Effect of nitric oxide donor and gamma irradiation on modifications of ERK and JNK in murine peritoneal macrophages

Mitogen activated protein kinases (MAPKs) play an important role in activation, differentiation and proliferation of macrophages. Macrophages, upon activation, produce large amounts of nitric oxide that inhibit the growth of variety of microorganisms and tumor cells. This nitric oxide which is known to interfere with tyrosine phosphorylation may result in changes in the pattern of activation of MAPKs. In a previous study we have found that tyrosine phosphorylation of MAPKs was completely abolished in the presence of nitric oxide donor and radiation but this did not affect the function of macrophages. In this study the other post translational modifications namely nitration and ubiquitination of JNK and ERK have been looked at. Both ERK and JNK were found to be nitrated. However, there was no increase in ubiquitination of ERK and JNK, indicating that ubiquitination, in this case was not a natural consequence of nitration and may serve in signaling. Additionally, when the nitration was extensive, phosphorylation was also inhibited. The activation of substrates of ERK and JNK were looked at to determine the consequences of such modifications. Inhibition of phosphorylation and extensive nitration of JNK did not prevent activation of its substrate, c-jun. This study indicates that ERK and JNK may be under regulation by different type of modifications in macrophages.     

Prevention of Encephalomyocarditis Virus-Induced Diabetes in Mice by Inhibition of the Tyrosine Kinase Signalling Pathway and Subsequent Suppression of Nitric Oxide Production in Macrophages

Macrophages comprise the major population of cells infiltrating pancreatic islets during the early stages of infection in DBA/2 mice by the D variant of encephalomyocarditis virus (EMC-D virus). Inactivation of macrophages prior to viral infection almost completely prevents EMC-D virus-induced diabetes. This investigation was initiated to determine whether a tyrosine kinase signalling pathway might be involved in the activation of macrophages by EMC-D virus infection and whether tyrosine kinase inhibitors might, therefore, abrogate EMC-D virus-induced diabetes in vivo. When isolated macrophages were infected with EMC-D virus, inducible nitric oxide synthase mRNA was expressed and nitric oxide was subsequently produced. Treatment of macrophages with the tyrosine kinase inhibitor tyrphostin AG126, but not tyrphostin AG556, prior to EMC-D virus infection blocked the production of nitric oxide. The infection of macrophages with EMC-D virus also resulted in the activation of the mitogen-activated protein kinases (MAPKs) p42(MAPK/ERK2)/p44(MAPK/ERK1), p38(MAPK), and p46/p54(JNK). In accord with the greater potency of AG126 than of AG556 in blocking EMC-D virus-mediated macrophage activation, the incidence of diabetes in EMC-D virus-infected mice treated with AG126 (25%) was much lower than that in AG556-treated (75%) or vehicle-treated (88%) control mice. We conclude that EMC-D virus-induced activation of macrophages resulting in macrophage-mediated beta-cell destruction can be prevented by the inhibition of a tyrosine kinase signalling pathway involved in macrophage activation.     

Particulate Matter Facilitates C6 Glioma Cells Activation and the Release of Inflammatory Factors Through MAPK and JAK2/STAT3 Pathways

It has been widely accepted that astrocytes, play a role in regulating almost every physiological system. In the present study, we investigated the role of particulate matter (PM) in regulating activation of astrocytes. The glial cell strain C6 was cloned from a rat glioma which was induced by N-nitrosomethylurea. The C6 cells were plated at a density of 5 × 10⁶ cells/10 cm diameter dish and incubated with different concentrations (0, 12, 25, 50, 100, 200, and 400 μg/mL) of PM for 24 h and different time (0, 1, 3, 6, 8,12, and 24 h) with 100 μg/mL at 37 °C. The study revealed that PM stimulated the expression of inducible nitric oxide synthase (iNOS) as well as the production of IL-1β in a dose- and time-dependent manner. Furthermore, activation of JAK2/STAT3 and p38/JNK/ERK MAPKs was found in astrocytes following PM treatment. Blockage of JAK and p38/JNK/ERK MAPKs with their specific inhibitors, AG490, SB202190, SP600125 and U0126 significantly reduced PM-induced iNOS expression and IL-1β production. In addition, it was demonstrated that inhibition of p38, JNK and JAK prevented STAT3 tyrosine phosphorylation induced by PM, while blocking ERK did not. MAPKs (p38 and JNK) could regulate tyrosine STAT3 phosphorylation, which suggested that the JAK2/STAT3 pathway might be the downstream of p38/JNK MAPK pathways.     

Effect of nitric oxide donor and gamma irradiation on MAPK signaling in murine peritoneal macrophages

Irradiation (IR) of cells is known to activate enzymes of mitogen activated protein kinase (MAPK) family. These are known to be involved in cellular response to stress and are determinants of cell death or survival. When radiotherapy is delivered to malignant cells, macrophages, being radioresistant, survive, get activated, and produce large amounts of nitric oxide. As a result of activation they recognize and phagocytose tumor and normal cell apoptotic bodies leading to tumor regression. In this study, the MAPK signaling in peritoneal macrophages was investigated which plays an important role in its various functions, in an environment which is predominantly nitric oxide, as is after IR. The behavior of macrophages in such an environment was also looked at. The three MAPK (ERK1/2, p38, and JNK) respond differently to Sodium nitroprusside (SNP) alone or IR alone. All the three were activated following IR but only JNK was activated following SNP treatment. Surprisingly, when both the stresses were given simultaneously or one after the other, this differential response was lost and there was a complete inhibition of phosphorylation of all the three MAPKs, irrespective of the order of the two insults (IR and SNP). The noteworthy observation was that despite the complete inhibition of MAPK signaling there was no effect on either the viability or the phagocytic efficiency of peritoneal macrophages.     

Cycloheximide-induced cPLA(2) activation is via the MKP-1 down-regulation and ERK activation

Extracellular signal-regulated kinase (ERK)-dependent phosphorylation is an important regulator for cytosolic phospholipase A(2) (cPLA(2)). In this study, we found that the protein synthesis inhibitor cycloheximide can potentiate thapsigargin-induced arachidonic acid (AA) release concomitant with ERK phosphorylation from murine RAW 264.7 macrophages. The cycloheximide effect is not due to the activation of p38 mitogen-activated protein kinase (MAPK) nor c-Jun NH(2)-terminal kinase (JNK), because the activator of both MAPKs anisomycin does not elicit AA release. Cycloheximide effect is additive to the tyrosine phosphatase inhibitor orthovanadate since these two stimuli induced sustained ERK activation respectively through inhibition of the translation and activity of MAPK phosphatase-1 (MKP-1).     

Immune-stimulating activity of water-soluble extracellular polysaccharide isolated from Rhizobium massiliae

The aim of this study is to investigate biochemical properties of water-soluble extracellular polysaccharide (WSP) from a novel bacterial strain designated to CA-1 and classified to Rhizobium massiliae by 16S rDNA sequence determination and homology analysis. The main composition of WSP was determined to be glucose by HPAEC. We evaluated immunomodulatory effects of WSP on RAW 264.7 macrophage activation. The results showed that the WSP dose-dependently induced the release of the pro-inflammatory cytokines such as TNF-α and IL-6. In addition, WSP induced nitric oxide synthase (iNOS) expression and increased the production of nitric oxide (NO). Intriguingly, WSP remarkably increased the mRNA expression of Toll-like receptor- 2 (TLR-2) and the phosphorylation of MAPKs (ERK, JNK and p38) in RAW 264.7 cells. These results indicated that WSP activates macrophages to secrete pro-inflammatory cytokines and induces iNOS expression via the activation of the TLR-2/MAPKs signaling pathways. Conclusively, we suggest that WSP of R. massiliae CA-1 can be a new immunomodulatory enhancing the early innate immunity.     

Tumour necrosis factor activates the mitogen-activated protein kinases p38alpha and ERK in the synovial membrane in vivo

Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane in vivo. We studied human TNF transgenic mice--an in vivo model of TNF-induced arthritis--to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKalpha in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-kappaB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKalpha and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKalpha was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKalpha and ERK, whereas inhibition of IL-1 only affected p38MAPKalpha and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKalpha and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKalpha and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.     

Nitric oxide inhibits angiotensin II-induced activation of the calcium-sensitive tyrosine kinase proline-rich tyrosine kinase 2 without affecting epidermal growth factor receptor transactivation.

In a previous study, we showed that nitric oxide donors and N-acetylcysteine, either alone or in combination, inhibited the activation of several mitogen-activated protein kinases by angiotensin II in rat cardiac fibroblasts (Wang, D., Yu, X., and Brecher, P. (1998) J. Biol. Chem. 273, 33027-33034). In the present study, we have focused on the mechanism by which nitric oxide exerts this effect on the activation of extracellular signal-regulated kinase (ERK). We contrasted the effects of nitric oxide on ERK activation by angiotensin II and epidermal growth factor (EGF), since the transactivation of the EGF receptor has been implicated as a response to angiotensin II. We found that nitric oxide inhibited ERK activation by angiotensin II but did not inhibit the relatively slight but significant transactivation of the EGF receptor by angiotensin II. The tyrphostin AG1478, known to inhibit EGF receptor phosphorylation, also inhibited the angiotensin II and EGF-induced activation of ERK, the phosphorylation of the EGF receptor, and the subsequent association of Shc and Grb2. Nitric oxide did not affect either EGF receptor phosphorylation or Shc-Grb2 activation induced by either Ang II or EGF. However, the activation of the calcium-sensitive tyrosine kinase PYK2, which occurred in response to angiotensin II, but not EGF, was inhibited by nitric oxide. The data suggested that PYK2 activation may be an important inhibitory site in signaling pathways affected by nitric oxide.     

Role of TAB1 in nitric oxide-induced p38 activation in insulin-producing cells

The aim of present study was to elucidate the role of TAB1 in nitric oxide-induced activation of p38 MAPK. For this purpose we over-expressed TAB1 in insulin-producing beta-TC6 cells. We observed in cells transiently over-expressing TAB1 that p38 activation was enhanced in response to DETA/NONOate. A lowering of TAB1 levels, using the siRNA technique, resulted in the opposite effect. The DETA/NONOate-induced cell death rate was increased in cells transiently overexpressing TAB1. In stable beta-TC6 cell clones with very high TAB1 levels p38 phosphorylation was enhanced also at basal conditions. DETA/NONOate increased also the phosphorylation of JNK and ERK in beta-TC6 cells, but these events were not affected by TAB1. Interestingly, the inhibitory effect of SB203580 on p38 phosphorylation was paralleled by a stimulatory effect on JNK phosphorylation and an inhibitory effect on ERK phosphorylation. In summary, we propose that TAB1 promotes nitric oxide-induced p38 autophosphorylation. In addition, nitric oxide-induced p38 activation seems to promote JNK inhibition and ERK activation, but this effect appears to not require TAB1. A better understanding of how the TAB1/p38 pathway promotes beta-cell death in response to nitric oxide might help in the development of novel pharmacological approaches in the treatment of diabetes.     

IFN-gamma + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(mapk) in a mouse macrophage cell line

Nitric oxide (NO.) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO. by interferon-gamma (IFN-gamma) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-gamma in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-gamma alone with respect to iNOS induction. In this RAW 264.7gammaNO(-) subclone, IFN-gamma and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38(mapk) inhibited IFN-gamma + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-gamma + LPS, also implicating the c-Jun NH(2)-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-gamma + LPS-induced tumor necrosis factor-alpha protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38(mapk) appears more complex and may be due to the ability of p38(mapk) to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO. expression by IFN-gamma + LPS.     

TRAF6 is functional in inhibition of TLR4-mediated NF-κB activation by resveratrol

Resveratrol was suggested to inhibit Toll-like receptor (TLR)4-mediated activation of nuclear factor-κB (NF-κB) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF)–(TANK)-binding kinase 1, but the myeloid differentiation primary response gene 88–tumor necrosis factor receptor-associated factor 6 (TRAF6) pathway is not involved in this effect. However, involvement of TRAF6 in this process is still elusive since cross talk between TRIF and TRAF6 has been reported in lipopolysaccharide (LPS)-induced signaling. Using RAW 264.7 macrophages, we determined the effect of resveratrol on LPS-induced TRAF6 expression, ubiquitination as well as activation of mitogen-activated protein (MAP) kinases and Akt in order to elucidate its involvement in TLR4 signaling. LPS-induced transient elevation in TRAF6 mRNA and protein expressions is suppressed by resveratrol. LPS induces the ubiquitination of TRAF6, which has been reported to be essential for Akt activation and for transforming growth factor-β activated kinase-1–NAP kinase kinase 6 (MKK6)-mediated p38 and c-Jun N-terminal kinase (JNK) activation. We found that resveratrol diminishes the effect of LPS on TRAF6 ubiquitination and activation of JNK and p38 MAP kinases, while it has no effect on the activation of extracellular-signal-regulated kinase (ERK)1/2. The effect of resveratrol on MAP kinase inhibition is significant since TRAF6 activation was reported to induce activation of JNK and p38 MAP kinase while not affecting ERK1/2. Moreover, Akt was identified previously as a direct target of TRAF6, and we found that, similarly to MAPKs, phosphorylation pattern of Akt followed the activation of TRAF6, and it was inhibited by resveratrol at all time points. Here, we provide the first evidence that resveratrol, by suppressing LPS-induced TRAF6 expression and ubiquitination, attenuates the LPS-induced TLR4–TRAF6, MAP kinase and Akt pathways that can be significant in its anti-inflammatory effects.     

Involvement of MAPK activation in chemokine or COX-2 productions by Toxoplasma gondii

This experiment focused on MAPK activation in host cell invasion and replication of T. gondii, as well as the expression of CC chemokines, MCP-1 and MIP-1 alpha , and enzyme, COX-2/prostaglandin E2 (PGE2) in infected cells via western blot, [3H]-uracil incorporation assay, ELISA and RT-PCR. The phosphorylation of ERK1/2 and p38 in infected HeLa cells was detected at 1 hr and/or 6 hr postinfection (PI). Tachyzoite proliferation was reduced by p38 or JNK MAPK inhibitors. MCP-1 secretion was enhanced in infected peritoneal macrophages at 6 hr PI. MIP-1 alpha mRNA was increased in macrophages at 18 hr PI. MCP-1 and MIP-1 alpha were reduced after treatment with inhibitors of ERK1/2 and JNK MAPKs. COX-2 mRNA gradually increased in infected RAW 264.7 cells and the secretion of COX-2 peaked at 6 hr PI. The inhibitor of JNK suppressed COX-2 expression. PGE2 from infected RAW 264.7 cells was increased and synthesis was suppressed by PD98059, SB203580, and SP600125. In this study, the activation of p38, JNK and/or ERK1/2 MAPKs occurred during the invasion and proliferation of T. gondii tachyzoites in HeLa cells. Also, increased secretion and expression of MCP-1, MIP-1 alpha , COX-2 and PGE2 were detected in infected macrophages, and appeared to occur via MAPK signaling pathways.     

Extracellular Signal-regulated Kinase Mediates Expression of Arginase II but Not Inducible Nitric-oxide Synthase in Lipopolysaccharide-stimulated Macrophages

The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype.     

Tumour necrosis factor activates the mitogen-activated protein kinases p38α and ERK in the synovial membrane in vivo

Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane in vivo. We studied human TNF transgenic mice – an in vivo model of TNF-induced arthritis – to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKα in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-κB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKα and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKα was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKα and ERK, whereas inhibition of IL-1 only affected p38MAPKα and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKα and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKα and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.     

Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases

The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways.     

Distinct effects of N-acetylcysteine and nitric oxide on angiotensin II-induced epidermal growth factor receptor phosphorylation and intracellular Ca(2+) levels

These studies describe inhibitory effects of N-acetylcysteine on several biochemical events associated with the activation of extracellular signal-regulated kinases (ERK) by angiotensin II in the cardiac fibroblast and compare these effects with those of the nitric oxide donor, S-nitroso-N-acetylpenicillamine, an agent we showed previously to inhibit angiotensin II-induced ERK activation and the concomitant phosphorylation of proline-rich tyrosine kinase 2 (Wang, D., Yu, X., and Brecher, P. (1999) J. Biol. Chem. 274, 24342-24348). The transactivation of the epidermal growth factor receptor by angiotensin II, a process required for the activation of ERK, was inhibited by N-acetylcysteine but not by nitric oxide. The transactivation of the epidermal growth factor receptor by angiotensin II was shown to be independent of intracellular calcium increases. Nitric oxide, but not N-acetylcysteine, inhibited the angiotensin II-induced increase in intracellular Ca(2+). Neither nitric oxide nor N-acetylcysteine inhibited either phospholipase C activation or inositol triphosphate generation in response to angiotensin II. N-Acetylcysteine did inhibit the phosphorylation of the calcium sensitive tyrosine kinases PYK2 and Src, effects that also occurred using nitric oxide. These studies describe a novel effect of N-acetylcysteine on cross-talk between a G protein-linked receptor and a tyrosine kinase receptor and offer additional molecular insight to explain how N-acetylcysteine and nitric oxide act at different sites and might have an additive effect on specific hormonal responses.     

MAPKs and Mst1/Caspase-3 pathways contribute to H2B phosphorylation during UVB-induced apoptosis

Apoptosis is a highly coordinated or programmed cell suicide mechanism in eukaryotes. Histone modification is associated with nuclear events in apoptotic cells. Specifically H2B phosphorylation at serine 14 (Ser14) catalyzed by Mst1 kinase has been linked to chromatin condensation during apoptosis. We report that activation of MAPKs (ERK1/2, JNK1/2 and p38) together with Mst1 and caspase-3 is required for phosphorylation of H2B (Ser14) during ultraviolet B light (UVB)-induced apoptosis. UVB can trigger activation of MAPKs and induce H2B phosphorylation at Ser14 but not acetylation in a time-dependent manner. Inhibition of ERK1/2, JNK1/2 or p38 activity blocked H2B phosphorylation (Ser14). Furthermore, caspase-3 was activated by UVB to regulate Mst1 activity, which phosphorylates H2B at Ser14, leading to chromatin condensation. Full inhibition of caspase-3 activity reduced Mst1 activation and partially inhibited H2B phosphorylation (Ser14), but ERK1/2, JNK1/2 and p38 activities were not affected. Taken together, these data revealed that H2B phosphorylation is regulated by both MAPKs and caspase-3/Mst1 pathways during UVB-induced apoptosis.     

Cobalt protoporphyrin inhibition of lipopolysaccharide or lipoteichoic acid-induced nitric oxide production via blocking c-Jun N-terminal kinase activation and nitric oxide enzyme activity

In the present study, low doses (0.5, 1, and 2 μM) of cobalt protoporphyrin (CoPP), but not ferric protoporphyrin (FePP) or tin protoporphyrin (SnPP), significantly inhibited lipopolysaccharide (LPS) or lipoteichoic acid (LTA)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages under serum-free conditions. IC₅₀ values of CoPP inhibition of NO and iNOS protein individually induced by LPS and LTA were around 0.25 and 1.7 μM, respectively. This suggests that CoPP is more sensitive at inhibiting NO production than iNOS protein in response to separate LPS and LTA stimulation. NO inhibition and HO-1 induction by CoPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). Decreasing iNOS/NO production and increasing HO-1 protein by CoPP were observed with CoPP pretreatment, CoPP co-treatment, and CoPP post-treatment with LPS and LTA stimulation. LPS- and LTA-induced NOS/NO productions were significantly suppressed by the JNK inhibitor, SP600125, but not by the ERK inhibitor, PD98059, through a reduction in JNK protein phosphorylation. Transfection of a dominant negative JNK plasmid inhibited LPS- and LTA-induced iNOS/NO production and JNK protein phosphorylation, suggesting that JNK activation is involved in LPS- and LTA-induced iNOS/NO production. Additionally, CoPP inhibition of LPS- and LTA-induced JNK, but not ERK, protein phosphorylation was identified in RAW264.7 cells. Furthermore, CoPP significantly reduced NO production in a cell-mediated, but not cell-free, iNOS enzyme activity assay accompanied by HO-1 induction. However, attenuation of HO-1 protein stimulated by CoPP via transfection of HO-1 siRNA did not affect NO's inhibition of CoPP against LPS stimulation. CoPP effectively suppressing LPS- and LTA-induced iNOS/NO production through blocking JNK activation and iNOS enzyme activity via a HO-1 independent manner is first demonstrated herein.