IGF-I stimulates IL-8 production in the promyelocytic cell line HL-60 through activation of extracellular signal-regulated protein kinase

Interleukin (IL)-8 serves as a major chemoattractant for neutrophils and has also been proposed to affect cancer progression. In the present study, we show that IGF-I stimulates IL-8 mRNA expression and IL-8 secretion in the leukemic cell line HL-60. Stimulation of IL-8 expression was completely attenuated by two inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK), which phosphorylates the MAPKs extracellular-regulated kinase (ERK)1 and ERK2, and by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. In contrast, inhibitors of p38 MAPK and phosphatidylinositol-3 kinase (PI3K) did not abrogate the effect of IGF-I. We also show that IGF-I stimulates the activation of ERK1 and ERK2, but we could not detect any effect of IGF-I on the phosphorylation of p38, JNK^p46 or JNK^p54. Collectively, our results suggest that basal JNK activity and activation of the MEK–ERK pathway are required for upregulation of IL-8 by IGF-I in HL-60 cells.     

Cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis: role in decreased responsiveness to inflammatory cytokines

Extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) are major signaling molecules activated in human neutrophils stimulated by cytokines. Both molecules were cleaved at the N-terminal portion in neutrophils undergoing apoptosis induced by in vitro culture alone or treatment with TNF and/or cycloheximide. The cleavage of both molecules was inhibited by G-CSF and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a caspase inhibitor, both of which can inhibit neutrophil apoptosis. In a cell-free system, ERK and p38 MAPK were not cleaved by recombinant caspase-3 or caspase-8 while gelsolin was cleaved by caspase-3 under the same condition. The cleavage of both molecules appears to be specific to mature neutrophils, since it was not detected in immature cells (HL-60 and Jurkat) undergoing apoptosis, indicating that proteases responsible for the cleavage of both molecules may develop during differentiation into mature neutrophils. Concomitant with the cleavage of ERK and p38 MAPK, GM-CSF- and TNF-induced superoxide release, adherence, and phosphorylation of ERK and p38 MAPK were decreased in neutrophils undergoing apoptosis. In addition, GM-CSF- and TNF-induced superoxide release and adherence were inhibited by PD98059 MAPK/ERK kinase inhibitor) as well as SB203580 (p38 MAPK inhibitor), suggesting possible involvement of ERK and p38 MAPK in superoxide release and adherence induced by these cytokines. These findings indicate that ERK and p38 MAPK are cleaved and degraded in neutrophils undergoing apoptosis in a caspase-dependent manner and the cleavage of both molecules may be partly responsible for decreased functional responsiveness to inflammatory cytokines.     

Realgar-induced differentiation is associated with MAPK pathways in HL-60 cells

The clinical efficacy and safety of realgar (arsenic sulfide, As(4)S(4)) in the treatment of acute promyelocytic leukemia in China have given rise to an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiation of HL-60 cells. Treatment with RNPs at 6 microM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductive ability, and elevation of CD11b expression at both mRNA and protein levels. The RNP-induced differentiation was synergized, enhanced and suppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways are closely related to the RNP-induced differentiation in HL-60 cells.     

House dust mite allergen Der f 1 induces IL-8 in human basophilic cells via ROS-ERK and p38 signal pathways

Der f 1, a major house dust mite allergen and member of the papain-like cysteine protease family, can provoke immune responses with its proteolytic activity. To understand the role of Der f 1 in inflammatory immune responses, we studied the mechanism of the regulation of interleukin (IL)-8 expressions in human basophilic cell KU812 by proteolytically active recombinant Der f 1. Not only production of IL-8 mRNA was induced but also the DNA binding activity of activator protein-1 (AP-1) and phosphorylation of NF-κB p65 were increased in Der f 1-treated KU812. Furthermore, Der f 1 induction of IL-8 expression was sensitive to pharmacological inhibition of ERK and p38 mitogen activated protein kinase (MAPK) pathways. Der f 1 also activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. The antioxidant N-acetyl-cysteine (NAC) inhibited phosphorylation of ERK, but not p38, suggesting that secretion of IL-8 in KU812 cells treated with Der f 1 is dependent on ROS, ERK MAPK and p38 MAPK. We describe the mechanism of Der f 1-induced IL-8 secretion from human basophilic cells, which are thought to be important for allergic inflammation independent of IgE antibodies. These findings improve our understanding of the inflammatory immune response in human basophils to protease allergens.     

Clostridium perfringens alpha-toxin induces the release of IL-8 through a dual pathway via TrkA in A549 cells

A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.     

Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils.

Stimulation of rat peritoneal neutrophils with staurosporine (64 nM) induced production of macrophage inflammatory protein-2 (MIP-2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase/MAP kinase (ERK/MAPK). The staurosporine-induced MIP-2 production at 4 h was inhibited by the highly specific p38 MAPK inhibitor SB 203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD 98059 in a concentration-dependent manner. By treatment with SB 203580 (1 microM) or PD 98059 (50 microM), the staurosporine-induced increase in the levels of mRNA for MIP-2 was only partially lowered, although the staurosporine-induced MIP-2 production was completely inhibited. Consistent with the inhibition by the protein synthesis inhibitor cycloheximide, SB 203580 and PD 98059 inhibited MIP-2 production at 4 h either when added simultaneously with staurosporine or 2 h after stimulation with staurosporine. In contrast, the DNA-dependent RNA polymerase inhibitor actinomycin D did not inhibit MIP-2 production at 4 h when it was added 2 h after staurosporine stimulation. Dot blot analysis demonstrated that treatment with SB 203580 or PD 98059 down-regulates the stability of MIP-2 mRNA. These results suggested that p38 MAPK and ERK/MAPK pathways are involved in translation of MIP-2 mRNA to protein and stabilization of MIP-2 mRNA.     

Role of SIRT1 in Streptococcus pneumoniae-induced human β-defensin-2 and interleukin-8 expression in A549 cell

Streptococcus pneumoniae is an important pathogen of pneumonia in human. Human alveolar epithelium acts as an effective barrier and is an active participant in host defense against invasion of bacterial by production of various mediators. Sirtuin 1 (SIRT1), the prototypic class III histone deacetylase, is involved in the molecular control of lifespans and immune responses. This study aimed at examining the role of SIRT1 in mediating S. pneumoniae-induced human β-defensin-2 (hBD2) and interleukin-8(IL-8) expression in the alveolar epithelial cell line A549 and the underlying mechanisms involved. A549 cells were infected with S. pneumoniae for indicated times. Exposure of A549 cells to S. pneumoniae increased the expressions of SIRT1 protein, hBD2 and IL-8 mRNA, and protein. The SIRT1 activator resveratrol enhanced S. pneumoniae-induced gene expression of hBD2 but decreased IL-8 mRNA levels. Blockade of SIRT1 activity by the SIRT1 inhibitors nicotinamide reduced S. pneumoniae-induced hBD2 mRNA expression but increased its stimulatory effects on IL-8 mRNA. S. pneumoniae-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SIRT1 expression was attenuated by selective inhibitors of ERK and p38 MAPK. The hBD2 mRNA production was decreased by pretreatment with p38 MAPK inhibitor but not with ERK inhibitor, whereas the IL-8 mRNA expression was controlled by phosphorylation of ERK. These results suggest that SIRT1 mediates the induction of hBD2 and IL-8 gene expression levels in A549 cell by S. pneumoniae. SIRT1 may play a key role in host immune and defense response in A549.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2(MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.     

Sj?gren’s Syndrome Antigen B Acts as an Endogenous Danger Molecule to Induce Interleukin-8 Gene Expression in Polymorphonuclear Neutrophils

Background

Sjögren’s syndrome antigen B is expressed in the nucleus and surface membrane of human polymorphonuclear neutrophils and is released after cell death. However, its biological role is not clear. This study is aimed to investigate the effect of Sjögren’s syndrome antigen B on human polymorphonuclear neutrophils.

Methods

Human recombinant Sjögren’s syndrome antigen B (rSSB) purified from E. coli was incubated with human polymorphonuclear neutrophils as well as retinoid acid-induced granulocytic differentiated HL-60 cells, HL-60 (RA). Interleukin (IL)-8 protein production and mRNA expressions were measured by enzyme-linked immunosorbent assay and quantitative-polymerase chain reaction, respectively. Uptake of fluorescein isothiocyanate (FITC)-rSSB was assessed by flow cytometry and fluorescence microscopy. Moreover, mitogen-activated protein kinase (MAPK) pathways and nuclear factor-kappaB activation were investigated.

Results

Human rSSB stimulated IL-8 production from normal human neutrophils and HL-60 (RA) cells in a time- and dose-dependent manner. This IL-8-stimulated activity was blocked by chloroquine and NH4Cl, indicating that endosomal acidification is important for this effect. We found rSSB activated both MAPK pathway and nuclear factor-kappaB signaling to transcribe the IL-8 gene expression of cells. Furthermore, tumor necrosis factor-α exerted an additive effect and rSSB-anti-SSB immune complex exhibited a synergistic effect on rSSB-induced IL-8 production.

Conclusions

Sjögren’s syndrome antigen B might act as an endogenous danger molecule to enhance IL-8 gene expression in human polymorphonuclear neutrophils.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

Regulation of Indian hedgehog mRNA levels in chondrocytic cells by ERK1/2 and p38 mitogen-activated protein kinases

Indian hedgehog (Ihh) is produced by growth plate pre-hypertrophic chondrocytes, and is an important regulator of endochondral ossification. However, little is known about the regulation of Ihh in chondrocytes. We have examined the role of integrins and mitogen-activated protein (MAP) kinases in Ihh mRNA regulation in CFK-2 chondrocytic cells. Cells incubated with the beta1-integrin blocking antibody had decreased Ihh mRNA levels, which was accompanied by decreases of activated extracellular signal-regulated kinases (ERK1/2) and activated p38 MAPK. Ihh mRNA levels were also inhibited by U0126, a specific MEK1/2 inhibitor, or SB203580, a specific p38 MAPK inhibitor. Cells transfected with constitutively active MEK1 or MKK3 had increased Ihh mRNA levels, which were diminished by dominant-negative MEK1, p38alpha or p38beta. Stimulation of the PTH1R with 10(-8) M rPTH (1-34) resulted in dephosphorylation of ERK1/2 that was evident within 15 min and sustained for 1 h, as well as transient dephosphorylation of p38 MAPK that was maximal after 25 min. PTH stimulation decreased Ihh mRNA levels, and this effect was blocked by transfecting the cells with constitutively active MEK1 but not by MKK3. These studies demonstrated that activation of ERK1/2 or p38 MAPK increased Ihh mRNA levels. Stimulation of the PTH1R or blocking of beta1-integrin resulted in inhibition of ERK1/2 and p38 MAPK and decreased levels of Ihh mRNA. Our data demonstrate the central role of MAPK in the regulation of Ihh in CFK-2 cells.     

NADPH oxidase-derived reactive oxygen species-mediated activation of ERK1/2 is required for apoptosis of human neutrophils induced by Entamoeba histolytica

The extracellular tissue penetrating protozoan parasite Entamoeba histolytica has been known to induce host cell apoptosis. However, the intracellular signaling mechanism used by the parasite to trigger apoptosis is poorly understood. In this study, we investigated the roles of reactive oxygen species (ROS), and of MAPKs in the Entamoeba-induced apoptosis of human neutrophils. The neutrophils incubated with live trophozoites of E. histolytica revealed a marked increase of receptor shedding of CD16 as well as phosphatidylserine (PS) externalization on the cell surface. The Entamoeba-induced apoptosis was effectively blocked by pretreatment of cells with diphenyleneiodonium chloride (DPI), a flavoprotein inhibitor of NADPH oxidase. A large amount of intracellular ROS was detected after exposure to viable trophozoites, and the treatment with DPI strongly inhibited the Entamoeba-induced ROS generation. However, a mitochondrial inhibitor rotenone did not attenuate the Entamoeba-induced ROS generation and apoptosis. Although E. histolytica strongly induced activation of ERK1/2 and p38 MAPK in neutrophils, the activation of ERK1/2 was closely associated with ROS-mediated apoptosis. Pretreatment of neutrophils with MEK1 inhibitor PD98059, but not p38 MAPK inhibitor SB202190, prevented Entamoeba-induced apoptosis. Moreover, DPI almost completely inhibited Entamoeba-induced phosphorylation of ERK1/2, but not phosphorylation of p38 MAPK. These results strongly suggest that NADPH oxidase-derived ROS-mediated activation of ERK1/2 is required for the Entamoeba-induced neutrophil apoptosis.