The role of RIP2 in p38 MAPK activation in the stressed heart

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.     

Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase

Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.     

p38 mitogen-activated protein kinase (MAPK) first regulates filamentous actin at the 8-16-cell stage during preimplantation development

BACKGROUND INFORMATION: The MAPK (mitogen-activated protein kinase) superfamily of proteins consists of four separate signalling cascades: the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPK); the ERKs (extracellular-signal-regulated kinases); the ERK5 or big MAPK1; and the p38 MAPK group of protein kinases, all of which are highly conserved. To date, our studies have focused on defining the role of the p38 MAPK pathway during preimplantation development. p38 MAPK regulates actin filament formation through the downstream kinases MAPKAPK2/3 (MAPK-activated protein kinase 2/3) or MAPKAPK5 [PRAK (p38 regulated/activated kinase)] and subsequently through HSP25/27 (heat-shock protein 25/27). We recently reported that 2-cell-stage murine embryos treated with cytokine-suppressive anti-inflammatory drugs (CSAIDtrade mark; SB203580 and SB220025) display a reversible blockade of development at the 8-16-cell stage, indicating that p38 (MAPK) activity is required to complete murine preimplantation development. In the present study, we have investigated the stage-specific action and role of p38 MAPK in regulating filamentous actin during murine preimplantation development. RESULTS: Treatment of 8-cell-stage embryos with SB203580 and SB220025 (CSAIDtrade mark) resulted in a blockade of preimplantation development, loss of rhodamine phalloidin fluorescence, MK-p (phosphorylated MAPKAPK2/3), HSP-p (phosphorylated HSP25/27) and a redistribution of alpha-catenin immunofluorescence by 12 h of treatment. In contrast, treatment of 2- and 4-cell-stage embryos with CSAIDtrade mark drugs resulted in a loss of MK-p and HSP-p, but did not result in a loss of rhodamine phalloidin fluorescence. All these effects of p38 MAPK inhibition were reversed upon removal of the inhibitor, and development resumed in a delayed but normal manner to the blastocyst stage. Treatment of 8-cell embryos with PD098059 (ERK pathway inhibitor) did not affect development or fluorescence of MK-p, HSP-p or rhodamine phalloidin. CONCLUSION: Murine preimplantation development becomes dependent on p38 MAPK at the 8-16-cell stage, which corresponds to the stage when p38 MAPK first regulates filamentous actin during early development.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.     

Adenosine A1 and A3 receptor agonists reduce hypoxic injury through the involvement of P38 MAPK

Activation of either the A₁ adenosine receptor (A₁R) or the A₃ adenosine receptor (A₃R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K_ATP) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K_ATP channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K_ATP channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A₁R or A₃R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K_ATP channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.     

Reduced Parasite Motility and Micronemal Protein Secretion by a p38 MAPK Inhibitor Leads to a Severe Impairment of Cell Invasion by the Apicomplexan Parasite Eimeria tenella

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

A selective inhibitor of p38 MAP kinase, SB202190, induced apoptotic cell death of a lipopolysaccharide-treated macrophage-like cell line, J774.1

A selective p38 MAP kinase (p38 MAPK) inhibitor, SB202190, induced apoptotic cell death of a macrophage-like cell line, J774.1, in the presence of lipopolysaccharide (LPS), as judged by DNA nicks revealed by terminal deoxy transferase (TdT)-mediated dUTP nick end labeling (TUNEL), activation of caspase-3, and subsequent release of lactate dehydrogenase. This cytotoxicity was dependent on both LPS and SB202190, and such inhibitors of the upstream LPS-signaling cascade as polymyxin B and TPCK blocked this macrophage cell death. SB202190 suppressed the kinase activity of p38, leading to inhibition of activation of MAPKAPK2 and then the subsequent phosphorylation of hsp27 in LPS-treated macrophages both in vitro and in vivo, but an inactive analog of SB202190, SB202474, did not. There was a threshold of the time of addition of SB202190 to LPS-treated macrophages to induce apoptosis, which was before full transmission of p38 activity to a direct downstream kinase, MAPKAPK2. Besides, localization of phosphorylated hsp27 in Golgi area of the LPS-treated macrophages was suppressed by SB202190, while it was not by SB202474. These results suggest that selective inhibition of p38 MAPK activity in LPS-induced MAP kinase cascade leads to apoptosis of macrophages.     

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

Differential requirement of members of the MAPK family for CCL2 expression by hepatic stellate cells

Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including CCL2 (formerly known as monocyte chemoattractant protein-1). In this study, we evaluated the role of different proteins of the MAPK family (ERK, p38(MAPK), and JNK) in the regulation of CCL2 expression by HSC, as an index of their proinflammatory activity. Several mediators activated all three MAPK, including TNF, IL-1, and PDGF. To assess the relative role of the different MAPKs, specific pharmacological inhibitors were used; namely, SB203580 (p38(MAPK)), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system were verified analyzing the enzymatic activity of the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation of downstream substrates. SB203580 and SP600125 dose-dependently inhibited CCL2 secretion and gene expression induced by IL-1 or TNF. In contrast, inhibition of ERK did not affect the upregulation of CCL2 induced by the two cytokines. Finally, activin A was also found to stimulate CCL2 expression and to activate ERK, JNK, p38, and their downstream targets. Unlike in cells exposed to proinflammatory cytokines, all three MAPKs were required to induce CCL2 secretion in response to activin. We conclude that members of the MAPK family differentially regulate cytokine-induced chemokine expression in human HSC.     

Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the Fc(epsilon)RI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following Fc(epsilon)RI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10 microM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for Fc(epsilon)RI-mediated cell migration.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Apoptosis of macrophages induced by Trichomonas vaginalis through the phosphorylation of p38 mitogen-activated protein kinase that locates at downstream of mitochondria-dependent caspase activation

Trichomonas vaginalis, a flagellated protozoan parasite, is the causative organism of trichomoniasis. We have recently demonstrated that T. vaginalis induces apoptotic cell death via a Bcl-x(L)-dependent pathway in RAW264.7 macrophages. In this study, we attempted to characterize in detail the signaling cascades resulting in T. vaginalis-induced macrophage apoptosis, focusing particularly on mitochondrial changes and the role of p38 mitogen-activated protein kinase (p38 MAPK) activation. We found that T. vaginalis induced mitochondrial changes including the release of cytochrome c and the serial activation of caspases, leading to the activation of p38 MAPK in macrophages. These biochemical changes culminated in the apoptosis of the host cells. Caspase inhibitors induced a significant inhibition of T. vaginalis-induced nuclear damage, as well as the activation of p38 MAPK. Treatment with the p38 MAPK inhibitor, SB203580, or the overexpression of kinase-inactive p38 MAPK, induced an attenuation of T. vaginalis-induced apoptosis but not cytochrome c release, the activation of caspase-9 and caspase-3, or PARP cleavage. Furthermore, SB203580 treatment to human macrophages consistently blocked T. vaginalis-induced apoptosis. Collectively, our findings indicate that p38 MAPK signaling cascade is requisite to apoptosis of T. vaginalis-infected macrophage, and this apoptotic process occurs via the phosphorylation of p38 MAPK, which is located downstream of mitochondria-dependent caspase activation, conferring insight into the plausible molecular mechanism of T. vaginalis-immune evasion from macrophage attack.     

HSP27 phosphorylation is correlated with ADP-induced platelet granule secretion

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. ADP has been reported to induce heat-shock protein (HSP) 27 phosphorylation in human platelets. However, the exact role of HSP27 phosphorylation in human platelets has not yet been clarified. In the present study, we investigated the mechanisms and the roles of ADP-induced HSP27 phosphorylation in human platelets. We showed for the first time that both of decreased phosphorylation levels of HSP27 by PD98059, a MEK1/2 inhibitor and SB203580, a p38 MAPK inhibitor were correlated with the suppressed levels of platelet granule secretion but not with platelet aggregation. Furthermore, the inhibition of either the p44/p42 MAPK or p38 MAPK pathways had no effect on ADP-induced platelet aggregation. These results strongly suggest that the ADP-induced phosphorylation of HSP27 via p44/p42 MAPK and/or p38 MAPK is therefore sufficient for platelet granule secretion but not for platelet aggregation in humans.     

The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research

In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/β inhibitors. In this “Letter to the editor” we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.     

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid

Human lung neoplasms frequently express mutations that down‐regulate expression of various tumor suppressor molecules, including mitogen‐activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti‐tumor therapies. We now report that 4‐phenyl‐3‐butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non‐cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell‐cell communication. H2009 human lung carcinoma cells and ras‐transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras‐transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell–cell communication and connexin 43 phosphorylation in ras‐transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up‐regulation of p38 MAPK activity, altered connexin 43 expression, and down‐regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down‐regulated p38 MAPK activity and/or activated JNK and altered cell–cell communication. J. Cell. Biochem. 113: 269–281, 2012. © 2011 Wiley Periodicals, Inc.     

A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration.

Smooth muscle cells are exposed to growth factors and cytokines that contribute to pathological states including airway hyperresponsiveness, atherosclerosis, angiogenesis, smooth muscle hypertrophy, and hyperplasia. A common feature of several of these conditions is migration of smooth muscle beyond the initial boundary of the organ. Signal transduction pathways activated by extracellular signals that instigate migration are mostly undefined in smooth muscles. We measured migration of cultured tracheal myocytes in response to platelet-derived growth factor, interleukin-1beta, and transforming growth factor-beta. Cellular migration was blocked by SB203580, an inhibitor of p38(MAPK). Time course experiments demonstrated increased phosphorylation of p38(MAPK). Activation of p38(MAPK) resulted in the phosphorylation of HSP27 (heat shock protein 27), which may modulate F-actin polymerization. Inhibition of p38(MAPK) activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38(MAPK), increased cell migration, whereas overexpression of p38alpha MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38(MAPK) pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction.