Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK) signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPK and p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provided cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 was found to abolish UV-induced SAPK and p38 MAPK activity, and inhibit UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 was also found to inhibit other biochemical events associated with apoptosis, including activation of caspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase. These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect on UV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it was found that MKP-1 inhibited UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit UV-induced activation of SAPK, did not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity.     

Stimulation of alpha1A-adrenoceptors in Rat-1 cells inhibits extracellular signal-regulated kinase by activating p38 mitogen-activated protein kinase

We report a patient with cholangiocellular carcinoma with tumor thrombi in the main portal trunk who has survived for 9.5 years after hepatic resection. A 57-year-old woman underwent an extended left lobectomy, and resection of the caudate lobe plus the main portal trunk for a liver tumor that had a portal tumor thrombus in the main portal trunk. The portal vein was reconstructed with an autologous vein graft obtained from the external iliac vein. Histological examination of the resected specimen revealed moderately differentiated tubular adenocarcinoma compatible with cholangiocellular carcinoma. Factors contributing to the patient's long-term survival are discussed. Aggressive surgical resection can be effective even for such an advanced case of cholangiocellular carcinoma.     

Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst

Bacterial LPS elicits both rapid activation of the stress-activated MAP kinase p38 in polymorphonuclear leukocytes (PMN) and rapid adhesion of the PMN to ligands for the leukocyte integrin CD11b/CD18. The functional correlation between these two events was examined. The time course for tyrosine phosphorylation of p38 in PMN in response to 10 ng/ml LPS in 1% normal human serum was consistent with participation in signaling for leukocyte integrin-dependent adhesion, with transient phosphorylation peaking at 10 to 20 min. The concentration dependence of p38 phosphorylation also resembled that for PMN adhesion, with <1 ng/ml LPS eliciting a response. Phosphorylation was inhibited by mAb 60b against CD14, but not by mAb 26ic, a nonblocking anti-CD14. The function of p38 in integrin-dependent adhesion and the adhesion-dependent oxidative burst was tested using a specific inhibitor of p38, SB203580. SB203580 inhibited adhesion by diminishing the initial rate of adherence in response to both LPS and TNF, with a half-maximal concentration in the range of 0.1 to 0.6 microM. It did not, however, block adhesion in response to formyl peptide or PMA. The p38 inhibitor also blocked the adhesion-dependent oxidative burst with a half-maximal concentration similar to that for adhesion. Timed delivery of the compound during the lag phase preceding H2O2 production suggested that p38 kinase activity was required throughout the lag but not after the oxidase was assembled. These results suggest that p38 functions in PMN to signal leukocyte integrin-dependent adhesion and the subsequent massive production of reactive oxygen intermediates.     

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.     

Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase

The changes in airway osmolarity have been described to contribute to the production of exercise- induced bronchoconstriction (EIB) and the development of the late-phase response (LPR). The mechanism has been investigated; however, the responsiveness of bronchial epithelial cells (BEC) to hyperosmolarity and the intracellular signals leading to cell activation have not been determined. In this study, we examined the effect of hyperosmolar medium on interleukin-8 (IL-8) expression and the role of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2 terminal kinase ( JNK) in human BEC in this response in order to clarify the intracellular signals regulating IL-8 expression in hyperosmolarity-stimulated BEC. The results showed that hyperosmolarity induced IL-8 expression in a concentration dependent manner, p38 MAP kinase phosphorylation and activation, and JNK activation whether NaCl or mannitol was used as the solute. SB 203580 as the specific p38 MAP kinase inhibitor inhibited hyperosmolarity-induced p38 MAP kinase activation and partially inhibited hyperosmolarity-induced IL-8 expression. These results indicate that p38 MAP kinase, at least in part, regulates hyperosmolarity-induced IL-8 expression in BEC. However, other signals such as JNK are possibly also involved. These results provide new evidence on the mechanism responsible for the development of the LPR induced by EIB, and a strategy for treatment with the specific p38 MAP kinase inhibitor.     

TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease

TL1 is a recently discovered novel member of the tumor necrosis factor (TNF) cytokine family. TL1 is abundantly expressed in endothelial cells, but its function is not known. The present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 72 ng/ml). The effect of TL1 was not inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas expression in BPAEC at 8 and 24 h after treatment, and significantly activated stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (p38 MAPK). The peak activities of SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold, respectively. TL1-induced apoptosis in the BPAEC was reduced by expression of a dominant-interfering mutant of c-Jun (62.8%, p < 0.05) or by a specific p38 inhibitor, SB203580 (1-10 microM) dose-dependently. TL1 also activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was significantly attenuated by the caspase inhibitor, ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC was caspase-3, which was based on substrate specificity and immunocytochemical analysis. These findings suggest that TL1 may act as an autocrine factor to induce apoptosis in endothelial cells via activation of multiple signaling pathways, including stress protein kinases as well as certain caspases.     

Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.     

Protein phosphatase 2Calpha inhibits the human stress-responsive p38 and JNK MAPK pathways

MAPK (mitogen-activated protein kinase) cascades are common eukaryotic signaling modules that consist of a MAPK, a MAPK kinase (MAPKK) and a MAPKK kinase (MAPKKK). Because phosphorylation is essential for the activation of both MAPKKs and MAPKs, protein phosphatases are likely to be important regulators of signaling through MAPK cascades. To identify protein phosphatases that negatively regulate the stress-responsive p38 and JNK MAPK cascades, we screened human cDNA libraries for genes that down-regulated the yeast HOG1 MAPK pathway, which shares similarities with the p38 and JNK pathways, using a hyperactivating yeast mutant. In this screen, the human protein phosphatase type 2Calpha (PP2Calpha) was found to negatively regulate the HOG1 pathway in yeast. Moreover, when expressed in mammalian cells, PP2Calpha inhibited the activation of the p38 and JNK cascades induced by environmental stresses. Both in vivo and in vitro observations indicated that PP2Calpha dephosphorylated and inactivated MAPKKs (MKK6 and SEK1) and a MAPK (p38) in the stress-responsive MAPK cascades. Furthermore, a direct interaction of PP2Calpha and p38 was demonstrated by a co-immunoprecipitation assay. This interaction was observed only when cells were stimulated with stresses or when a catalytically inactive PP2Calpha mutant was used, suggesting that only the phosphorylated form of p38 interacts with PP2Calpha.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

Activation of the mitogen-activated protein kinase pathway in U937 leukemic cells induces phosphorylation of the amino terminus of the TATA-binding protein

Quality-adjusted life-years (QALYs) and willingness to pay (WTP) are two preference-based measures of health-related outcomes. In this article, we compare these two measures in eliciting individuals' preferences for health outcomes associated with shingles. To collect the necessary preference data, we administered computer-interactive interviews to a sample of 65- to 70-year-olds. We found no significant correlation between QALYs and WTP across individuals. We discuss our findings and argue that our results raise questions about whether QALYs and WTP are equivalent preference-based measures of health outcomes.     

Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms

The p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that are induced in response to lipopolysaccharide, hyperosmolarity, and interleukin 1. p38 MAP kinase appears to play a role in regulating inflammatory responses, including cytokine secretion and apoptosis. Here we show that diverse classes of DNA-damaging agents such as cisplatinum, 1-beta-D-arabinofuranosylcytosine, UV light, ionizing radiation, and methyl methanesulfonate activate p38 MAP kinase. We also demonstrate that cells deficient in c-Abl fail to activate p38 MAP kinase after treatment with cisplatinum and 1-beta-D-arabinofuranosylcytosine but not after exposure to UV and methyl methanesulfonate. Reconstitution of c-Abl in the Abl-/- cells restores that response. Similar results were obtained for induction of the Jun-NH2-kinase/stress-activated protein kinase. These findings indicate that p38 MAP and Jun-NH2-kinase/stress-activated protein kinases are differentially regulated in response to different classes of DNA-damaging agents.     

Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells

Between 1985 and 1987, 1837 primary total knee arthroplasty (TKA) prostheses were implanted in 1503 patients. Group I included 843 knee with metal-backed patellar components (MBPC), and group II included 994 knees with all polyethylene patellar components (APPC). Follow-up averaged 5.7 years (range, 2 to 11 years). Twenty-four MBPC (2.9%) and 16 APPC TKA cases (1.6%) developed deep infection. In the time interval between arthroplasty and 2-year follow-up, eight MBPC and 11 APPC knees developed deep periprosthetic infection. The difference in the cumulative probability of infection between the two groups during this time interval was not significant (relative risk, 0.9; 95% confidence interval [CI], 0.3-2.1; P = 0.73). However, after the 2-year follow-up, 16 MBPC and 5 APPC knees developed late infection, and the difference in the cumulative probability of infection between the MBPC and APPC knees during this time interval was significant (relative risk, 3.1; 95% CI, 1.1-8.5; P = 0.02). Although the mechanism for this increased risk of these late infections is not well understood, the attendant synovitis, effusion, and relative hyperemia of these knees in the presence of the particulate metal and polyethylene debris may increase the potential of bacterial seeding to these prostheses. Particulate metal debris has been previously shown to suppress bacterial phagocytosis and may play a role in the pathogenesis of these infections. We propose that the presence of metal-backed patellar failure represents a "prosthesis at risk" for the development of late prosthetic infection.     

Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB

We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.