Stem cell factor-induced migration of mast cells requires p38 mitogen-activated protein kinase activity.

Stem cell factor (SCF) can be considered a cardinal cytokine in mast cell biology as it affects mast cell differentiation, survival, and migration. The objective of this study was to investigate the role of two mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) and p38, in SCF-induced cell migration. This was examined in mouse mast cells by using PD 098059 and SB203580, which are specific inhibitors of mitogen-induced extracellular kinase (MEK) and p38 MAP kinase, respectively. SCF induced a rapid and transient activation of ERK and p38 in a dose-dependent manner. Inhibition of p38 activity by SB203580 was paralleled with a marked reduction of migration toward SCF, whereas the effect of the MEK inhibitor was less pronounced. This is the first report of a physiological function of SCF-dependent activation of p38. Whether p38-mediated mast cell migration is a possible target for suppression of mast cell hyperplasia remains to be determined.     

Alpha(v) integrin, p38 mitogen-activated protein kinase, and urokinase plasminogen activator are functionally linked in invasive breast cancer cells

We reported previously that endogenous p38 MAPK activity is elevated in invasive breast cancer cells and that constitutive p38 MAPK activity is important for overproduction of uPA in these cells (Huang, S., New, L., Pan, Z., Han, J., and Nemerow, G. R. (2000) J. Biol. Chem. 275, 12266-12272). However, it is unclear how elevated endogenous p38 MAPK activity is maintained in invasive breast cancer cells. In the present study, we found that blocking alpha(v) integrin functionality with a function-blocking monoclonal antibody or down-regulating alpha(v) integrin expression with alpha(v)-specific antisense oligonucleotides significantly decreased the levels of active p38 MAPK and inhibited cell-associated uPA expression in invasive breast cancer MDA-MB-231 cells. These results suggest a function link between alpha(v) integrin, p38 MAPK activity, and uPA expression in invasive tumor cells. We also found that vitronectin/alpha(v) integrin ligation specifically induced p38 MAPK activation and uPA up-regulation in invasive MDA-MB-231 cells but not in non-invasive MCF7 cells. Finally, using a panel of melanoma cells, we demonstrated that the cytoplasmic tail of alpha(v) integrin subunit is required for alpha(v) integrin ligation-induced p38 MAPK activation.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

Active mutants of the human p38alpha mitogen-activated protein kinase

Mitogen-activated protein (MAP) kinases compose a family of serine/threonine kinases that function in many signal transduction pathways and affect various cellular phenotypes. Despite the abundance of available data, the exact role of each MAP kinase is not completely defined, in part because of the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358), we designed and constructed single and multiple mutants of human MAP kinase p38alpha. Single (p38D176A, p38F327L, and p38F327S) and subsequent double (p38D176A/F327L and p38D176A/F327S) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38alpha. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38gamma active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.     

Involvement of p38alpha mitogen-activated protein kinase in lung metastasis of tumor cells

To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38alpha(+/-) mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38alpha(+/-) mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38alpha(+/-) mice. Platelets of p38alpha(+/-) mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38alpha(+/-) mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38alpha(+/-) mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38alpha(+/-) mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38alpha(+/-) mice was similar to that of WT mice. These results suggest that p38alpha plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis.     

Interferon alpha and ribavirin collaboratively regulate p38 mitogen-activated protein kinase signaling in hepatoma cells

Signaling events triggered by interferon alpha (IFN-α) and ribavirin are involved in anti-hepatitis C virus (HCV) action. The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in HCV pathogenesis. Effects of IFN-α and ribavirin on p38 MAPK signaling were investigated in human hepatoma cells. Type I IFN receptor 2 (IFNAR2) mediated IFN-α-induced p38 MAPK phosphorylation. Also, p38 MAPK phosphorylation was enhanced by ribavirin. Treatment for 48 h with a combination of IFN-α and ribavirin increased p38 MAPK phosphorylation, whereas the treatment for 72 h reduced p38 MAPK phosphorylation. Cell culture-derived HCV (HCVcc) infection dramatically increased p38 MAPK phosphorylation and such phosphorylation was inhibited by IFN-α or ribavirin. Moreover, siRNA-mediated knockdown of p38 MAPK resulted in enhancement of ribavirin-dependent HCV RNA replication. These results suggest that regulation of p38 MAPK signaling by IFN-α and ribavirin might contribute to anti-HCV action.     

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.     

p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression

The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.     

Nitric oxide activation of p38 mitogen-activated protein kinase in 293T fibroblasts requires cGMP-dependent protein kinase

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.     

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

Cocaine up-regulation of the norepinephrine transporter requires threonine 30 phosphorylation by p38 mitogen-activated protein kinase

The norepinephrine (NE) transporter (NET) regulates NE signaling by rapidly clearing synaptic NE. Cocaine binds NET and modulates NE transport. These actions contribute to rewarding effects and abuse liability of cocaine. Activation of mitogen-activated protein kinase (MAPK) cascades is implicated in cocaine-induced neuroadaptations. However, the role of MAPK and the mechanisms involved in cocaine modulation of NET are not clear. Acute intra-peritoneal injections of cocaine (20 mg/kg body weight) to rats resulted in increased NE uptake by prefrontal cortex (PFC) synaptosomes with a parallel increase in the surface expression of endogenous NET. Cocaine also enhanced the immunoreactivity of phospho-p38 MAPK in the PFC synaptosomes without affecting the total p38 MAPK. In vitro cocaine (30-50 μM) treatment of rat PFC synaptosomes increased native NET function, surface expression, and phosphorylation in a manner sensitive to p38 MAPK inhibition by PD169316. We next examined cocaine-elicited effects on wild-type human NET (hNET) expressed heterologously in human placental trophoblast cells to gain more insights into the mechanisms involved. Cocaine treatment of hNET expressing human placental trophoblast cells up-regulated the function, surface expression, and phosphorylation of hNET in a PD169316-sensitive manner. In addition, cocaine inhibited constitutive endocytosis of hNET. Mutational analysis of serine and threonine residues revealed that substitution of threonine 30, located at the amino terminus of hNET with alanine (T30A-hNET), abolished cocaine-induced up-regulation of NET function, surface expression, and phosphorylation. Furthermore, cocaine did not alter T30A-hNET endocytosis. These studies identify a novel molecular mechanism that cocaine-activated p38 MAPK-mediated phosphorylation of NET-T30 dictates surface NET availability, and hence, NE transport.     

Endothelin-1 activates p38 mitogen-activated protein kinase via endothelin-A receptor in rat myocardial cells

In myocardial cells (MCs), endothelin-1 (ET-1) exerts various effects such as hypertrophy, and causes cellular injury. Long-term treatment with an endothelin-A (ET_A) receptor antagonist improves the survival of rats with heart failure, suggesting that myocardial endothelin system contributes to the progression of heart failure. p38 mitogen-activated kinase (MAPK) is a member of the MAPK family and activated by several forms of environmental stresses. We show here the effect of ET-1 on p38 MAPK activation and the role of ET-1-activated p38 MAPK on morphological changes in MCs. ET-1-stimulated p38 MAPK phosphorylation was detectable within 2 min and maximal at 5 min and was concentration dependent. The maximum effect was obtained at 10 nM. An ET_A receptor antagonist, BQ-123, but not an endothelin-B receptor antagonist, BQ-788, inhibited these reactions. A p38 MAPK inhibitor, SB203580, failed to inhibit the morphological changes associated with ET-1-induced myocardial cell hypertrophy. These results indicate that p38 MAPK is activated by ET-1 but does not contribute to the development of ET-1-induced myocardial cell hypertrophy.     

Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase

p38 mitogen-activated protein (MAP) kinases play an important role in the regulation of cellular responses to all kinds of stresses. The most abundant and broadly expressed p38 MAP kinase is p38alpha, which can also control the proliferation, differentiation, and survival of several cell types. Here we show that the absence of p38alpha correlates with the up-regulation of one of its upstream activators, the MAP kinase kinase MKK6, in p38alpha(-/-) knockout mice and in cultured cells derived from them. In contrast, the expression levels of the p38 activators MKK3 and MKK4 are not affected in p38alpha-deficient cells. The increase in MKK6 protein concentration correlates with increased amounts of MKK6 mRNA in the p38alpha(-/-) cells. Pharmacological inhibition of p38alpha also up-regulates MKK6 mRNA levels in HEK293 cells. Conversely, reintroduction of p38alpha into p38alpha(-/-) cells reduces the levels of MKK6 protein and mRNA to the normal levels found in wild-type cells. Moreover, we show that the MKK6 mRNA is more stable in p38alpha(-/-) cells and that the 3'untranslated region of this mRNA can differentially regulate the stability of the lacZ reporter gene in a p38alpha-dependent manner. Our data indicate that p38alpha can negatively regulate the stability of the MKK6 mRNA and thus control the steady-state concentration of one of its upstream activators.