and Woodgett, 1999, Davis, 1999, Cobb, 1999). MKK4 activation is regulated in turn by phosphorylation by the upstream MAPK kinase kinase MKKK1 (MEKK1) (Lange-Carter et al., 1993).

In this study, we examine the effects of VIP/PACAP on the MKKK1/MKK4/JNK cascade. We conclude that VIP and PACAP inhibit MKKK1, MKK4, and JNK activity, and the subsequent binding of the transcriptional factor AP-1, by changing the composition of the AP-1 complex from c-Jun/c-Fos to JunB/c-Fos. The VIP/PACAP effect is mediated through the VIP/PACAP receptor VPAC1 and is cAMP-dependent.