Extracellular Response Kinase Research Articles

Resistance to CpG DNA-induced autoimmunity through tolerogenic B cell antigen receptor ERK signaling.

CpG sequences in self-DNA are an important potential trigger for autoantibody secretion in systemic lupus and other systemic autoimmune disorders. It is not known how this ubiquitous threat may be controlled by active mechanisms for maintaining self tolerance. Here we show that two distinct mechanisms oppose autoantibody secretion induced by CpG DNA in anergic B cells that are constantly binding self-antigen. Uncoupling of the antigen receptor (BCR) from a calcineurin-dependent pathway prevents signals that synergize with CpG DNA for proliferation. The BCR does not become desensitized by activating the extracellular response kinase (ERK) MAP kinase pathway, however, and continuous self-antigen signaling to ERK inhibits CpG DNA-induced plasma cell differentiation. These two mechanisms seem to act as a general control against autoantibody production elicited by Toll-like receptors, and their regulation of T cell-independent responses to Toll-like receptor 9 (TLR9) is probably crucial for resistance to systemic autoimmunity.

Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils

Heterophils are the predominant polymorphonuclear leukocytes (PMNs) in poultry. The oxidative burst of activated heterophils, which generates reactive oxygen species (ROS), is one of the first line cellular defenses against invading microorganisms. In this report, the oxidative response of heterophils from neonatal chicks to in vitro stimulation by various inflammatory agonists was investigated using a fluorescence microplate assay. Both non-opsonized formalin-killed Salmonella enteritidis and Staphylococcus aureus were able to stimulate heterophil oxidative burst. The phorbol myristate acetate (PMA) was the most potent stimulant for the chicken heterophil oxidative response, whereas, the bacterial cell surface components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were less effective. Protein kinase C (PKC) is an essential signaling component regulating heterophil oxidative response to stimulation by PMA, LPS, LTA and S. enteritidis. However, inhibition of PKC did not affect the oxidative response to stimulation by S. aureus, suggesting differential signaling pathway responsible for the activation of oxidative burst by Gram-negative S. enteritidis and Gram-positive S. aureus. Inhibition of mitogen activated protein (MAP) kinase p38 and extracellular response kinase (ERK) by SB 203580 and PD 098059, respectively, did not inhibit activated oxidative burst.

Effects of inhibition of myocardial extracellular-responsive kinase and P38 mitogen-activated protein kinase on mechanical function of rat hearts after prolonged hypothermic ischemia.

Mitogen-activated protein kinases (MAPKs), including extracellular-responsive kinase (ERK) and p38 MAPK, are activated by stresses associated with hypothermia-rewarming and ischemia-reperfusion. Their activation in heart is associated with beneficial (preconditioning) and adverse effects (apoptosis and impaired contractility). This study determined whether ERK and p38 MAPK activities are altered by hypothermic ischemia and normothermic reperfusion and the consequences of their inhibition on recovery of myocardial function. Left ventricular work (L x min(-1) x mm Hg) was assessed during normothermic perfusion (30 min) of isolated rat hearts that were either freshly excised or previously subjected to hypothermic storage (8 hr, 3 degrees C) and rewarming (10 min, 37 degrees C) before normothermic reperfusion (30 min). Phospho-specific immunoblot analysis of p38 MAPK was performed in hearts and various cultured cells. Compared with fresh hearts, hearts subjected to hypothermia and rewarming demonstrated impaired left ventricular work (1.96+/-0.53, n=12 vs. 8.37+/-0.46, n=4, <0.05) during reperfusion. The ERK inhibitor, PD98059 (20 microM), present during storage and rewarming, caused modest improvement (3.66+/-0.75, n=9, <0.05). The p38 MAPK inhibitor, SB202190 (10 microM), when present during reperfusion, improved recovery (to 6.12+/-0.75, n=6, <0.05); it was ineffective if present only during rewarming (1.52+/-0.88, n=4). In rat2 fibroblasts, hypothermia and rewarming activated p38 MAPK and its downstream kinase MAPK-activated protein kinase 2, but not c-Jun N-terminal kinase/stress-activated protein kinase. Myocardial p38 MAPK and MAPK-activated protein kinase 2 are stimulated by hypothermia, ischemia, and rewarming and are detrimental to recovery of mechanical function of hearts subjected to prolonged hypothermic storage. Inhibition of p38 MAPK may be useful in protocols to improve the recovery of mechanical function of cold-stored hearts.

Formylpeptides trigger selective molecular pathways that are required in the physiological functions of human neutrophils

For-Met-Δ zLeu-Phe-OMe ([Δ zLeu 2]) is a conformationally restricted for-Met-Leu-Phe-OMe (fMLP-OMe) analogue able to discriminate between different responses of human neutrophils. In contrast, [Δ zLeu 2] significantly activates the transduction pathways—involving Ca 2+, inositol phosphate, and cyclic AMP (cAMP) enhancement, as is the case with the full agonist fMLP-OMe. Here, we have studied the specific involvement of protein kinase C (PKC) isoforms and mitogen activated protein kinases (MAPKs) in the presence or absence of extracellular Ca 2+, being the cation clearly involved in the activation of neutrophils by fMLP. A strong correlation has been found between PKC isoforms, MAPKs and the selective physiological functions by [Δ zLeu 2]-activated neutrophils. In a calcium-free condition, our data suggest that the failure of PKC β1 translocation and of p38 MAPK phosphorylation by the analogue refers to its inability to induce chemotaxis, and that the failure by both fMLP-OMe and [Δ zLeu 2] to evoke extracellular response kinase 1 and 2 (ERK1/2) phosphorylation would suggest a reduction in superoxide anion production.

Inorganic lead activates the mitogen-activated protein kinase kinase-mitogen-activated protein kinase-p90(RSK) signaling pathway in human astrocytoma cells via a protein kinase C-dependent mechanism.

We have previously reported that lead acetate activates protein kinase Calpha (PKCalpha) and induces DNA synthesis in human 1321N1 astrocytoma cells. In this study, we investigated the ability of lead to activate the mitogen-activated protein kinase (MAPK) cascade. We found that exposure to lead acetate (1-50 microM) resulted in concentration- and time-dependent activation of MAPK (extracellular signal responsive kinase 1/2), as shown by increased phosphorylation and increased kinase activity. This effect was significantly reduced by the PKC-specific inhibitor bisindolylmaleimide (GF109203X), by down-regulation of PKC with 12-O-tetradecanoyl-phorbol 13-acetate, by a pseudosubstrate to PKCalpha, and by selective down-regulation of PKCalpha by prior lead exposure. Lead was also shown to activate MAPK kinase (MEK1/2), and this effect was mediated by PKC. Two MEK inhibitors, 2-(2'-amino-3'-methoxyphenol)-oxanaphthalen-4-one (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (UO126), blocked lead-induced MAPK activation and inhibited lead-induced DNA synthesis, as measured by incorporation of [methyl-3H]thymidine into cell DNA. The 90 kDa ribosomal S6 protein kinase, p90(RSK), a substrate of MAPK, was also found to be activated by lead in a PKC- and MAPK-dependent manner. Stimulation of DNA synthesis by lead in astrocytoma cells may be of interest in light of the observed association between exposure to lead and an increased risk of astrocytomas.

Expression of functional chemokine receptors by rat cerebellar neurons

In this study, we examined chemokine receptor expression and function in rat cerebellar neurons. Calcium imaging experiments demonstrated that a wide variety of chemokines elicited [Ca 2+] i transients in acutely isolated and cultured cerebellar Purkinje and granule neurons. In many cases, these chemokine responses were pertussis toxin (PTX) insensitive. In addition, chemokines activated the Ca 2+ and cAMP-dependent transcription factor CREB and the extracellular response kinases ERK1/ERK2. Chemokines increased the survival of Purkinje neurons deprived of their trophic support. Thus, the presence of chemokine receptors and the signaling pathways they activate suggest that chemokines play a role in the control of cerebellar neuron survival and development and may mediate communication between the CNS and the immune system.

Erk 1/2 differentially regulates the expression from the 1G/2G single nucleotide polymorphism in the MMP-1 promoter in melanoma cells

Matrix metalloproteinase-1 (MMP-1) breaks down interstitial collagens, a major component of stromal tissue and a barrier for invading tumor cells. The degradation of collagen by MMP-1 may, therefore, provide one mechanism for facilitating tumor invasion and metastasis. Because of the potential for excessive matrix degradation, the expression of MMP-1 is tightly regulated, often by the mitogen-activated protein kinase (MAPK) pathway. The MAPK signal cascade consists of three separate pathways, the extracellular response kinase (ERK), p38 and Jun N-terminal kinase, which target proteins of the AP-1 and ETS families transcription of the gene. The MMP-1 promoter contains a single nucleotide polymorphism (SNP) at −1607 bp, which creates an ETS binding site by the addition of a guanine (5′-GGAT-3′ or ‘2G SNP’) compared to the 1G SNP (5′-GAT-3′), and enhances MMP-1 transcription. A2058 melanoma cells represent one tumor cell line that is homozygous for the 2G allele and that produces constitutively high levels of MMP-1. Thus, we used these cells to define the mechanism(s) responsible for this high level of expression. We show that inhibition of ERK 1/2 leads to the repression of MMP-1 transcription, and that both the 2G polymorphism and the adjacent AP-1 site at −1602 bp are necessary for high levels of MMP-1 transcription and for the inhibition of MMP-1 expression by PD098059, a specific ERK inhibitor. Furthermore, restoration of MMP-1 levels after ERK 1/2 inhibition requires de novo protein synthesis of a factor necessary for MMP-1 expression. Thus, this study suggests that the ERK 1/2 pathway targets the 2G polymorphism, and that the continuous synthesis of a protein(s) is necessary for the constitutive expression of MMP-1.

Variation in the kinetics of caspase-3 activation, Bcl-2 phosphorylation and apoptotic morphology in unselected human ovarian cancer cell lines as a response to docetaxel

Paclitaxel is able to cause cell death through the induction of apoptosis. Cell death characteristics for docetaxel have not yet been described in detail. We investigated four unselected human ovarian cancer cell lines for the sensitivity to a 1 hr exposure to docetaxel and calculated the concentrations inhibiting 50% ( ic 50) and 90% ( ic 90) of cell growth. Of the cell lines A2780, H134, IGROV-1 (all wild-type p53) and OVCAR-3 (mutant, mt p53) A2780 was most sensitive and OVCAR-3 least sensitive. Equitoxic drug concentrations representing ic 90 values (25–510 nM) were applied for 1 hr to measure cell cycle distribution, DNA degradation, and to count apoptotic cell bodies and cells with multifragmented nuclei at various time-points after drug exposure. H134, IGROV-1 and OVCAR-3 showed a continued mitotic block up to at least 72 hr and prolonged presence of cells with multifragmented nuclei. High percentages of apoptosis were calculated at 48 hr and at later time-points. In contrast, A2780 cells accumulated in the S-phase of the cell cycle and apoptosis was hardly present. The changes in the expression levels of p53, p21/WAF1, Bax and Bcl-2, were not predictive for docetaxel-induced apoptosis. Caspase-3 activation occurred only in cells with accumulation in the G2/M phase starting as early as 8 hr in OVCAR-3. Prolonged Bcl-2 phosphorylation was evident in OVCAR-3, visible at 24 hr in H134 and IGROV-1, while this phenomenon did not occur in A2780. The mitogen-activated protein kinase pathway (JNKs/SAPKs or c-Jun N-terminal kinases/stress-activated protein kinases, JNK1/2; extracellular response kinase, ERK1/2; p38) did not seem to be directly involved in Bcl-2 phosphorylation or apoptosis. We conclude that docetaxel is able to activate caspase-3, induce Bcl-2 phosphorylation and apoptosis in cells that show a prolonged G2/M arrest, but cells may also die by a caspase-3-independent cell death mechanism.

Basic fibroblast growth factor protects cardiac myocytes from iNOS-mediated apoptosis.

Basic fibroblast growth factor (bFGF) is an important angiogenic factor produced by hearts subjected to ischemia. However, the direct effects of bFGF on myocardial cells are unknown. Primary cultured cardiac myocytes from neonatal rats were stimulated with lipopolysaccharide (LPS), a potent inducer of inducible nitric oxide synthase (iNOS), in the presence or the absence of bFGF. LPS induced the expression of iNOS in cardiac myocytes, demonstrated at both mRNA and protein levels. We showed that LPS activated the apoptotic pathway, evidenced by TUNEL staining, DNA ladder formation, and morphologic features. LPS-induced apoptosis was blocked by the administration of L-NAME, an inhibitor of NOS. This indicates that LPS induces apoptosis via an iNOS-dependent pathway. Administration of bFGF completely inhibited myocardial cell apoptosis induced by hydrogen peroxide or acidic medium as well as LPS. To determine signaling pathways for this inhibitory effect, we utilized PD098059, an MEK-1-specific inhibitor. PD098059 blocked bFGF-induced activation of ERK (extracellularly responsive kinase)-1/2 and neutralized the apoptotic inhibitory effect of bFGF. These findings demonstrate that LPS induces myocardial cell apoptosis in an iNOS-dependent manner. The results also suggest that bFGF is a protective factor against myocardial cell apoptosis and that this protection requires the MEK-1-ERK pathway.

Enamel matrix derivative (EMDOGAIN) rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells.

In our previous study, we demonstrated that porcine enamel matrix derivative (EMD) induces p21WAF1/cip1 within 8 hours and subsequently arrests the cell cycle of human oral epithelial cells in G1 phase. In contrast, EMD markedly stimulates the proliferation of gingival fibroblasts without inducing p21WAF1/cip1. To investigate the mechanism of how EMD produces these differential effects, we have focused on the initial response of these two cell types to EMD. In epithelial cell cultures, EMD stimulated cytoskeletal actin polymerization within 30 min and promoted cell adhesion in our experimental system. EMD failed to stimulate either intracellular Ca2+ mobilization or cAMP production in either cell type. In both epithelial and fibroblastic cells, EMD (25-100 microgram/ml) rapidly produced dose-dependent phosphorylation of the mitogen-activated protein kinase (MAPK) family: extracellular signal response kinase (ERK), p38-MAPK (p38-K), and c-Jun-terminal kinase/stress-activated protein kinase (JNK). However, neither inhibitors of MEK (ERK kinase) nor p38-K could block EMD's anti-proliferative action on epithelial cells. On the other hand, EMD rapidly stimulated translocation of smad2 into the nucleus in both cell types. Spurred by this finding, we assayed for TGF-beta1, a ligand for one receptor associated with smad2 activation, and detected significant levels in EMD preparations. The sum of these pharmacological findings indicates that EMD contains at least one bioactive factor, which is most probably TGF-beta1 (or TGF-beta-like substances). In conjunction with the similarities in the differential growth-modulating actions between EMD and what is known for TGF-beta, we suggest that TGF-beta might act as the principal growth regulating agent of oral fibroblastic and epithelial cell types in EMD despite being present in only low levels.

The expression of the γ subunit of Na-K-ATPase is regulated by osmolality via C-terminal Jun kinase and phosphatidylinositol 3-kinase-dependent mechanisms

The alpha and beta subunits of Na-K-ATPase are up-regulated by hypertonicity in inner-medullary collecting duct cells adapted to survive in hypertonic conditions. We examined the regulation of the gamma subunit by hypertonicity. Although cultured inner-medullary collecting duct cells lacked the gamma subunits, both variants gamma(a) and gamma(b) were expressed in cells adapted to 600 and 900 mosmol/KgH(2)O. This expression was reversible with a half-time of 17.2 +/- 0.5 h. The message of the gamma subunit was absent in isotonic conditions and increased with higher tonicity in adapted cells. In acute experiments the appearance of the gamma subunit was found to be both time-dependent (> or =24 h) and osmolality-dependent (> or =500 mosmol/KgH(2)O). No induction was noted with urea and only minimal induction with mannitol. Increasing concentrations of the phosphatidylinositol 3-kinase inhibitor LY294002 resulted in a dose-dependent decrement in the expression of the gamma subunit with total abolition at 10 microM. This was associated with a decrease in cell viability as <20% survived the treatment with 10 microM of LY294002. Neither inhibition of extracellular response kinase nor p38 mitogen-activated protein kinase inhibited osmotic induction of the gamma subunit. In contrast, cells transfected with a dominant negative c-Jun N-terminal kinase 2-APF construct displayed complete inhibition of the gamma subunit. Such cells have accelerated loss of viability in hypertonic conditions. This study describes the regulation of the gamma subunit of Na-K-ATPase by hypertonicity. This regulation is transcriptionally regulated and involves signaling mediated by phosphatidylinositol 3-kinase and c-Jun N-terminal kinase 2 pathways.

Overexpression of Calbindin-D28K Induces Neurite Outgrowth in Dopaminergic Neuronal Cells via Activation of p38 MAPK

An MN9D dopaminergic neuronal cell line overexpressing calbindin-D28K (MN9D/Calbindin) was established in order to investigate directly the potential role of calcium-binding protein in neuronal differentiation. Overexpression of calbindin-D28K in MN9D cells resulted in significant increases in the number of neurites, the length of primary neurites, and the total extent of neurites. This robust neurite outgrowth occurred without cessation of cell division. Analysis of immunoblots revealed that this morphological differentiation was accompanied by increased expression of such markers of maturation as the synaptosomal protein SNAP-25. During calbindin-D28K-evoked neurite outgrowth in MN9D cells, phosphorylation of p38 mitogen-activated protein kinase (MAPK) dramatically increased while the levels and extent of phosphorylation of such other MAPKs as c-Jun N-terminal kinase (JNK) or extracellular response kinase (ERK) were not altered. Consequently, calbindin-D28K-induced neurite outgrowth was largely abolished by treatment with a p38 inhibitor, PD 169316, while the level of SNAP-25 in MN9D/Calbindin cells was not altered by this treatment. These data support an idea that calbindin-D28K and its associated p38 signaling pathway play a role in dopaminergic neuronal differentiation.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-beta.

Epithelial-mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-beta (TGF-beta) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-beta-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-beta within 4 hours after treatment. TGF-beta-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell-matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-beta. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Troglitazone and rosiglitazone induce apoptosis of vascular smooth muscle cells through an extracellular signal-regulated kinase-independent pathway

Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands thiazolidinediones have been reported to induce apoptosis in macrophages and in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis in VSMC, cultured rat VSMC were treated with increasing doses of the thiazolidinedione analogues troglitazone (TRO) and rosiglitazone (RSG). Both ligands induced cell death in a concentration-dependent manner (EC50 12.1+/-3.3 microM and 1.43+/-0.39 microM, respectively), causing almost complete cell death at the highest concentrations (100 microM and 10 microM for TRO and RSG, respectively), along with an expected parallel decrease in [3H]thymidine uptake into cell DNA (EC50 6.7+/-2.4 microM and 0.75+/-0.19 microM, respectively). The cell count was determined by the coulter counter principle. Furthermore two apoptotic markers were measured, the caspase 3 activity and the cytoplasmic histone-associated DNA fragments, both of which were significantly increased when the aforementioned high concentrations were used. This indicates that apoptosis is involved in the TRO- and RSG-induced VSMC growth suppression. The same concentrations of TRO and RSG caused an unexpected stimulation of the extracellular signal-regulated response kinases 1 and 2 (ERK1/2) and stimulated the p38 mitogenic-activated protein (MAP) kinase as determined by Western blotting. In order to establish whether the proapoptotic effects of TRO and RSG are mediated through ERK1/2 activation, we used the selective MAP kinase kinase (MEK) inhibitor PD98059 (20 microM), which suppressed the TRO- and RSG-induced ERK1/2 activation but did not abolish their proapoptotic effects. We conclude that the thiazolidinedione analogues TRO and RSG induce cell death due to apoptosis in VSMC through an ERK1/2-independent pathway.

Overexpression of p97Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97Eps8-mediated ERK activation.

Two isoforms of Eps8, p97Eps8 and p68Eps8, have been identified as the substrates for receptor tyrosine kinases. Our previous studies indicated that both tyrosyl phosphorylation and protein expression of Eps8 were elevated in v-Src transformed cells. In an attempt to examine the role played by p97Eps8 in tumorigenesis, we have first obtained cells overexpressing p97Eps8 and its pleckstrin homology (PH)-truncated variant. We then demonstrated that cells overexpressing p97Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. Furthermore, elevated serum-induced extracellular responsive kinase (ERK) activation was observed in p97Eps8 overexpressors. This enhanced ERK activation was sensitive to a MEK1 specific inhibitor PD98059 and was important for p97Eps8-mediated transformation, since transfection of vectors expressing dominant negative MEK1 and p97Eps8 abrogated focus formation by p97Eps8. In contrast, PH-truncated p97Eps8 failed to localize at the plasma membrane and that the truncated variant also did not elevate ERK activation and cellular transformation in response to serum stimulation. Our results thus indicated that: (i) the gene encoding p97Eps8 was an oncogene; (ii) p97Eps8-induced oncogenesis was partly mediated by ERK activation; and (iii) the PH domain of p97Eps8 was critical for its cellular localization, ERK activation and its ability to transform cells. Oncogene (2001) 20, 106 - 112.

FMLP-induced formation of F-actin in HL60 cells is dependent on PI3-K but not on intracellular Ca2+, PKC, ERK or p38 MAPK.

To further understand the mechanisms of signal transduction pathways for the formation of F-actin (polymerization of actin) and the activation of NADPH oxidase in phagocytic cells, the effects of various inhibitors on them were studied. Differentiated HL60 cells were studied to examine their N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated formation of F-actin and activation of NADPH oxidase following treatment with various inhibitors. These included a protein kinase C (PKC) inhibitor (GF 109203X), a phosphatidylinositide 3 kinase (PI3-K) inhibitor (wortmannin), an extracellular response kinase (ERK) inhibitor (PD 98059), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB 203580) and an intracellular Ca2+ -chelator (BAPTA-AM). The treatment with wortmannin suppressed the formation of F-actin, with less suppression of the activation of NADPH oxidase. BAPTA-AM and GF 109203X did not attenuate the formation of F-actin but completely inhibited the activation of NADPH oxidase. PD 98059 and SB 203580 partially inhibited the activation of NADPH oxidase without influence on the formation of F-actin. Furthermore, wortmannin but not BAPTA-AM and GF 109203X inhibited the fMLP-induced activation of Akt, which is known to regulate NADPH oxidase. These results suggest that the formation of F-actin is dependent on PI3-K and independent of PKC, ERK and p38 MAPK as well as the increase in intracellular Ca2+, whereas the activation of NADPH oxidase is partly dependent on ERK, p38 MAPK, Akt regulated by PI3-K, and strongly dependent on the activation of PKC and the increase in intracellular Ca2+.

Immunohistochemical localization of mitogen-activated protein kinase (MAPK) family and morphological changes in rat heart after ischemia-reperfusion injury.

The mitogen-activated protein kinase (MAPK) family is considered to be activated by stress, but the role of the MAPK family is still unknown in cardiac pathology. In the present study, not only the localization of MAPKs such as the extracellular responsive kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK (p38), but also ultrastructural changes were investigated in the ischemia-reperfusion model of Wistar rats. At 5, 10, 30, 60, and 180 min reperfusion after 30 min ischemia by occluding the coronary artery, the expression of these MAPKs was increased in blood vessels and cardiomyocytes by Western blotting and immunohistochemical methods. In addition, after ischemia reperfusion, various ultrastructural changes such as decreased glycogen granules, mitochondrial swelling, and myolysis were observed in the blood vessels and cardiomyocytes. These results suggest that protein kinases may regulate numerous biological processes, including the regulation of contraction and ion transport.

Mitogen-Activated Protein Kinases Are Differently Activated in Early Hypertrophic Response to Acute Pressure Overload in Rat Myocardium

P55 Mitogen-activated protein kinases (MAPKs) have been reported as important signaling molecules mediating cardiomyocyte hypertrophic response to various stimuli. Four subfamilies of MAPKs have been described in mammalian cells: c-jun NH2 terminal kinases (JNKs), p38-MAPKs (p38), extracellular response kinases (ERKs) and big MAP-kinase (BMK). Our objective was to study the activation of these kinases in the early phases of hypertrophic response, which is currently obscure. Transverse aorta constriction (TAC) was performed in male Wistar rats (160-200g) and left ventricles were studied up to 3 h following the surgery. To study the activation of ERKs, JNKs and p38, heart homogenates were separated into cytosolic (C) and nuclear (N) fractions and their kinase activities were determined by Western Blot using anti p-ERK, p-JNK and p-p38 antibodies. The membranes were then stripped and blotted with anti-ERK, anti-JNK and anti-p38 to determine the protein contents. The amount of BMK was studied in total homogenates using anti-BMK antibody and its activity was studied by measuring its in vitro autophosphorylation. ERK1/2 were transiently activated in both subcellular fractions, reaching a maximum 30 min after TAC (ERK1 - 236% in C and 196% in N; ERK2 - 280% in C and 248% in N) and returning toward basal levels after 1 h. p38 showed a sustained increase in its activity only in C (to 259%) but not in N after 1 h of TAC. JNKs showed a biphasic response with an early and major increase in their activities after 10 min (to 313% for JNK1 and to 288% for JNK2 in C) and a second and minor one following 3 h of TAC. In parallel, TAC induced a significant nuclear translocation of JNK1. The total amount of BMK remained stable during the studied period. We observed a progressive increase in its kinase activity which was directly related to TAC time course (45% in 10 min; 159% in 1 h and 173 % in 2h). These results indicate that pressure overload induces an early activation of the four subfamilies of MAPKs. Differences in their time course activation suggest that they might play different roles in the early hypertrophic response in adult rat myocardium.

Mitogen-Activated Protein Kinases Are Differently Activated in Early Hypertrophic Response to Acute Pressure Overload in Rat Myocardium

P55 Mitogen-activated protein kinases (MAPKs) have been reported as important signaling molecules mediating cardiomyocyte hypertrophic response to various stimuli. Four subfamilies of MAPKs have been described in mammalian cells: c-jun NH2 terminal kinases (JNKs), p38-MAPKs (p38), extracellular response kinases (ERKs) and big MAP-kinase (BMK). Our objective was to study the activation of these kinases in the early phases of hypertrophic response, which is currently obscure. Transverse aorta constriction (TAC) was performed in male Wistar rats (160-200g) and left ventricles were studied up to 3 h following the surgery. To study the activation of ERKs, JNKs and p38, heart homogenates were separated into cytosolic (C) and nuclear (N) fractions and their kinase activities were determined by Western Blot using anti p-ERK, p-JNK and p-p38 antibodies. The membranes were then stripped and blotted with anti-ERK, anti-JNK and anti-p38 to determine the protein contents. The amount of BMK was studied in total homogenates using anti-BMK antibody and its activity was studied by measuring its in vitro autophosphorylation. ERK1/2 were transiently activated in both subcellular fractions, reaching a maximum 30 min after TAC (ERK1 - 236% in C and 196% in N; ERK2 - 280% in C and 248% in N) and returning toward basal levels after 1 h. p38 showed a sustained increase in its activity only in C (to 259%) but not in N after 1 h of TAC. JNKs showed a biphasic response with an early and major increase in their activities after 10 min (to 313% for JNK1 and to 288% for JNK2 in C) and a second and minor one following 3 h of TAC. In parallel, TAC induced a significant nuclear translocation of JNK1. The total amount of BMK remained stable during the studied period. We observed a progressive increase in its kinase activity which was directly related to TAC time course (45% in 10 min; 159% in 1 h and 173 % in 2h). These results indicate that pressure overload induces an early activation of the four subfamilies of MAPKs. Differences in their time course activation suggest that they might play different roles in the early hypertrophic response in adult rat myocardium.

Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptides Stimulate Mitogen-Activated Protein Kinase in the Pituitary Cell Line GH4C1 by a 3′,5′-Cyclic Adenosine Monophosphate Pathway

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP38) regulate anterior pituitary cell secretion and proliferation. In the somatolactotrope GH4C1 cell line, these effects are mediated through the type-II-like PACAP receptor (VPAC2) coupled to the cAMP pathway. In this study, the control of the extracellularly responsive kinases (ERKs) by VIP and PACAP38 was investigated in GH4C1 cells. VIP and PACAP38 increased ERK1 and ERK2 phosphorylation and were equipotent stimulators of both kinases. ERK activation was mimicked by cholera toxin, forskolin and 8bromo-cAMP. VIP and PACAP38 activation of ERK2 was blocked by the protein kinase A inhibitor H89, whereas the protein kinase C inhibitor GF109203X, or prior PMA-induced depletion of the protein kinases C, failed to inhibit VIP and PACAP38 activation of ERK2. In contrast, thyrotropin-releasing hormone (TRH) elicited ERK activation by a PKC-dependent process. ERK activation by VIP or PACAP38 and TRH were additive and both sensitive to the MEK inhibitors PD98059 and U0126. In parallel, U0126 reduced prolactin (PRL) mRNA levels induced by VIP. These results demonstrate for the first time that VIP and PACAP38 activate ERK in GH4C1 cells. Cyclic AMP increase is sufficient to elicit ERK activation in these cells and thus likely to represent the transduction pathway underlying VIP- and PACAP38-dependent ERK activation. This mechanism seems to be involved in VIP-induced PRL gene regulation.