Opposite effects of inhibitors of mitogen-activated protein kinase pathways on the egr-1 and beta-globin expression in erythropoietin-responsive murine erythroleukemia cells

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.     

U0126 and PD98059, specific inhibitors of MEK,accelerate differentiation of RAW264.7 cells into osteoclast-like cells

Osteoclasts are multinucleated cells that differentiate from hematopoietic cells and possess characteristics responsible for bone resorption. To study the involvement of mitogen-activated protein kinases (MAPKs) in osteoclastogenesis of the murine monocytic cell line RAW264.7, which can differentiate into osteoclast-like cells in the presence of the receptor activator of nuclear factor kappa B ligand (RANKL), we treated the cells with specific inhibitors of p38 MAPK, PD169316 and SB203580, and specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK), U0126 and PD98059. Each inhibitor blocked differentiation into osteoclast-like cells when the cells were plated at the standard cell density (2000-4000 cells per well (96-well)). However, the effect of MEK inhibitors on osteoclastogenesis varied according to the initial cell density during culture, because cell growth was clearly inhibited by them. When the cells were plated at more than 8000 cells per well, marked enhancement and acceleration of the differentiation were observed. In addition, immunoblot analysis revealed that phosphorylation of ERK was increased by treatment with the p38 inhibitors, whereas the MEK inhibitors increased phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteoclastogenesis is regulated under a balance between ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteoclastogenesis while the p38 pathway does so positively. This is the first report that an inhibitor of signal transduction enhanced osteoclastogenesis.     

p38 differentially regulates ERK,p21, and mitogenic signalling in two pancreatic carcinoma cell lines

Whereas the p38 MAP kinase has largely been associated with anti-proliferative functions, several observations have indicated that it may also have positive effects on proliferation. In hepatocytes, we have found that p38 has opposing effects on DNA synthesis when activated by EGF and HGF. Here we have studied the function of p38 in EGF- and HGF-induced DNA synthesis in the two pancreatic carcinoma cell lines AsPC-1 and Panc-1. In Panc-1 cells, the MEK inhibitor PD98059 reduced EGF- and HGF-induced DNA synthesis, while the p38 inhibitor SB203580 strongly increased the basal DNA synthesis and reduced expression of the cyclin-dependent kinase inhibitor (CDKI) p21. In contrast, in AsPC-1 cells, EGF- and HGF-induced DNA synthesis was not significantly reduced by PD98059 but was inhibited by SB203580. Treatment with SB203580 amplified the sustained ERK phosphorylation induced by these growth factors and caused a marked upregulation of the expression of p21, which could be blocked by PD98059. These results suggest that while DNA synthesis in Panc-1 cells is enhanced by ERK and strongly suppressed by p38, in AsPC-1 cells, p38 exerts a pro-mitogenic effect through MEK/ERK-dependent downregulation of p21. Thus, p38 may have suppressive or stimulatory effects on proliferation depending on the cell type, due to differential cross-talk between the p38 and MEK/ERK 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.     

IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and intercellular adhesion molecule-1/LFA-1/ERK1/2-dependent pathways

IL-27, a novel member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in positive and negative regulations of immune responses. We recently demonstrated that IL-27 induces Th1 differentiation through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-independent mechanism. In this study, we further investigated the molecular mechanisms by focusing on p38 MAPK and ERK1/2. IL-27-induced Th1 differentiation was partially inhibited by lack of T-bet expression or by blocking ICAM-1/LFA-1 interaction with anti-ICAM-1 and/or anti-LFA-1, and further inhibited by both. Similarly, the p38 MAPK inhibitor, SB203580, or the inhibitor of ERK1/2 phosphorylation, PD98059, partially suppressed IL-27-induced Th1 differentiation and the combined treatment completely suppressed it. p38 MAPK was then revealed to be located upstream of T-bet, and SB203580, but not PD98059, inhibited T-bet-dependent Th1 differentiation. In contrast, ERK1/2 was shown to be located downstream of ICAM-1/LFA-1, and PD98059, but not SB203580, inhibited ICAM-1/LFA-1-dependent Th1 differentiation. Furthermore, it was demonstrated that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK, and that IL-27 directly induces mRNA expression of growth arrest and DNA damage-inducible 45gamma, which is known to mediate activation of p38 MAPK. Finally, IL-12Rbeta2 expression was shown to be up-regulated by IL-27 in both T-bet- and ICAM-1/LFA-1-dependent mechanisms. Taken together, these results suggest that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only p38 MAPK/T-bet-dependent pathway.     

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.     

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of MAPKs after angiotensin II stimulation

Angiotensin II (AII, 100 nM) stimulation of bovine adrenal chromaffin cells (BACCs) produced angiotensin II receptor subtype 1 (AT1)-mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress-activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1-50 microM) and UO126 (0.1-10 microM), dose-dependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 microM, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 microM SB203580 did not affect AII-stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 microM SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 microg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non-specific effects of 20 microM SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.     

Activin A induces erythroid gene expressions and inhibits mitogenic cytokine-mediated K562 colony formation by activating p38 MAPK

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in the regulation of erythroid differentiation. Previous studies have shown that activin A inhibited the colony-forming activity of mouse Friend erythroleukemia cells, however, the mechanism remains unknown. First, we show herein that activin A induced the expression and activated the promoters of alpha-globin and zeta-globin in K562 cells, confirming that activin A induces erythroid differentiation in K562 cells. The p38 mitogen activated protein kinase (MAPK) inhibitor, SB203580, inhibited and the extracellular signal regulated kinase (ERK) inhibitor, PD98059, enhanced the expression and promoter activities of alpha-globin and zeta-globin by activin A, indicating that p38 MAPK and ERK are crucial for activin A-induced erythroid genes expression. Second, SB203580 inhibited the inhibitory effect of activin A on the colony-forming activity of K562 cells using the methylcellulose colony assay, indicating that activin A inhibits K562 colony formation by activating p38 MAPK. In addition, mitogenic cytokines SCF, IL-3, and GM-CSF induced colony formation of K562 cells that could be inhibited by PD98059 or enhanced by SB203580, respectively, indicating that these mitogenic cytokines induce K562 colony formation by activating ERK and inactivating p38 MAPK. Furthermore, activin A reduced the induction effect of these mitogenic cytokines on K562 colony formation in a dose-dependent manner. The inhibition of p38 MAPK reverted the inhibitory effect of activin A on mitogenic cytokine-mediated K562 colony formation. We conclude that activin A can regulate the same pathway via p38 MAPK to coordinate cell proliferation and differentiation of K562 cells.     

Cytokine-induced apoptosis inhibitor-1 causes dedifferentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways

Cytokine-induced apoptosis inhibitor-1 (CIAPIN-1, formally named anamorsin) is a well-known regulator of apoptosis in many different cell types. Recently, it has been reported that some anti-apoptotic proteins are involved with the regulation of cell differentiation. However, relatively little is known about the role of CIAPIN-1 on rabbit articular chondrocytes differentiation. In this study, we investigated the effects of CIAPIN-1 in chondrocytes, focusing on extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling. CIAPIN-1 caused dedifferentiation, as determined by the inhibition of type II collagen expression and sulfated-proteoglycan synthesis. CIAPIN-1 activated ERK-1/2 and inactivated p38 kinase, as determined by the phosphorylation level of each protein. CIAPIN-1-induced ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which also prevented the CIAPIN-1-induced loss of type II collagen expression. Inhibition of p38 kinase with SB203580 enhanced the decrease in type II collagen expression. Our findings collectively suggest that ERK-1/2 and p38 kinase regulate CIAPIN-1-induced dedifferentiation in rabbit articular chondrocytes.     

Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. However, the intracellular death signaling mechanisms by which Cin inhibits tumor cell growth are poorly understood. In this study, we investigated the effect of mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the stress-responsive MAPK pathway induced by Cin in PLC/PRF/5 cells. Trypan blue staining assay indicated that Cin was cytotoxic to PLC/PRF/5 cells. Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and accumulation of sub-G1 peak. It down-regulated the Bcl-2 and Mcl-1 expression, and up-regulated Bax protein in a time-response manner. Treatment with 1 microM Cin resulted in an activation of caspase-8 and cleavage of Bid to its truncated form in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin-induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK. These results conclude that Cin triggers apoptosis in PLC/PRF/5 cells could be through the activation of pro-apoptotic Bcl-2 family (Bax and Bid) proteins and MAPK signaling pathway.     

Shiga toxin activates p38 MAP kinase through cellular Ca(2+) increase in Vero cells

We examined whether the mitogen-activated protein kinase (MAPK) pathway is involved in Shiga toxin (Stx)-induced Vero cell injury. Consonant with cell injury, Stx caused a transient extracellular signal-regulated kinase1/2 (ERK1/2) and a sustained p38 MAPK phosphorylation. p38 MAPK inhibitors (SB 203580 and PD 169316), but not an ERK1/2 kinase inhibitor (PD 98059), partially inhibited the Stx-induced cell death. BAPTA-AM, a Ca(2+) chelator, reduced both cell injury and p38 MAPK phosphorylation. Antioxidants reduced Stx1-induced p38 MAPK phosphorylation. These data indicate that Stx activates p38 MAPK through an increase in intracellular Ca(2+) and reactive oxygen species, and this signaling is involved in Stx-induced cell death.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

Ligand-independent activation of the androgen receptor by insulin-like growth factor-i and the role of the MAPK pathway in skeletal muscle cells

In this study, the roles of the p38 MAPK, ERK1/2 and JNK signaling pathway in IGF-I-induced AR induction and activation were examined. C2C12 cells were treated with IGF-I in the absence or presence of various inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK (SP600125). Inhibition of the MAPK pathway with SB203580, PD98059, or SP600125 significantly decreased IGF-I-induced AR phosphorylation and total AR protein expression. IGF-I-induced nuclear fraction of total AR and phosphorylated AR were significantly inhibited by SB203580, PD98059, or SP600125. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by those inhibitors in dose-dependent manner. Confocal images showed that IGF-I-induced AR nuclear translocation from cytosol was significantly blocked by SB203580, PD98059, or SP600125, suggesting that the MAPK pathway regulates IGF-I-induced AR nuclear localization in skeletal muscle cells. The present results suggest that the MAPK pathways are required for the ligand-independent activation of AR by IGF-I in C2C12 skeletal muscle cells.     

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.     

Nicotine induces the expression of C-reactive protein via MAPK-dependent signal pathway in U937 macrophages

Atherosclerosis is an inflammatory disease in the vessel wall. Nicotine, a major component of cigarette smoke, is an independent risk factor for cardiovascular diseases including atherosclerosis. As an inflammatory molecule, C- reactive protein (CRP) participates in atherogenesis. Although it has been confirmed that CRP level in smoking patient is significantly higher than non-smokers and cigarette withdrawal, it is unknown whether nicotine induces CRP expression in macrophages. The present study was to observe effect of nicotine on CRP production and the related signal pathway in U937 macrophages. The results showed that nicotine significantly increased mRNA and protein expression of CRP in U937 macrophages in time- and concentration-dependent ways. Nicotinic acetylcholine receptor (nAChR) blocker hexamethonium, MEK1/2 inhibitor PD98059, p38 MAPK inhibitor SB203580 and NF-κB inhibitor PDTC almost completely abolished nicotineinduced CRP expression in mRNA and protein levels in U937 macrophages. The further study indicated that hexamethonium, PD98059, and SB203580 significantly inhibited ERK1/2 and p38 MAPK phosphorylation. These demonstrate that nicotine has ability to induce CRP expression in macrophages through nAChR-ERK1/2/p38 MAPK-NF-κB signal pathway, which contributes to better understanding of the pro-inflammatory and pro-atherosclerotic effects of nicotine in cigarette smokers.