LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase

Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.     

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.     

Specific regulation of cytokine-dependent p38 MAP kinase activation by p62/SQSTM1

We have previously shown that p62/SQSTM1 binds to p38. In this study, we identified two association domains of p62 to p38 by conducting co-immunoprecipitation experiments. One domain comprises the amino acids 173-182, named N-terminal p38 interaction (NPI) domain, and the other domain comprises the amino acids 335-344, named C-terminal p38 interaction (CPI) domain. An aspartic acid tripeptide located at 335-337 was required for their association. However, the direct interaction was only observed between the recombinant p38 and the peptide of the NPI domain, but not that of the CPI domain in the surface plasmon resonance analyses. These results suggest that the CPI domain may serve to form a certain conformation suitable for the association with p38. Furthermore, we showed that knockdown of p62 expression by siRNA led to impaired p38 phosphorylation only when HeLa cells were stimulated by cytokine. The critical role of p62 in cytokine-dependent p38 signalling pathway was further confirmed by measuring IL-8 mRNA. Cytokine mRNA is often stabilized via p38 pathway. In the absence of p62, IL-8 mRNA induced by IL-1beta became more fragile. These data show that p62 specifically regulates cytokine-dependent p38 signalling pathway.     

p38 MAP kinase regulates benzo(a)pyrene-induced apoptosis through the regulation of p53 activation

Polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), are widespread in the environment and cause untoward effects, including carcinogenesis, in mammalian cells. However, the molecular mechanism of apoptosis by BaP is remained to be elusive. Pharmacological inhibition of p38 kinase markedly inhibited the BaP-induced cytotoxicity, which was proven as apoptosis characterized by an increase in sub-G(0)/G(1) fraction of DNA content, ladder-pattern fragmentation of genomic DNA, and catalytic activation of caspase-3 with PARP cleavage. Our data also demonstrated that activation of caspase-3 was accompanied with activation of caspase-9 and mitochondrial dysfunction, which was also apparently suppressed by pretreatment with p38 kinase inhibitors. Also, pharmacological inhibition of p38 markedly inhibited the phosphorylation, accumulated expression, and transactivation activity of p53 in BaP-treated cells. Adenoviral overexpression of human p53 (wild-type) further augmented in increase of PARP cleavage and the sub-G(0)/G(1) fraction of DNA content. Furthermore, p53 mediated apoptotic activity in BaP-treated cells was inhibited by p38 kinase inhibitor. The current data collectively indicate that BaP induces apoptosis of Hepa1c1c7 cells via activation of p53-related signaling, which was, in part, regulated by p38 kinase.     

Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP kinase

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) or of Akt induces myoblast differentiation. Activation of p38 MAP kinase also triggers myogenic differentiation. The current paper shows that PI 3-kinase and p38 MAP kinase signalling are activated by two separate pathways during myogenic differentiation; both are required for muscle differentiation. p38-induced myogenic differentiation can be inhibited by the PI 3-kinase inhibitor LY294002 without affecting p38 activity. Similarly, a constitutively active form of Akt, myristylated c-Akt (Myr-Akt), induces myogenic differentiation that is inhibited by the p38 inhibitor SB203580. An analysis of the two forms of p38, p38 and p38beta, shows that the activity of both is required for myogenic differentiation. These data suggest that PI 3-kinase and p38 signalling are essential and parallel pathways for myogenic differentiation. They may either affect different downstream targets required for myogenesis or they may converge on shared targets that require input from both signalling pathways.     

Inhibitors of unactivated p38 MAP kinase

Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.     

'Reverse' alpha-ketoamide-based p38 MAP kinase inhibitors

We have identified a second series of potent p38 inhibitors. As with our first generation series, these compounds are based on an alpha-ketoamide scaffold. The reversal of the ketoamide order, however, introduces more chemical flexibility and in addition results in improve potencies against p38.     

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.     

p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65

The stress-activated c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) regulate apoptosis induced by several forms of cellular insults. Potential targets for these kinases include members of the Bcl-2 family proteins, which mediate apoptosis generated through the mitochondria-initiated, intrinsic cell death pathway. Indeed, the activities of several Bcl-2 family proteins, both pro- and anti-apoptotic, are controlled by JNK phosphorylation. For example, the pro-apoptotic activity of Bim(EL), a member of the Bcl-2 family, is stimulated by JNK phosphorylation at Ser-65. In contrast, there is no reported evidence that p38-induced apoptosis is due to direct phosphorylation of Bcl-2 family proteins. Here we report evidence that sodium arsenite-induced apoptosis in PC12 cells may be due to direct phosphorylation of Bim(EL) at Ser-65 by p38. This conclusion is supported by data showing that ectopic expression of a wild type, but not a non-phosphorylatable S65A mutant of Bim(EL), potentiates sodium arsenite-induced apoptosis and by experiments showing direct phosphorylation of Bim(EL) at Ser-65 by p38 in vitro. Furthermore, sodium arsenite induced Bim(EL) phosphorylation at Ser-65, which was blocked by p38 inhibition. This study provides the first example whereby p38 induces apoptosis by phosphorylating a member of the Bcl-2 family and illustrates that phosphorylation of Bim(EL) on Ser-65 may be a common regulatory point for cell death induced by both JNK and p38 pathways.     

p85 beta-PIX is required for cell motility through phosphorylations of focal adhesion kinase and p38 MAP kinase

Lysophosphatidic acid (LPA) mediates diverse biological responses, including cell migration, through the activation of G-protein-coupled receptors. Recently, we have shown that LPA stimulates p21-activated kinase (PAK) that is critical for focal adhesion kinase (FAK) phosphorylation and cell motility. Here, we provide the direct evidence that p85 beta-PIX is required for cell motility of NIH-3T3 cells by LPA through FAK and p38 MAP kinase phosphorylations. LPA induced p85 beta-PIX binding to FAK in NIH-3T3 cells that was inhibited by pretreatment of the cells with phosphoinositide 3-kinase inhibitor, LY294002. Furthermore, the similar inhibition of the complex formation was also observed, when the cells were transfected with either p85 beta-PIX mutant that cannot bind GIT or dominant negative mutants of Rac1 (N17Rac1) and PAK (PAK-PID). Transfection of the cells with specific p85 beta-PIX siRNA led to drastic inhibition of LPA-induced FAK phosphorylation, peripheral redistribution of p85 beta-PIX with FAK and GIT1, and cell motility. p85 beta-PIX was also required for p38 MAP kinase phosphorylation induced by LPA. Finally, dominant negative mutant of Rho (N19Rho)-transfected cells did not affect PAK activation, while the cells stably transfected with p85 beta-PIX siRNA or N17Rac1 showed the reduction of LPA-induced PAK activation. Taken together, the present data suggest that p85 beta-PIX, located downstream of Rac1, is a key regulator for the activations of FAK or p38 MAP kinase and plays a pivotal role in focal complex formation and cell motility induced by LPA.     

Imidazopyrimidines,potent inhibitors of p38 MAP kinase

The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohn's disease. A novel series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-alpha in vivo.     

Benzothiazole based inhibitors of p38alpha MAP kinase

Rational design, synthesis, and SAR studies of a novel class of benzothiazole based inhibitors of p38alpha MAP kinase are described. The issue of metabolic instability associated with vicinal phenyl, benzo[d]thiazol-6-yl oxazoles/imidazoles was addressed by the replacement of the central oxazole or imidazole ring with an aminopyrazole system. The proposed binding mode of this new class of p38alpha inhibitors was confirmed by X-ray crystallographic studies of a representative inhibitor (6a) bound to the p38alpha enzyme.     

Estrogen prevents cardiomyocyte apoptosis through inhibition of reactive oxygen species and differential regulation of p38 kinase isoforms

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.     

Preconditioning enhanced glucose uptake is mediated by p38 MAP kinase not by phosphatidylinositol 3-kinase

Ischemia is reported to stimulate glucose uptake, but the signaling pathways involved are poorly understood. Modulation of glucose transport could be important for the cardioprotective effects of brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning. Previous work indicates that preconditioning reduces production of acid and lactate during subsequent sustained ischemia, consistent with decreased glucose utilization. However, there are also data that preconditioning enhances glucose uptake. The present study examines whether preconditioning alters glucose transport and whether this is mediated by either phosphatidylinositol 3-kinase (PI3K) or p38 MAP kinase. Langendorff-perfused rat hearts were preconditioned with 4 cycles of 5 min of ischemia and 5 min of reperfusion, with glucose as substrate. During the last reflow, glucose was replaced with 5 mM acetate and 5 mM 2-deoxyglucose (2DG), and hexose transport was measured from the rate of production of 2-deoxyglucose 6-phosphate (2DG6P), using (31)P nuclear magnetic resonance. Preconditioning stimulated 2DG uptake; after 15 min of perfusion with 2DG, 2DG6P levels were 165% of initial ATP in preconditioned hearts compared with 96% in control hearts (p < 0.05). Wortmannin, an inhibitor of PI3K, did not block the preconditioning induced stimulation of 2DG6P production, but perfusion with SB202190, an inhibitor of p38 MAP kinase, did attenuate 2DG6P accumulation (111% of initial ATP, p < 0. 05 compared with preconditioned hearts). SB202190 had no effect on 2DG6P accumulation in nonpreconditioned hearts. Preconditioning stimulation of translocation of GLUT4 to the plasma membrane was not inhibited by wortmannin. The data demonstrate that ischemic preconditioning increases hexose transport and that this is mediated by p38 MAP kinase and is PI3K-independent.     

p38 MAP kinase-dependent regulation of endothelial cell permeability

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.