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.     

Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release

Neisseria meningitidis traversal across the blood-cerebrospinal fluid barrier is an essential step in the pathogenesis of bacterial meningitis. We have previously shown that invasion of human brain microvascular endothelial cells (HBMEC) by meningococci is mediated by bacterial outer membrane protein Opc that binds fibronectin, thereby anchoring the bacterium to the integrin alpha 5 beta 1-receptor on the endothelial cell surface. However, subsequent signal transduction mechanisms essential for or regulated by N. meningitidis adhesion and invasion, or HBMEC responses to N. meningitidis are unknown. In this report we investigated the role of c-Jun N-terminal kinases 1 and 2 (JNK1 and JNK2), p38 mitogen-activated (MAP) kinase and protein tyrosine kinases in endothelial-N. meningitidis interaction. Binding of meningococci to HBMEC phosphorylated and activated JNK1 and JNK2 and p38 MAPK as well as their direct substrates c-Jun and MAP kinase activated kinase-2 (MAPKAPK-2), respectively. Non-invasive meningococcal strains lacking opc gene (opc mutants and sequence type 11 complex meningococci) still activated p38 MAPK, however, failed to activate JNK. Inhibition of JNK1 and JNK2 significantly reduced internalization of N. meningitidis by HBMEC without affecting its adherence. Blocking the endothelial integrin alpha 5 beta 1 also decreased N. meningitidis-induced JNK activation in HBMEC. These findings indicate the crucial role of JNK signalling pathway in N. meningitidis invasion in HBMEC. In contrast, p38 MAPK pathway was important for the control of interleukin-6 (IL-6) and IL-8 release by HBMEC. Genistein, a protein tyrosine kinase inhibitor, decreased both invasion of N. meningitidis into HBMEC and IL-6 and IL-8 release, indicating that protein tyrosine kinases, which link signals from integrins to intracellular signalling pathways are essential for both bacterial internalization and cytokine secretion by HBMEC.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

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.     

Involvement of small GTPases in Mycoplasma fermentans membrane lipoproteins-mediated activation of macrophages.

Mycoplasma fermentans lipoproteins (LAMPf) are capable of activating macrophages and inducing the secretion of proinflammatory cytokines. We have recently reported that mitogen-activated protein kinase (MAPK) pathways and NF-kappaB and activated protein 1 (AP-1) play a crucial role in the activation induced by this bacterial compound. To further elucidate the mechanisms by which LAMPf mediate the activation of macrophages, we assessed the effects of inhibiting small G proteins Rac, Cdc42, and Rho. The Rho-specific inhibitor C3 enzyme completely abolished the secretion of tumor necrosis factor alpha by macrophages stimulated with LAMPf and also inhibited the activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 kinase. In addition, we have shown that LAMPf stimulate Cdc42 and that inhibition of Cdc42 or Rac by dominant negative mutants abrogates LAMPf-mediated activation of JNK and transactivation of NF-kappaB and AP-1 in the murine macrophage cell line RAW 264.7. These results indicate that small G proteins Rho, Cdc42, and Rac are involved in the cascade of events leading to the macrophage activation by mycoplasma lipoproteins.     

p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mphi). We used synthetic inhibitors and dominant-negative Mphi mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3(-/-) Mphi, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38alpha MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38alpha MAPK association with adaptor protein, TGF-beta-activated protein kinase 1-binding protein-1. We observed TGF-beta-activated protein kinase 1-binding protein-1-p38alpha MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mphi. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38alpha MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappaB activation: TAO2 regulates TAK1 pathways

Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IkappaB kinase (IKK) that leads to degradation of IkappaB and subsequent NF-kappaB activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IkappaB or activation of NF-kappaB. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-kappaB but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.     

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.     

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.