Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.     

ERK activation and mitogenesis in human airway smooth muscle cells

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE(2) and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.     

PKC-delta/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE(2) production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.     

Fluvastatin attenuates IGF-1-induced ERK1/2 activation and cell proliferation by mevalonic acid depletion in human mesangial cells

AimsInsulin-like growth factor (IGF)-1 is a major mitogenic growth factor for mesangial cells (MCs). Statins slow the progression of chronic kidney disease by affecting inflammatory cell signaling pathways, in addition to improving lipid profile, however, no studies have investigated the effects of fluvastatin on mitogen-activated protein (MAP) kinase activity or MC proliferation in kidney cells. We investigated the effects of fluvastatin on IGF-1-induced activation of intracellular signal pathways and MC proliferation, and examined the inhibitory mechanisms of fluvastatin.Main methodsWestern blotting and cell proliferation assay were used.Key findingsIGF-1 induced phosphorylation of extracellular-related kinase (ERK)1/2, MAP or ERK kinase (MEK)1/2, and Akt, expression of cyclin D1, and MC proliferation in cultured human MCs. Fluvastatin or PD98059, an MEK1 inhibitor, completely abolished IGF-1-induced MEK1/2 and ERK1/2 phosphorylation and MC proliferation, whereas inhibition of Akt had no effect on MC proliferation. Mevalonic acid prevented fluvastatin inhibition of IGF-1-induced MEK1/2 and ERK1/2 phosphorylation, cyclin D1 expression, and MC proliferation.SignificanceFluvastatin inhibits IGF-1-induced activation of the MAP kinase pathway and MC proliferation by mevalonic acid depletion, and might have renoprotective effects by inhibiting IGF-1-mediated MC proliferation.     

The expression and activation of ERK/MAPK pathway in human esophageal cancer cell line EC9706

While there have been more and more studies concerning mitogen-activated protein kinases (MAPKs) signaling pathways, which control many cellular complex programmes, such as cell proliferation, differentiation, cell death and embryogenesis. However, few studies are carried out about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) in human esophageal cancer cell line. Therefore, in the present study, we investigated the expression and activation of ERK1/2 in human esophageal cancer cell line EC9706 and human normal esophageal epithelial cell line Heepic, which is as control. This study showed that ERK1/2 was transiently phosphorylated both in EC9706 and Heepic, the kinetics of which were slightly different. To further study the ERK/MAPK signaling pathway in EC9706 and Heepic cell line, U0126 a kind of specific inhibitor of MEK was used. This study showed that U0126 can block the phosphorylation of ERK1/2 in a short time, the complete inhibition concentration for EC9706 and Heepic cell line is 50 and 20 ??M, respectively. Incidentally, to further investigate the different roles of ERK1 and ERK2, vector-based short hairpin interference vectors targeted on ERK1/2 was constructed. Moreover, the effective interference target sequence was screened out in a transient transfection manner. MTT experiment showed that ERK2 is more important than ERK1 in the proliferation of EC9706 cells.     

Peroxisome proliferator-activated receptor-beta/delta inhibits epidermal cell proliferation by down-regulation of kinase activity

Recent work has shown that peroxisome proliferator-activated receptor beta (PPARbeta) attenuates cell proliferation and skin carcinogenesis, and this is due in part to regulation of ubiquitin C expression. In these studies, the role of PPARbeta in modulating ubiquitin-dependent protein kinase Calpha (PKCalpha) levels and phosphorylation signaling pathways was evaluated. Intracellular phosphorylation analysis showed that phosphorylated PKCalpha and other kinases were lower in wild-type mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) as compared with PPARbeta-null mouse skin. No differences in expression levels of other PKC isoforms present in skin were observed. Lower ubiquitination of PKCalpha was found in TPA-treated PPARbeta-null skin as compared with wild-type, and inhibition of ubiquitin-dependent proteasome degradation prevented TPA-induced down-regulation of PKCalpha. The activity of PKCalpha and downstream signaling kinases is enhanced, and expression of cyclooxygenase-2 (COX-2) is significantly greater, in PPARbeta-null mouse skin in response to TPA compared with wild-type mouse skin. Inhibition of PKCalpha or COX-2 reduced cell proliferation in TPA-treated PPARbeta-null keratinocytes in a dose-dependent manner, whereas it only slightly influenced cell proliferation in wild-type keratinocytes. Combined, these studies provide strong evidence that PPARbeta attenuates cell proliferation by modulating PKCalpha/Raf1/MEK/ERK activity that may be due in part to reduced ubiquitin-dependent turnover of PKCalpha.     

Novel suppression mechanism operating in early phase of adipogenesis by positive feedback loop for enhancement of cyclooxygenase-2 expression through prostaglandin F2α receptor mediated activation of MEK/ERK-CREB cascade

Prostaglandin (PG) F(2α) suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. In this study, we identified a novel suppression mechanism, operating in the early phase of adipogenesis, that increased the production of anti-adipogenic PGF(2α) and PGE(2) by enhancing cyclooxygenase (COX) 2 expression through the PGF(2α) -activated FP receptor/extracellular-signal-regulated kinase (ERK)/cyclic AMP response element binding protein (CREB) cascade. COX-2 expression was enhanced with a peak at 1 h for the mRNA level and at 3 h for the protein level after the addition of Fluprostenol, an FP receptor agonist. The Fluprostenol-derived elevation of COX-2 expression was suppressed by the co-treatment with an FP receptor antagonist, AL8810, with a mitogen-activated protein kinase (MEK; ERK kinase) inhibitor, PD98059. ERK was phosphorylated within 10 min after the addition of Fluprostenol, and its phosphorylation was inhibited by the co-treatment with AL8810 or PD98059. Moreover, FP receptor mediated activation of the MEK/ERK cascade and COX-2 expression increased the production of PGF(2α) and PGE(2) . An FP receptor antagonist and each inhibitor for MEK and COX-2 suppressed the PGF(2α) -derived induction of synthesis of these PGs. Furthermore, promoter-luciferase and chromatin immunoprecipitation assays demonstrated that PGF(2α) -derived COX-2 expression was activated through binding of CREB to the promoter region of the COX-2 gene in 3T3-L1 cells. These results indicate that PGF(2α) suppresses the progression of the early phase of adipogenesis by enhancing the binding of CREB to the COX-2 promoter via FP receptor activated MEK/ERK cascade. Thus, PGF(2α) forms a positive feedback loop that coordinately suppresses the early phase of adipogenesis through the increased production of anti-adipogenic PGF(2α) and PGE(2) .     

PKC isoenzymes differentially modulate the effect of thrombin on MAPK-dependent RPE proliferation

Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation, differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders, such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation, transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated protein kinase and MEK is MAPK/ERK kinase) through the activation of G(alpha) and G(betagamma) heterotrimeric G-proteins, and the downstream stimulation of the PLC (phospholipase C)-beta/PKC (protein kinase C) and PI3K (phosphoinositide 3-kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis. Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation of 'conventional' PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2 requires the activation of atypical PKCzeta by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell proliferation were prevented completely by PI3K or PKCzeta inhibition. These results suggest that thrombin induces RPE cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCzeta in this process.     

A3 adenosine receptor activation inhibits cell proliferation via phosphatidylinositol 3-kinase/Akt-dependent inhibition of the extracellular signal-regulated kinase 1/2 phosphorylation in A375 human melanoma cells

Adenosine exerts its effects through four subtypes of G-protein-coupled receptors: A(1), A(2A), A(2B), and A(3). Stimulation of the human A(3) receptor has been suggested to influence cell death and proliferation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Due to their importance, the cross-talk between these two pathways has been investigated. Here, we show that the A(3) adenosine receptor agonist Cl-IB-MECA stimulates PI3K-dependent phosphorylation of Akt leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation. The response to Cl-IB-MECA was not blocked by A(1), A(2A), or A(2B) receptor antagonists, although it was abolished by A(3) receptor antagonists. Furthermore, the response to Cl-IB-MECA was generated at the cell surface, since the inhibition of A(3) receptor expression, by using small interfering RNA, abolished agonist effects. Using A375 cells, we show that A(3) adenosine receptor stimulation results in PI3K-dependent phosphorylation of Akt, leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation.     

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AimsStress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved.Main methodsThe effect of adrenaline on HT-29 cell proliferation was determined by [³H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E₂ (PGE₂) release were determined by zymography and enzyme immunoassay, respectively.Key findingsAdrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE₂ release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β₁- and β₂-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE₂ release in HT-29 cells.SignificanceThese results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β₁- and β₂-adrenoceptors by a COX-2 dependent pathway.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

Differential regulation of IL-1beta and TNF-alpha RNA expression by MEK1 inhibitor after focal cerebral ischemia in mice

Activation of the extracellular-signal-responsive kinase (ERK 1/2) by MAP kinase/ERK kinase (MEK1/2) following ischemia/reperfusion in the brain has been associated with cell death since inhibition of MEK1/2 provides neuroprotection in cerebral ischemia injury. Since inflammation has been implicated in ischemic brain injury, the present study investigated whether MEK1/2 modifies expression of two key inflammatory cytokines, IL-1beta and TNFalpha, that have been shown to exacerbate ischemic brain injury. A mouse model of transient cerebral ischemia was deployed to test the effect of selective MEK1/2 inhibitor (SL327) on infarct size and cytokine expression. SL327 (100 mg/kg, i.p.) administered 15 min prior to ischemia resulted in 64% reduction in infarct size over controls (n = 8, P < 0.01). Under the same condition, SL327 significantly reduced peak expression of IL-1beta mRNA (59% reduction compared to vehicle, P < 0.01, n = 4) but not TNF-alpha mRNA. A parallel reduction in IL-1beta protein (67%, P < 0.05, n = 6) was also observed using ELISA analysis. These data suggest that the neuroprotective effect of MEK1/2 inhibition may be mediated by suppression of IL-1beta. The study also demonstrates for the first time that these two cytokines are differentially regulated by kinase mediated signaling pathways.     

MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.     

Loss of active MEK1-ERK1/2 restores epithelial phenotype and morphogenesis in transdifferentiated MDCK cells

Constitutive activation of the MAPK/ERK kinase (MEK)1-ERK2 signaling module in Madin-Darby canine kidney (MDCK)-C7 cells disrupts their ability to form cystlike structures in collagen gels and induces an invasive, myofibroblastlike phenotype. However, the reversibility of these cellular events, as well as the relative role of both MEK isoforms (MEK1 and MEK2) and both ERK isoforms (ERK1 and ERK2) during these processes, has not yet been investigated. We now report that loss of constitutively active MEK1 (caMEK1) and, thus, loss of active ERK1/2 in C7caMEK1 cells is associated with increased MEK2 protein expression, reexpression of ERK1 protein, and epithelial redifferentiation of these cells. The morphological changes toward an epithelial phenotype in these revertant cell lines (C7rev4, C7rev5, C7rev7) are reflected by the upregulation of epithelial marker proteins, such as E-cadherin, -catenin, and cytokeratin, by the loss of -smooth muscle actin expression, and by the ability of these epithelial revertants to form well-organized spherical cysts when grown in three-dimensional collagen gels. Further evidence for a role of the MEK1-ERK1/2 module in epithelial-mesenchymal transition was obtained from the analysis of two novel, spontaneously transdifferentiated MDCK-C7 cell clones (C7e1 and C7e2 cells). In these clones, increased MEK1/2-ERK1/2 phosphorylation, reduced MEK2 protein expression, and loss of ERK1 protein expression is associated with phenotypic alterations similar to those observed in transdifferentiated C7caMEK1 cells. C7e1 cells at least partially regained some of their epithelial characteristics at higher passages. In contrast, C7e2 cells maintained a transdifferentiated phenotype at high passage, were unable to generate cystlike epithelial structures, and retained invasive properties when grown on a three-dimensional collagen matrix. We conclude that in renal epithelial MDCK-C7 cells, stable epithelial-to-mesenchymal transition (EMT) is associated with loss of ERK1 protein expression, reduced MEK2 protein expression, and increased basal ERK2 phosphorylation. In contrast, loss of active MEK1-ERK1/2 results in increased MEK2 protein expression and reexpression of ERK1 protein, concomitant with the restoration of epithelial phenotype and the ability to form cystic structures. mitogen-activated protein kinase; extracellular signal-regulated kinase; epithelial differentiation; epithelial-to-mesenchymal transition; invasion; Madin-Darby canine kidney cells     

Beta-adrenergic receptors of the normal heart and in heart failure

The heart is often refereed to as an "beta-adrenergic organ" because beta-adrenergic agonists are powerful stimulants of cardiac contractility. Catecholamines acting through beta-adrenoceptors produce both positive inotropic and chronotropic effects in human heart. It is now generally accepted that in human heart both beta 1- and beta 2-adrenoceptors coexist. beta-Adrenergic transduction system consist of membrane-bound beta-receptors, the effector enzyme adenylyl cyclase and guanine nucleotide-binding transduction (G) proteins. Repeated long-lasting agonist stimulus evokes homologous or heterologous desensitization of transduction system. Chronic heart failure accompanies with decreased responsiveness to beta-adrenoceptor agonists and is thought to exacerbate the loss of cardiac contractility. Depending on the etiology of heart failure abnormalities of the beta-receptor-G protein-adenylyl cyclase system result from a reduced of beta 1-receptors, uncoupling of beta 1- or beta 2-receptors, alteration of G-protein function, or decreased catalytic subunit activity of adenylyl cyclase and enhanced expression of beta-adrenoceptor kinase. The model most widely used is that of circulating lymphocytes that contain a homogeneous population of beta 2-adrenoceptors. The biochemical and pharmacological properties of human lymphocyte beta 2-adrenoceptors are quite comparable to those of heart beta 2-receptors. The analysis of lymphocyte beta 2-adrenoceptor-adenylyl cyclase system can be used as a model for long-term regulation of human cardiac beta 1- and beta 2-adrenoceptors only if serial changes in response to administration of non-selective beta-adrenergic agonists or antagonists are being investigated. This review concentrates on beta-adrenoceptors in human healthy heart and in heart failure and also on lymphocyte beta 2-adrenoceptors and on the changes of these receptors properties under the influence of some cardiotropic drugs.