Activation of discoidin domain receptor 1 facilitates the maturation of human monocyte-derived dendritic cells through the TNF receptor associated factor 6/TGF-beta-activated protein kinase 1 binding protein 1 beta/p38 alpha mitogen-activated protein kinase signaling cascade

Maturation of dendritic cells (DCs) is critical for their ability to stimulate resting naive T cells in primary immune responses. Previous studies demonstrated that collagen, such as type I collagen, could facilitate DC maturation; however, the basis of collagen-mediated DC maturation remains unclear. Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor constitutively expressed in a variety of epithelial cells, including tumor cells, and is inducible in leukocytes. In this study, we evaluated the role of DDR1 in DC maturation using human monocyte-derived DCs. Two DDR1 isoforms, DDR1a and DDR1b, were expressed in both immature and mature DCs. Activation of DDR1 on immature DCs resulted in their partial maturation; however, DDR1 activation markedly amplified TNF-alpha- and LPS-induced phenotypic and functional maturation of DCs through activation of p38 mitogen-activated protein kinase (MAPK), suggesting the involvement of DDR1b in this process. Activation of DDR1b on differentiated DDR1b-overexpressing THP-1 cells or DDR1 on mature DCs induced the formation of TNFR associated factor 6 (TRAF6)/TGF-beta-activated kinase 1 binding protein 1beta/p38alpha MAPK complex and p38alpha autophosphorylation. Transfection of differentiated DDR1b-overexpressing THP-1 cells with dominant negative TRAF6 completely abrogated DDR1b-mediated p38 MAPK phosphorylation, indicating a critical role of TRAF6 in DDR1b-mediated p38 MAPK activation. Taken together, our data suggest that DDR1b-collagen interaction augments the maturation of DCs in a tissue microenvironment through a unique TRAF6/TGF-beta-activated kinase 1 binding protein 1beta/p38alpha MAPK signaling cascade and contributes to the development of adaptive immune responses.     

Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages

Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-alpha or IFN-gamma, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not beta1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-kappaB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-kappaB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.     

PKA-dependent activation of PKC, p38 MAPK and IKK in macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl cAMP

In this study, we examined the signal transduction of dibutyryl cyclic adenosine monophosphate (dBcAMP) to stimulate the release of nitric oxide (NO) and interleukin-6 (IL-6) from J774 macrophages. These actions of dBcAMP were diminished by the presence of the inhibitors of protein kinase A (PKA), protein kinase C (PKC), p38 MAPK and nuclear factor-kappa B (NF-kappaB). In contrast, Go 6976 and PD98059 had no significant effects. Consistently, dBcAMP caused membrane translocation of PKCbetaII, delta, mu, lambda and zeta isoforms, and increased atypical protein kinase C (aPKC) and p38 MAPK activities. The nuclear translocation and DNA-binding study revealed that dBcAMP stimulated NF-kappaB, activator protein-1 (AP-1), and CAAT/enhancer-binding protein (c/EBPbeta). Via PKA, PKC and p38 MAPK-dependent signals, dBcAMP also induced inhibitory subunit of NF-kappaB (IkappaB) degradation, IkappaB kinase (IKK) activation, nuclear translocation of NF-kappaB subunit p65 and its association with the CREB-binding protein (CBP). These results illustrate that PKA activation in macrophages is able to stimulate PKC and p38 MAPK, which lead to IKK-dependent NF-kappaB activation and contribute to the induction of inducible nitric oxide synthase (iNOS) and IL-6 genes.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis

Two key features of atherosclerotic plaques that precipitate acute atherothrombotic vascular occlusion ("vulnerable plaques") are abundant inflammatory mediators and macrophages with excess unesterified, or "free," cholesterol (FC). Herein we show that FC accumulation in macrophages leads to the induction and secretion of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The increases in TNF-alpha and IL-6 mRNA and protein were mediated by FC-induced activation of the IkappaB kinase/NF-kappaB pathway as well as activation of MKK3/p38, Erk1/2, and JNK1/2 mitogen-activated protein kinases (MAPK). Activation of IkappaB kinase and JNK1/2 was needed for the induction of both cytokines. However, MKK3/p38 signaling was specifically involved in TNF-alpha induction, and Erk1/2 signaling was required for IL-6. Most interestingly, activation of all of the signaling pathways and induction of both cytokines required cholesterol trafficking to the endoplasmic reticulum (ER). The CHOP branch of the unfolded protein response, an ER stress pathway, was required for Erk1/2 activation and IL-6 induction. In contrast, one or more other ER-related pathways were responsible for activation of p38, JNK1/2, and IkappaB kinase/NF-kappaB and for the induction of TNF-alpha. These data suggest a novel scenario in which cytokines are induced in macrophages by endogenous cellular events triggered by excess ER cholesterol rather than by exogenous immune cell mediators. Moreover, this model may help explain the relationship between FC accumulation and inflammation in vulnerable plaques.     

TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2^−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1^−/− macrophages. In contrast, although TNFR2^−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.     

Activation mechanisms of endothelial NF-kappaB, IKK, and MAP kinase by tert-butyl hydroperoxide

Lipid peroxidation plays a major role in vascular dysfunction and age-related cardiovascular diseases. A major product of lipid peroxidation, tert-butyl hydroperoxide (t-BHP), has been reported to modulate vascular reactivity and cellular signaling. To better understand vascular abnormality, we set out to delineate the activation mechanism of nuclear factor kappa B (NF-kappaB) by t-BHP and the regulation of MAPK in endothelial cells. The results showed that t-BHP induces NF-kappaB activation by an inhibitor of kappaB (IkappaB) phosphorylation through IkappaB kinase (IKK) activation. Our data from this t-BHP study also showed increased p38 MAP kinase and ERK activity; however, interestingly, t-BHP showed no influence on JNK. Pretreatment with the p38 MAP kinase inhibitor, SB203580 and the ERK1/2 inhibitor, PD98059, prevented t-BHP-induced increases in p65 translocation, NF-kappaB luciferase activity, and phospho-IKKalpha/beta. Data suggested that t-BHP induces NF-kappaB activation through the IKK pathway, which involves p38 MAPK and ERK activation. This study illustrates a role of t-BHP in NF-kappaB activation and MAPK related-signaling pathways. The t-BHP-induced activation of NF-kappaB and MAPK could be a major player in vascular dysfunctions, as seen in oxidative stressed responses and the vascular inflammatory process.     

Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors

ADP-ribosylation is involved in nuclear factor kappaB (NF-kappaB)-dependent gene expression induced by lipopolysaccharide in murine macrophages. Here we have investigated the mechanism by which ADP-ribosylation inhibitors block signaling pathways induced in macrophages. In RAW264.7 macrophages the inducers of NF-kappaB activate the production of reactive oxygen species and three mitogen-activated protein kinases (MAPK), the extracellular signal regulated kinase (ERK), the c-jun N-terminal kinase/stress-activated protein kinase (JNK), and p38. We demonstrate that ADP-ribosylation inhibitors specifically inhibit ERK MAPK activation and reduce the release of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), IL-6 and nitrite.     

Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway

Articular adipose tissue is a ubiquitous component of human joints, and adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. We investigated the signaling pathway involved in IL-6 production caused by adiponectin in both rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts. Rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts expressed the AdipoR1 and AdipoR2 isoforms of the adiponectin receptor. Adiponectin caused concentration- and time-dependent increases in IL-6 production. Adiponectin-mediated IL-6 production was attenuated by AdipoR1 and 5'-AMP-activated protein kinase (AMPK)alpha1 small interference RNA. Pretreatment with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor, IkappaB protease inhibitor, and NF-kappaB inhibitor peptide also inhibited the potentiating action of adiponectin. Adiponectin increased the kinase activity and phosphorylation of AMPK and p38. Stimulation of synovial fibroblasts with adiponectin activated IkappaB kinase alpha/beta (IKK alpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser (276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Adiponectin-mediated an increase of IKK alpha/beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element and was inhibited by compound C, SB203580 and AdipoR1 small interference RNA. Our results suggest that adiponectin increased IL-6 production in synovial fibroblasts via the AdipoR1 receptor/AMPK/p38/IKKalphabeta and NF-kappaB signaling pathway.     

Rac1 negatively regulates lipopolysaccharide-induced IL-23 p19 expression in human macrophages and dendritic cells and NF-kappaB p65 trans activation plays a novel role

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and of a p40 subunit that is also common to IL-12. We defined the distinct signaling mechanisms that regulate the LPS-mediated induction of IL-23 p19 and p40 in human macrophages and dendritic cells. We found that the overexpression of dominant-negative Rac1 (N17Rac1) enhanced LPS-induced IL-23 p19 expression but did not alter p40 expression or IL-12 p70 production in PMA-treated THP-1 macrophages and in human monocyte-derived dendritic cells. Although the inhibition of either p38 MAPK or JNK enhanced LPS-induced p19 expression, N17Rac1 did not influence either p38 MAPK or JNK activation. By contrast, N17Rac1 augmented both NF-kappaB gene expression and p65 trans activation stimulated by LPS without affecting the degradation of IkappaB-alpha or DNA binding to NF-kappaB. Furthermore, small interference RNA of NF-kappaB p65 attenuated cellular amounts of p65 and suppressed LPS-induced p19 expression but did not affect p40 expression. Our findings indicate that Rac1 negatively controls LPS-induced IL-23 p19 expression through an NF-kappaB p65 trans activation-dependent, IkappaB-independent pathway and that NF-kappaB p65 regulates LPS-induced IL-23 p19, but not p40, expression, which causes differences in the control of IL-23 p19 and p40 expression by Rac1.