Mitogen-activated protein kinases and NF-kappa B are involved in TNF-alpha responses to group B streptococci

TNF-alpha is a mediator of lethality in experimental infections by group B streptococcus (GBS), an important human pathogen. Little is known of signal transduction pathways involved in GBS-induced TNF-alpha production. Here we investigate the role of mitogen-activated protein kinases (MAPKs) and NF-kappa B in TNF-alpha production by human monocytes stimulated with GBS or LPS, used as a positive control. Western blot analysis of cell lysates indicates that extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well as I kappa B alpha, became phosphorylated, and hence activated, in both LPS- and GBS-stimulated monocytes. The kinetics of these phosphorylation events, as well as those of TNF-alpha production, were delayed by 30-60 min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-kappa B (caffeic acid phenetyl ester (CAPE)) could all significantly reduce TNF-alpha production, although none of the inhibitors used alone was able to completely prevent TNF-alpha release. However, this was completely blocked by combinations of the inhibitors, including PD98059-SB203580, PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and GBS-stimulated monocytes. In conclusion, our data indicate that the simultaneous activation of multiple pathways, including NF-kappa B, ERK 1/2, and p38 MAPKs, is required to induce maximal TNF-alpha production. Accordingly, in septic shock caused by either GBS or Gram-negative bacteria, complete inhibition of TNF-alpha release may require treatment with drugs or drug combinations capable of inhibiting multiple activation pathways.     

Role of p38 MAPK in the regulation of apoptosis signaling induced by TNF-alpha in differentiated PC12 cells

TNF-alpha elicits various responses including apoptosis, proliferation, and differentiation according to cell type. In neuronal PC12 cells, TNF-alpha induces moderate apoptosis while lipopolysarccaharide or trophic factor deprivation can potentiate apoptosis that is induced by TNF-alpha. TNF-alpha initiates various signal transduction pathways leading to the activation of the caspase family, NF-subk;B, Jun N-terminal kinase, and p38 MAPK via the death domain that contains the TNF-alpha receptor. Inhibition of translation using cycloheximide greatly enhanced the apoptotic effect of TNF-alpha. This implies that the induction of anti-apoptotic genes for survival by TNF-alpha may be able to protect PC12 cells from apoptosis. Accordingly, Bcl-2, an anti-apoptotic Bcl-2 family member, was highly expressed in response to TNF-alpha. In this study, we examined the anti-apoptotic role of p38 MAPK that is activated by TNF-alpha in neuronal PC12 cells. The phosphorylation of p38 MAPK in response to TNF-alpha slowly increased and lasted several hours in the PC12 cell and DRG neuron. This prolonged and slow phosphorylation of p38 MAPK was distinct from other non-neuronal cells. The specific inhibitor of p38 MAPK, SB202190, significantly enhanced the apoptosis that was induced by TNF-alpha in PC12 cells. This indicates that the activation of p38 MAPK could protect PC12 cells from apoptosis since there is no known role of p38 MAPK in response to TNF-alpha in neuron. This discovery could be evidence for the neuroprotective role of the p38 MAPK.     

Downregulation of angiotensin converting enzyme by TNF-alpha in differentiating human macrophages

OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.     

Quantitative role of p42/44 and p38 in the production and regulation of cytokines TNF-alpha, IL-1beta and IL-12 by murine peritoneal macrophages in vitro by concanavalin A

In the present study the quantitative role of p42/44 and p38 in the production of TNF-alpha, IL-1beta and IL-12 by murine peritoneal macrophages, in vitro, on treatment with Concanavalin A (ConA) has been investigated. Maximum expression/production of cytokines TNF-alpha, IL-1beta and IL-12 was observed after 16 h by RT-PCR and 24 h by ELISA, on in vitro treatment with ConA. To investigate the role of MAP kinases in the production of cytokines, pharmacological inhibitors of MAP kinases--PD98059, SB202190 and SP600125, were used. The expression of TNF-alpha, IL-1beta and IL-12 was down regulated in the presence of PD98059 and SB202190 in a dose dependent manner, suggesting the involvement of p42/44 and p38 in ConA induced production of TNF-alpha, IL-1beta and IL-12 by macrophages. It was observed that SP600125 did not have any effect on the expression of TNF-alpha, IL-1beta and IL-12. Using different combinations of MAPK inhibitors, it was found that 45% signal is conveyed via p42/44 and 25% via p38 in the production of these cytokines, by ConA treated macrophages while 30% signal passes through unidentified pathways.     

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.     

Ethanol induces the production of cytokines via the Ca2+, MAP kinase, HIF-1alpha, and NF-kappaB pathway

In the present study, we sought to investigate the signal transduction pathways of expression of cytokines in the ethanol-stimulated human mast cell line, HMC-1. Ethanol significantly increased the intracellular calcium level in HMC-1. Ethanol also significantly enhanced IL-6, TNF-alpha, and TGF-beta1 production compared with media control, but did not significantly affect the IL-1beta production. After 8 h of stimulation, ethanol increased mRNA and protein expression levels of TNF-alpha and TGF-beta1 in HMC-1. The increased cytokine level was significantly inhibited by BAPTA-AM, PD98059, and SB203580. These inhibitors also inhibited ethanol-induced ERK and p38 MAPK phosphorylation. Ethanol resulted in a great increase in protein levels and promoter activity driving luciferase expression of HIF-1alpha and NF-kappaB in HMC-1 cells, but it did not affect on HIF-1alpha mRNA expression. Our observations show that calcium, MAPK activation, HIF-1alpha, and NF-kappaB are necessary for ethanol-induced TNF-alpha and TGF-beta1 expression. These results may have important implications for the study of alcohol-related diseases.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

Tumor necrosis factor-alpha induction of endothelial ephrin A1 expression is mediated by a p38 MAPK- and SAPK/JNK-dependent but nuclear factor-kappa B-independent mechanism

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that induces a broad spectrum of responses including angiogenesis. Angiogenesis promoted by TNF-alpha is mediated, at least in part, by ephrin A1, a member of the ligand family for Eph receptor tyrosine kinases. Although TNF-alpha induces ephrin A1 expression in endothelial cells, the signaling pathways mediating ephrin A1 induction remain unknown. In this study, we investigated the signaling mechanisms of TNF-alpha-dependent induction of ephrin A1 in endothelial cells. Both TNFR1 and TNFR2 appear to be involved in regulating ephrin A1 expression in endothelial cells, because neutralizing antibodies to either TNFR1 or TNFR2 inhibited TNF-alpha-induced ephrin A1 expression. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by a trans-dominant inhibitory isoform of mutant IkappaBalpha did not affect ephrin A1 induction, suggesting that NF-kappaB proteins are not major regulators of ephrin A1 expression. In contrast, ephrin A1 induction was blocked by inhibition of p38 mitogen-activated protein kinase (MAPK) or SAPK/JNK, but not p42/44 MAPK, using either selective chemical inhibitors or dominant-negative forms of p38 MAPK or TNF receptor-associated factor 2. These findings indicate that TNF-alpha-induced ephrin A1 expression is mediated through JNK and p38 MAPK signaling pathways. Taken together, the results of our study demonstrated that induction of ephrin A1 in endothelial cells by TNF-alpha is mediated through both p38 MAPK and SAPK/JNK, but not p42/44 MAPK or NF-kappaB, pathways.     

EV71 induces VCAM-1 expression via PDGF receptor, PI3-K/Akt, p38 MAPK, JNK and NF-kappaB in vascular smooth muscle cells

Enterovirus 71 (EV71) is a widespread virus that causes severe and fatal diseases in patients, including circulation failure. The mechanisms underlying EV71-initiated intracellular signaling pathways to influence host cell functions remain unknown. In this study, we identified a requirement for PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB in the regulation of VCAM-1 expression by rat vascular smooth muscle cells (VSMCs) in response to viral infection. EV71 induced VCAM-1 expression in a time- and viral concentration-dependent manner. Infection of VSMCs with EV71 stimulated VCAM-1 expression and phosphorylation of PDGFR, Akt, and p38 MAPK which were attenuated by AG1296, wortmannin, and SB202190, respectively. The phosphorylation of JNK stimulated by EV71 was not detected under present conditions. In contrast, JNK inhibitor SP600125 inhibited EV71-induced VCAM-1 expression. Furthermore, VCAM-1 expression induced by EV71 was significantly attenuated by a selective NF-kappaB inhibitor (helenalin). Consistently, EV71-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha as well as VCAM-1 mRNA expression was blocked by helenalin, AG1296, SB202190, SP600125, wortmannin, and LY294002. Moreover, the involvement of p38 MAPK, PI3-K/Akt, and NF-kappaB in EV71-induced VCAM-1 expression was reveled by that transfection with dominant negative plasmids of p38 MAPK, p85, Akt, NIK, IKK-alpha, and IKK-beta attenuated these responses. These findings suggest that in VSMCs, EV71-induced VCAM-1 expression was mediated through activation of PDGFR, PI3-K/Akt, p38 MAPK, JNK, and NF-kappaB pathways.     

Induction of tumor necrosis factor-alpha (TNF-alpha) by interleukin-12 p40 monomer and homodimer in microglia and macrophages

The present study was undertaken to explore the role of interleukin-12 (IL-12) p40 in the expression of TNF-alpha in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-alpha and the expression of TNF-alpha mRNA in BV-2 microglial cells. In addition to BV-2 microglial cells, p70, p402 and p40 also induced the production of TNF-alpha in mouse primary microglia and peritoneal macrophages. As the activation of both NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) is important for the expression of TNF-alpha in microglial cells, we investigated the effect of p40 on the activation of NF-kappaB as well as C/EBPbeta. Activation of NF-kappaB as well as C/EBPbeta by p40 and inhibition of p40-induced expression of TNF-alpha by Deltap65, a dominant-negative mutant of p65, and DeltaC/EBPbeta, a dominant-negative mutant of C/EBPbeta, suggests that p40 induces the expression of TNF-alpha through the activation of NF-kappaB and C/EBPbeta. In addition, we show that p40 induced the activation of both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40-induced expression of TNF-alpha through the inhibition of C/EBPbeta, but not that of NF-kappaB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40-induced expression of TNF-alpha through the inhibition of both NF-kappaB and C/EBPbeta. This study delineates a novel biological function of p40 in inducing TNF-alpha in microglia and macrophages.