Targeting mitogen-activated protein kinase phosphatase-1 (MKP-1): structure-based design of MKP-1 inhibitors and upregulators

Mitogen-activated protein kinase phosphatases (MKPs) are dual specificity protein phosphatases (DUSPs) that dephosphorylate both phospho-tyrosine and phospho-threonine residues on mitogen-activated protein kinases (MAPKs). Because the MAPK family of signalling molecules (phospho-p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)) play essential roles in cell signalling pathways that regulate cell growth and inflammation, controlling MAPK-mediated pathways is a therapeutically attractive strategy. While small molecule MAPK inhibitors have utility, in this review we will focus on exploring the potential of targeting the endogenous MAPK deactivator--MKP-1. Importantly, there is a strong justification for developing both inhibitors and upregulators of MKP-1 because of the diverse roles played by MAPKs in disease: for example, in cancer, MKP-1 inhibitors may prove beneficial, as MKP-1 is overexpressed and is considered responsible for the failure of JNK-driven apoptotic pathways induced by chemotherapeutics; conversely, in inflammatory diseases such as asthma and arthritis, MKP-1 reduces MAPK-mediated signalling and developing novel ligands to upregulate MKP-1 levels would be a therapeutically attractive anti-inflammatory strategy. Thus, in this review we utilise MKP-1 homology modeling to highlight the structural features of MKP-1 inhibitors that permit potent and selective inhibition, and to provide insights into the structural requirements for selective MKP-1 upregulators.     

Dual-specificity MAP kinase phosphatases as targets of cancer treatment

The protein tyrosine phosphatase family (PTP) contains a group of dual-specificity phosphatases (DUSPs) that regulate the activivity of MAP kinases (MAPKs), which are key effectors in the control of cell growth and survival in physiological and pathological processes, including cancer. These phosphatases, named as MKP-DUSPs, include the MAPK phosphatases (MKPs) as well as a group of small-size atypical DUSPs structurally and functionally related to the MKPs. MKP-DUSPs, in most of the cases, are direct inactivators of MAPKs by dephosphorylation of both the Thr and the Tyr regulatory residues at the MAPKs catalytic loop. In some other cases, MKP-DUSPs regulate the activity of MAPKs indirectly, acting through upstream MAPK pathways components. The active involvement of MKP-DUSPs in oncogenesis or resistance to cancer therapies is now well documented, making the search and validation of MKP-DUSPs inhibitors a prominent area in clinical cancer research. Here, we review the current knowledge on the role of MKP-DUSPs in human cancer, the status of the preclinical development and validation of specific MKP-DUSP inhibitors, and the potential of MKP-DUSPs as targets for anti-cancer drugs.     

Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dual-specificity protein phosphatase inhibitors

We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dual-specificity phosphatase inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for inhibitors of MKP-1 activity at 10 microM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by > or =50% at 10 microM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent inhibitors of MKP-1 with 50% inhibitory concentration (IC(50)) values of <50 microM; four exhibited IC(50) values <1.0 microM, six produced IC(50) values in the 1-10 microM range, and 36 produced IC(50) values in the 10-50 microM range. A clustering and classification analysis of the compound structures of the 46 confirmed MKP-1 inhibitors produced 29 singleton structures and seven clusters of related structures. Some MKP-1 inhibitors were members of structural classes or contained substructure pharmacophores that previously were reported to inhibit either MKP-1 or other protein tyrosine phosphatases, validating the HTS assay. Importantly, we have identified several attractive and novel MKP-1 inhibitor structures that warrant further investigation as potential probes to study the biology of MKP-1 and its role in controlling the amplitude and/or duration of MAPK signaling, cell survival, and tumor progression.     

Transforming growth factor-beta 1 impairs endothelin-1-mediated contraction of brain vessels by inducing mitogen-activated protein (MAP) kinase phosphatase-1 and inhibiting p38 MAP kinase

Brain levels of transforming growth factor-beta1 (TGF-beta1) are increased in Alzheimer's disease and have been implicated in the associated cerebrovascular pathology. We recently reported that transgenic mice that overexpress TGF-beta1 (TGF+ mice) display, with aging, selectively reduced endothelin-1 (ET-1)-mediated contractions. Because ET-1 is a key regulator of cerebrovascular tone and homeostasis, we investigated how increased levels of TGF-beta1 could selectively alter this contractile response. We found that ETA receptors, via activation of p38 mitogen-activated protein (MAP) kinase, mediate the ET-1-induced contraction in mouse cerebral arteries, a response significantly decreased in aged TGF+ mice (-39%; p < 0.01) despite unaltered ETA receptor levels or affinity. In cerebrovascular smooth muscle cell cultures, long-term treatment with TGF-beta1 significantly decreased (>50%; p < 0.05) the ET-1-induced activation of the p38 MAPK/27-kDa heat shock protein (HSP27) signaling pathway. This occurred with no effect upstream to p38 MAP kinase but with the concomitant induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. Inhibition of MKP-1 expression with Ro-31-8220 or suppression of MKP-1 expression by short interfering RNA restored the ET-1-mediated p38 MAP kinase response. These results disclose a new role for long-term increases of TGF-beta1 in modulating cerebrovascular tone by dampening ET-1-mediated activation of the p38 MAPK/HSP27 signaling pathway. Such changes in ET-1-mediated signaling may help maintain vascular wall homeostasis by compensating for the diminished dilatory function induced by TGF-beta1 and amyloid-beta; brain levels of these two molecules are increased in patients with Alzheimer's disease.     

A superoxide-mediated mitogen-activated protein kinase phosphatase-1 degradation and c-Jun NH(2)-terminal kinase activation pathway for luteolin-induced lung cancer cytotoxicity

Although luteolin is identified as a potential cancer therapeutic and preventive agent because of its potent cancer cell-killing activity, the molecular mechanisms by which its cancer cell cytotoxicity is achieved have not been well elucidated. In this report, luteolin-induced cellular signaling was systematically investigated, and a novel pathway for luteolin's lung cancer killing was identified. The results show that induction of superoxide is an early and crucial step for luteolin-induced apoptotic and nonapoptotic death in lung cancer cells. The c-Jun N-terminal kinase (JNK) was potently activated after superoxide accumulation. Suppression of superoxide completely blocked luteolin-induced JNK activation, which was well correlated to alleviation of luteolin's cytotoxicity. Although luteolin slightly stimulated the JNK-activating kinase mitogen-activated protein kinase kinase 7, the latter was not dependent on superoxide. We further found that luteolin triggers a superoxide-dependent rapid degradation of the JNK-inactivating phosphatase mitogen-activated protein kinase phosphatase-1 (MKP-1). Introduction of a degradation-resistant MKP-1 mutant effectively attenuated luteolin-induced JNK activation and cytotoxicity, suggesting that inhibition of the JNK suppressor MKP-1 plays a major role in luteolin-induced lung cancer cell death. Taken together, our results unveil a novel pathway consisting of superoxide, MKP-1, and JNK for luteolin's cytotoxicity in lung cancer cells, and manipulation of this pathway could be a useful approach for applying luteolin for lung cancer prevention and therapy.     

Suppression of mitogen-activated protein kinase phosphatase-1 (MKP-1) by heparin in vascular smooth muscle cells

Heparin inhibits vascular smooth muscle cell (VSMC) proliferation, but mechanisms remain elusive. Because heparin inhibits signaling through multiple kinase cascades, we investigated the possibility that phosphatases could be involved. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was the predominant MKP detected in VSMC lines. MKP-1 protein was increased by serum stimulation of quiescent cells, and this increase was diminished by heparin (1 microg/mL). Increased MKP-1 expression was dependent on the mitogen-activated protein kinase, Erk. Decreased Erk activity in the presence of heparin preceded, and may account for, decreased MKP-1. The antimitogenic effects of heparin are therefore unlikely to act through a shift in the kinase/phosphatase balance, but rather through direct kinase suppression. However, because MKP-1 is known to cause an increase in activity of kinases upstream of Erk, that may signal through additional pathways, the decrease in MKP-1 activity may paradoxically enhance heparin's antiproliferative effects. VSMC selected to grow in the presence of heparin express decreased levels of MKP-1 that are unresponsive to heparin, and Erk activity becomes unresponsive to heparin in one cell line. We conclude that phosphatase activation is not a direct mechanism of suppression of multiple kinase cascades by heparin.     

Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses

Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases, many of which dephosphorylate threonine and tyrosine residues on mitogen-activated protein kinases (MAPKs), and hence are also referred to as MAPK phosphatases (MKPs). The regulated expression and activity of DUSP family members in different cells and tissues controls MAPK intensity and duration to determine the type of physiological response. For immune cells, DUSPs regulate responses in both positive and negative ways, and DUSP-deficient mice have been used to identify individual DUSPs as key regulators of immune responses. From a drug discovery perspective, DUSP family members are promising drug targets for manipulating MAPK-dependent immune responses in a cell-type and disease-context-dependent manner, to either boost or subdue immune responses in cancers, infectious diseases or inflammatory disorders.     

Marker genes to predict sensitivity to FK228, a histone deacetylase inhibitor

In this study, we detected genes sensitive to an histone deacetylase inhibitor, FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] in vitro and identified marker genes to predict sensitivity to FK228 in vivo using Affymetrix GeneChip. Three percent of genes (205/7070) were sensitive to FK228 in vitro, 105 and 100 genes, were up- and down-regulated, respectively, by FK228. Commonly up-regulated genes included p21(WAF1/Cip1), interleukin-8 (IL-8), histone family, JunB, caspase 9, mitogen-activated protein kinase phosphatase 1 (MKP-1) and mitogen-activated protein kinase (MAPK) family, and commonly down-regulated genes included cyclin A and MAPK family. One percent of genes (76/7070) showed native differences in patterns of expression, when FK228-sensitive (PC-3 prostate and SC-6-JCK (SC-6) stomach) and FK228-resistant (ACHN and A-498 renal) tumors implanted in BALB/c nu/nu mice were compared. Twenty-seven and forty nine of those genes were expressed at high or low levels, respectively, in FK228-sensitive tumors. Caspase 9 and MKP-1 genes showed distinct differences in patterns of expression between FK228-sensitive and resistant tumors and have been known to have roles in apoptosis and chromatin remodeling. The expression of caspase 9 gene was higher in FK228-sensitive tumors and the expression of MKP-1 gene was higher in FK228-resistant tumors. Caspase 9 and MKP-1 genes in the other FK228-sensitive tumors had the same patterns of expression as they did in PC-3 and SC-6 tumors. Our results present profiles of gene expression related to FK228 and marker genes to predict sensitivity to FK228, such as caspase 9 and MKP-1 genes.     

p38 MAP激酶在山冈橐吾碱肝细胞毒性中的作用

目的探讨丝裂原活化蛋白激酶p38 (p38MAP激酶)在山冈橐吾碱肝细胞毒性中的作用。方法采用western杂交方法观察山冈橐吾碱(100μmol·L~(-1),分别作用1,5,15,30及60 min)对p38MAP激酶激活的影响;10μmol·L~(-1) p38MAP激酶抑制剂SKF86002预处理15 min后,分别观察其对山冈橐吾碱(100μmol·L~(-1))诱导p38MAP激酶磷酸化(30 min,western杂交法)及细胞毒性(MTT法,36及48 h;台盼蓝染色法,36 h)的影响。结果western blot结果显示,100μmol·L~(-1)山冈橐吾碱明显诱导p38 MAP激酶的磷酸化激活,5～30 min时处于较高水平;SKF86002预处理可以明显抑制山冈橐吾碱诱导的p38 MAP激酶磷酸化;MTT染色法和台盼蓝染色实验均发现SKF86002预处理能部分降低山冈橐吾碱诱导的肝细胞毒性[MTT,36 h:(0.210±0.008) vs (0.170±0.003),48 h:(0.33±0.03) vs (0.200±0.003);台盼蓝染色(80±2)% vs (72±7)%;n=8,P<0.05]。结论p38MAP激酶可能参与了山冈橐吾碱诱导的肝细胞毒性作用。     

Comprehensive analysis of gene expression in rat and human hepatoma cells exposed to the peroxisome proliferator WY14,643

Peroxisome proliferators (PPs) are an important class of chemicals that act as hepatic tumor promoters in laboratory rodents. The key target for PPs is the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) and these chemicals cause cancer by altering the expression of a subset of genes involved in cell growth regulation. The purpose of the present study was to utilize high-density gene expression arrays to examine the genes regulated by the potent PP Wy14,643 (50 microM, 6 h) in both rat (FaO) and human (HepG2) hepatoma cells. Treatment of FaO cells, but not HepG2, revealed the expected fatty acid catabolism genes. However, a larger than expected number of protein kinases, phosphatases, and signaling molecules were also affected exclusively in the FaO cells, including MAPK-phosphatase 1 (MKP-1), Janus-activated kinases 1 and 2 (JAK1 and 2), and glycogen synthetase kinase alpha and beta (GSKalpha and beta). The mRNA accumulation of these genes as well as the protein level for GSK3alpha, JAK1, and JAK2 and MKP-1 activity was corroborated. Due to the importance of MKP-1 in cell signaling, this induction was examined further and was found to be controlled, at least in part, at the level of the gene's promoter. Interestingly, overexpression of MKP-1 in turn affected the constitutive activity of PPARalpha. Taken together, the gene expression arrays revealed an important subset of PP-regulated genes to be kinases and phosphatases. These enzymes not only would affect growth factor signaling and cell cycle control but also could represent feedback control mechanisms and modulate the activity of PPARalpha.     

The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells

We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8?h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E(2), a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.     

The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells

We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8?h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E₂, a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.     

MAPK-specific tyrosine phosphatases: new targets for drug discovery?

Protein tyrosine phosphatases (PTPs) have key roles in a diverse range of cellular processes, and their dysregulation is associated with several human diseases. Many PTPs are recognized as potential drug targets; however, inhibitor development has focused only on a small number of enzymes, most notably PTP1B for type II diabetes and obesity, and MKP1 and CDC25 for cancer. The future challenge of selective-inhibitor development for PTPs will be significantly facilitated by the recent rapid progress in the structural biology of the 'PTPome'. In this article, we focus on the family of mitogen-activated protein kinase (MAPK)-specific tyrosine phosphatases--PTPN5 [also called striatal-enriched phosphatase (STEP)], PTPN7 (also called hematopoietic PTP) and PTPRR (also called PC12 PTP or STEP-like PTP)--and discuss approaches for achieving selectivity for the MAPK-PTPs at the molecular level using recently determined high-resolution X-ray crystal structures. We believe that the development of specific inhibitors would provide a valuable set of experimental pharmacological tools for investigating the physiological role of these phosphatases and exploring their emerging role in human disease.     

Regionally selective activation and differential regulation of ERK, JNK and p38 MAP kinase signalling pathway by protein kinase C in mood modulation

A growing body of evidence indicates that the extracellular signal-regulated kinase (ERK) pathway may participate in the neuronal modulation of depression. p38MAPK and c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) also belong to the MAPK family which mainly function as mediators of cellular stresses. Since increasing evidence implicates stress as an important factor in vulnerability to depressive illnesses, the involvement of ERK, JNK and p38MAPK pathways in the modulation of mood was investigated in the forced swim test (FST) and tail suspension test (TST). The effect produced by a single acute session of FST and TST on hippocampal and cortical MAPK expression and phosphorylation was investigated by immunoblotting experiments. In the hippocampus of animals exposed to FST and TST, an intensive, PKC-dependent, ERK1, ERK2, JNK, and p38MAPK phosphorylation was observed. In the frontal cortex, the FST and TST produced a PKC-dependent increase of ERK2 and p38MAPK phosphorylation, a PKC-independent activation of JNK and cAMP response element-binding protein (CREB) whereas any involvement of ERK1 was detected. The PKC blocker calphostin C (0.05-0.1 μg i.c.v.), the MEK inhibitor U0126 (10-20 μg i.c.v.), the p38MAPK inhibitor SB203580 (5-20 μg i.c.v.) and the JNK inhibitor II (0.5-5 μg i.c.v.), produced antidepressant-like behaviour without altering locomotor activity. These results illustrate a differentially mediated activation of MAPK in hippocampus and frontal cortex of animals exposed to behavioural despair paradigms. An antidepressant-like phenotype produced by acute blockade of MAPK signalling was also demonstrated.     

Electroconvulsive seizures increase the expression of MAP kinase phosphatases in limbic regions of rat brain.

The mitogen-activated protein (MAP) kinase cascades regulate a variety of cellular activities, including cell growth, proliferation, and apoptosis, and are reported to play a role in the actions of antidepressant treatment. There are a number of different classes of protein phosphatases that could influence the MAP kinase cascade. One of these, the MAP kinase phosphatase (MKP) family, is known to play a key role in dephosphorylation of activated MAP kinase. In the present study, we analyzed the expression of the MKP1, MKP2, and MKP3 isoforms in rat brain after electroconvulsive seizure (ECS), considered the most effective treatment for depression. In situ hybridization analysis demonstrates that ECS differentially regulates the expression of the MKP isoforms. Expression of MKP1 mRNA is robustly increased by acute ECS in the major cell layers of the hippocampus, including the dentate gyrus granule cell layer and the CA1 and CA3 pyramidal cell layers. In contrast, MKP2 is induced mainly in the dentate gyrus and MKP3 is preferentially increased in the CA1 and CA3 cell layers. In the prefrontal cortex, all three MKP isoforms are upregulated by acute ECS administration. Chronic ECS resulted in a similar pattern of induction for each of the MKP subtypes, demonstrating that there is little or no desensitization of the response to repeated ECS. The induction of MKP expression serves as negative feedback control for the MAP kinase cascades. Upregulation of MKP expression could dampen the actions of ECS, indicating that blockade of the MKPs could enhance the actions of antidepressant treatment.     

Role of MKP-1 (DUSP1) in clozapine-induced effects on the ERK1/2 signaling pathway in the rat frontal cortex

Rationale

Clozapine affects the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in the brain, which plays an important role in its antipsychotic action. However, previous findings are inconsistent, and related molecular mechanisms require further clarification.

Objectives

Time- and dose-dependent effects of clozapine on the ERK1/2 pathway and its regulatory mechanism were investigated in rat frontal cortex.

Methods and results

At 15, 30, 60, and 120 min after intraperitoneal injection of clozapine (5, 10, and 20 mg/kg), changes in ERK1/2, its upstream canonical kinases (Raf1 and mitogen-activated protein kinase kinase 1/2 [MEK1/2]), and its downstream molecule (p90 ribosomal S6 kinase [p90RSK]) were investigated in rat frontal cortex. At 15 min, p-Raf1, p-MEK1/2, p-ERK1/2, and p-p90RSK all increased dose-dependently. At 30 min, p-ERK1/2 and p-p90RSK showed no significant changes, while dose-dependent increases in p-Raf1 and p-MEK1/2 were found. At 60 and 120 min, although p-ERK1/2 and p-p90RSK decreased, increases in p-Raf1 and p-MEK1/2 were maintained. A clozapine-induced reduction in ERK1/2 phosphorylation was evident at both tyrosine and threonine residues, suggesting the involvement of dual specificity phosphatases (DUSPs; mitogen-activated protein kinase phosphatases [MKPs]). mRNA expression of seven Dusps that can dephosphorylate ERK1/2 were examined; Mkp-1 (Dusp1) mRNA increased following clozapine treatment. Moreover, MKP-1 protein and phosphatase activity increased, and binding of MKP-1 to ERK1/2 was also upregulated by clozapine administration.

Conclusions

In rat frontal cortex, clozapine regulates ERK1/2 phosphorylation via MKP-1, which induces uncoupling between Raf1-MEK1/2 and ERK1/2-p90RSK activity. These findings suggest an important role of MKP-1 in the mechanism of action of clozapine.     

Modification of tight junction function by protein kinase C isoforms

The regulation of tight junction permeability by a variety of signal transduction pathways is summarized. An emphasis is placed on regulation of paracellular permeability by the protein kinase C family of isoforms, which involves the reporting of a large number of studies using the phorbol ester family of protein kinase C activators. The ability of protein kinase C activation to open epithelial barriers to a very wide range of solutes is emphasized, but then countered with discussion of the role of phorbol esters and protein kinase C activation in epithelial carcinogenesis. The ability of protein kinase C activation to enable growth factors to leak from luminal fluid compartments of epithelial tissues into lateral intercellular and interstitial fluid spaces may play a role in this carcinogenic action. An examination of protein kinase C effects on the phosphorylation states of tight junctional proteins suggests that downstream kinases and/or phosphatases mediate protein kinase C's effect on tight junction permeability. A role for protein kinase C in transepithelial drug delivery is questioned herein. The tight junctional leakiness associated with protein kinase C activation and apparently intrinsic to transformed epithelia suggests a potentially useful role for tight junction leakiness as a marker for early cancer diagnosis.     

SB203580, a pharmacological inhibitor of p38 MAP kinase transduction pathway activates ERK and JNK MAP kinases in primary cultures of human hepatocytes

Mitogen-activated protein kinases (MAPKs) were extensively studied in cancer-derived cell lines; however, studies in non-transformed human cells are scarce. In the current paper, we studied the effect of SB203580, a pharmacological inhibitor of p38 MAPK, on activation and inhibition of p38 MAPK transduction partway in primary human hepatocytes (in vitro model of differentiated cells) in comparison with several tumor cell lines (proliferating non-differentiated in vitro model). In addition, we analyzed the effect of SB203580 on extracellular-regulated protein kinase (ERK) and c-jun-N-terminal kinase (JNK) pathways both in primary human hepatocytes and tumor cell lines employing primary antibodies detecting phosphorylated kinases. We show that SB203580 activates ERK and JNK in primary cultures of human hepatocytes. The levels of ERK-P(Thr202/Tyr204), JNK-P(Thr183/Tyr185) and c-Jun-P(Ser63/73), a target down-stream protein of JNK, were increased by SB203580. In contrast, SB203580 activated ERK but not JNK in HepG2, HL-60, Saos-2 and HaCaT human cancer cell lines. We tested, whether the effects of SB203580 are due to metabolism. Using liquid chromatography/mass spectrometry, we found one minor metabolite in human liver microsomes but not in HepG2 cells. These data imply that biotransformation could be responsible for the effects of SB203580 in human hepatocytes. This study is the first report on the effects of MAPK activators (sorbitol, anisomycin, EGF) and MAPK inhibitors in primary human hepatocytes. We observed differential effects of these compounds in primary human hepatocytes and in cancer cells, implying the cell-type specificity and the essential differences between the role and function of MAPKs in normal and cancer cells.     

Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling

Extracellular signals transduced via receptor tyrosine kinases, G-protein-coupled receptors or integrins activate Ras, a key switch in cellular signalling. Although Ras can activate multiple downstream effectors (PI3K, Ral em leader ) one of the major activated pathway is a conserved sequential protein kinase cascade referred to as the mitogen activated protein (MAP) kinase module: Raf>MEK>ERK. The fidelity of signalling among protein kinases and the spatio-temporal activation are certainly key determinants for generating precise biological responses. The fidelity is ensured by scaffold proteins, a sort of protein kinase "insulators" and/or specific docking sites among the members of the signalling cascade. These docking sites are found in upstream and downstream regulators and MAPK substrates [Nat Cell Biol 2 2000 110]. The duration and the intensity of the response are in part controlled by the compartmentalisation of the signalling molecules. Growth factors promote nuclear accumulation and persistent activation of ERK (p42/p44 MAP kinases) during the entire G1 period with an extinction during S-phase. These features are exquisitely well controlled by (i) the temporal induction of the MAP kinase phosphatases, MKP1-3, and (ii) the compartmentalisation of the signalling molecules. We have shown that MKP1-2 induction is strictly controlled by the activation of the MAP kinase module providing evidence for an autoregulatory mechanism. This negative regulatory loop was further enhanced by the capacity of ERK to phosphorylate MKP1 and 2. This action reduced the degradation rate of these MKPs through the ubiquitin-proteasomal system [Science 286 1999 2514].Whereas the two upstream kinases of the module, Raf and MEK remained cytoplasmic, ERK anchored to MEK in the cytoplasm of resting cells, rapidly translocated to the nucleus upon mitogenic stimulation. This process was rapid, reversible, and controlled by the strict activation of the MAPK cascade. Prevention of this nuclear translocation, by overexpression of a cytoplasmic ERK-docking molecule (inactive MKP3) prevented growth factor-stimulated DNA replication [EMBO J 18 1999 664]. Following long term stimulation, ERK progressively accumulated in the nucleus in an inactive form. This nuclear retention relied on the neosynthesis of short-lived nuclear anchoring proteins. Nuclear inactivation and sequestration was likely to be controlled by MAP kinase phosphatases 1 and 2. Therefore we propose that the nucleus represents a site for ERK action, sequestration and signal termination [J Cell Sci 114 2001 3433].In addition, with the generation of mice invalidated for each of the ERK isoforms, we will illustrate that besides controlling cell proliferation the ERK cascade also controls cell differentiation and cell behaviour [Science 286 1999 1374].