Comparison of effects of protein kinase A, mitogen-activated protein kinase, and cyclin-dependent kinase blockers on rabbit ovarian granulosa cell functions

The aim of the present study was to define the role of protein kinase A (PKA)-, mitogen-activated protein kinase (MAPK)-, and cyclin-dependent kinase (CDK)-dependent pathways in the control of ovarian cell functions. The effects of PKA, MAPK, and CDK blockers (KT 5720, PD 98059, and olomoucine, respectively), given at doses of 0.001-10.0 μg/ml medium on functions of cultured rabbit granulosa cells were examined. Expression of PKA, MAPK/ERK1,2, secretory activity (IGF-I output), and proliferation (proliferating cell nuclear antigen, PCNA) in these cells were determined by RIA, immunocytochemistry and Western blotting. A PKA inhibitor, KT 5720 suppressed the expression of PKA and MAPK/ERK1,2, the IGF-I release, and the ratio of PCNA-positive cells in granulosa cells. A MAPK blocker, PD 98059 reduced the expression of MAPK/ERK1,2 (but not PKA), the IGF-I release, and percentage of PCNA-positive cells. A CDK blocker, olomoucine, increased the PKA expression, decreased the expression of MAPK/ERK1,2 and PCNA, but did not affect the IGF-I release. These observations confirm the involvement of PKs in control of basic ovarian functions and demonstrate the involvement of PKA in stimulation of ovarian cell proliferation and MAPK (but not CDK) and in promotion of ovarian IGF-I release. Different activity and specificity of the PKA, MAPK, and CDK blockers in their effects on PCNA and IGF-I suggests different biological role of these PKs in control of proliferative and secretory functions of rabbit ovarian cells.     

Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38(MAPK) phosphorylation. Phosphorylation of ERK1/2 or p38(MAPK) was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38(MAPK) was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38(MAPK) inhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90(Rsk)), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90(Rsk) and MAPKAP-K2 most closely reflects ERK and p38(MAPK) stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38(MAPK). These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.     

4-Aminopyridine-induced epileptogenesis depends on activation of mitogen-activated protein kinase ERK

Extracellular signal-regulated kinases such as ERK1 [p44 mitogen-activated protein kinase (MAPK)] and ERK2 (p42 MAPK) are activated in the CNS under physiological and pathological conditions such as ischemia and epilepsy. Here, we studied the activation state of ERK1/2 in rat hippocampal slices during application of the K(+) channel blocker 4-aminopyridine (4AP, 50 micro m), a procedure that enhances synaptic transmission and leads to the appearance of epileptiform activity. Hippocampal slices superfused with 4AP-containing medium exhibited a marked activation of ERK1/2 phosphorylation that peaked within about 20 min. These effects were not accompanied by changes in the activation state of c-Jun N-terminal kinase (JNK), another member of the MAP kinase superfamily. 4AP-induced ERK1/2 activation was inhibited by the voltage-gated Na(+) channel blocker tetrodotoxin (1 micro m). We also found that application of the ERK pathway inhibitors U0126 (50 micro m) or PD98059 (100 micro m) markedly reduced 4AP-induced epileptiform synchronization, thus abolishing ictal discharges in the CA3 area. The effects induced by U0126 or PD98059 were not associated with changes in the amplitude and latency of the field potentials recorded in the CA3 area following electrical stimuli delivered in the dentate hylus. These data demonstrate that activation of ERK1/2 accompanies the appearance of epileptiform activity induced by 4AP and suggest a cause-effect relationship between the ERK pathway and epileptiform synchronization.     

Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059.

The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation.     

Opposite effects of inhibitors of mitogen-activated protein kinase pathways on the egr-1 and beta-globin expression in erythropoietin-responsive murine erythroleukemia cells

The effect of erythropoietin (Epo) on the expression of mitogen-activated protein kinase (MAPK) target genes egr-1 and c-fos was investigated in Epo-responsive murine erythroblastic cell line ELM-I-1. Epo induced a transient rise in egr-1 mRNA without a similar effect on c-fos expression. The induction of egr-1 correlated with a rapid ERK1/2 phosphorylation and was prevented with MEK1/2 inhibitors PD 98059 and UO126. The p38 inhibitor SB 203580 enhanced ERK1/2 phosphorylation and egr-1 mRNA levels. Longer incubations of ELM-I-1 cells with Epo revealed a second later phase of increase in egr-1 expression which was also prevented by MEK1/2 inhibitors, whereas SB 203580 had a stimulatory effect. In contrast, the beta-globin mRNA production was enhanced in the presence of PD 98059 and UO126 and reduced by SB 203580. The results suggest a regulatory role of egr-1 expression in Epo signal transduction and provide pharmacological evidence for the negative modulation of differentiation-specific gene expression by the ERK1/2 pathway in murine erythroleukemia cells.     

Requirement of extracellular signal-regulated kinase/mitogen-activated protein kinase for long-term potentiation in adult mouse anterior cingulate cortex

Long-term potentiation (LTP) in the anterior cingulate cortex (ACC) is believed to be critical for higher brain functions including emotion, learning, memory and chronic pain. N-methyl-D-aspartate (NMDA) receptor-dependent LTP is well studied and is thought to be important for learning and memory in mammalian brains. As the downstream target of NMDA receptors, the extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) cascade has been extensively studied for its involvement in synaptic plasticity, learning and memory in hippocampus. By contrast, the role of ERK in cingulate LTP has not been investigated. In this study, we examined whether LTP in ACC requires the activation of ERK. We found that P42/P44 MAPK inhibitors, PD98059 and U0126, suppressed the induction of cingulate LTP that was induced by presynaptic stimulation with postsynaptic depolarization (the pairing protocol). We also showed that cingulate LTP induced by two other different protocols was also blocked by PD98059. Moreover, we found that these two inhibitors had no effect on the maintenance of cingulate LTP. Inhibitors of c-Jun N-terminal kinase (JNK) and p38, other members of MAPK family, SP600125 and SB203850, suppressed the induction of cingulate LTP generated by the pairing protocol. Thus, our study suggests that the MAPK signaling pathway is involved in the induction of cingulate LTP and plays a critical role in physiological conditions.     

ERK1/2 and p38 mitogen-activated protein kinase mediate iNOS-induced spinal neuron degeneration after acute traumatic spinal cord injury

The enhanced production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of neuronal apoptosis after acute traumatic spinal cord injury (SCI). In the present study, to further characterize the pathways mediating the synthesis and release of NO, we examined activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) in microglia/macrophages in the injured area of adult rats subjected to a complete transection at the T10 vertebrae level and assessed their role in NO production and survival of neurons by using immunohistochemistry, Western blot, RT-PCR and pharmacological interventions. Results showed activation of microglia/macrophages featured by morphological changes, as visualized immunohistochemically with the marker OX-42, in the areas adjacent to the lesion epicenter 1 h after surgery. Concomitantly, iNOS mRNA and its protein in the activated microglia/macrophages were also significantly upregulated at early hours after surgery. Their levels were maximal at 6 h, persisted for at least 24 h, and returned to basal level 72 h after SCI. Furthermore, phosphorylated ERK1/2 and p38 MAPK were activated as well in microglia/macrophages in injured area with a similar time course as iNOS. With administration of L-NAME, a NOS inhibitor, the number of apoptotic neurons was clearly decreased, as assessed with TUNEL method at 24 h after SCI. In parallel, loss of neurons induced by SCI, assessed with NeuN immunohistochemistry, was also diminished. Moreover, the effect of inhibition of phosphorylation ERK1/2 and p38 MAPK by corresponding inhibitors PD98059 and SB203580 administered before and after SCI was also investigated. Inhibition of p38 effectively reduced iNOS mRNA expression and rescued neurons from apoptosis and death in the area adjacent to the lesion epicenter; whereas the inhibition of ERK1/2 had a smaller effect on decrease of iNOS mRNA and no long-term protective effect on cell loss. These results indicate the ERK1/2 and p38 MAPK signaling pathway, especially the latter, play an important role in NO-mediated degeneration of neuron in the spinal cord following SCI. Strategies directed to blocking the initiation of this cascade prove to be beneficial for the treatment of acute SCI.     

Insulin induces expression of adenosine kinase gene in rat lymphocytes by signaling through the mitogen-activated protein kinase pathway

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.     

Activation of p38 mitogen-activated protein kinase abolishes insulin-mediated myocardial protection against ischemia-reperfusion injury

Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU.min(-1).kg(-1)) was begun either 10 min before ischemia (InsulinBI), 5 min before reperfusion (InsulinBR), or 30 min after the onset of reperfusion (InsulinAR), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both InsulinBI (P < 0.001) and InsulinBR (P < 0.02) rats but not in InsulinAR rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in InsulinBI and InsulinBR rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 min before reperfusion enabled insulin-mediated myocardial protection in InsulinAR rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect.     

Anoxic preconditioning up-regulates 14-3-3 protein expression in neonatal rat cardiomyocytes through extracellular signal-regulated protein kinase 1/2

Anoxic preconditioning (APC) attenuates myocardial injury caused by ischemia/reperfusion. The protective mechanisms of APC involve up-regulation of the protective proteins and inhibition of apoptosis. 14-3-3 protein, as a molecular chaperone, plays an important role in regulating cell survival and apoptosis. However, the role of 14-3-3 protein in cardioprotection of APC and the pathways determining 14-3-3 protein expression during APC are not clear. In this work, Western blotting analysis was used to detect the 14-3-3 protein expression and activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2) in cardiomyocytes subjected to anoxia-reoxygenation injury with and without APC and control. The cardiomyocytes from APC group were more resistant to injury induced by anoxia-reoxygenation and had much stronger phosphorylation of ERK1/2 than the control. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in cardiomyocytes induced by APC. The results indicate that APC up-regulates 14-3-3 protein expression through the ERK1/2 signaling pathways.     

Roles of mitogen-activated protein kinase pathways during <Emphasis Type="Italic">Escherichia coli-</Emphasis>induced apoptosis in U937 cells

Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 cells.     

Anchorage-independent activation of mitogen-activated protein kinase through phosphatidylinositol-3 kinase by insulin-like growth factor I

Insulin-like growth factor I (IGF-I) is a well-established mitogen in human breast cancer cells. We show here that human breast cancer MCF-7 cells, which were prevented from attaching to the substratum and were floating in medium, responded to IGF-I and initiated DNA synthesis. The addition of IGF-I to floating cells induced activation of protein kinase B (PKB)/Akt, as to cells attached to the substratum. In addition, mitogen-activated protein kinase (MAPK)/extracellular response kinase (ERK) and its upstream kinases, ERK kinase (MEK) and Raf-1, were activated by IGF-I in floating cells. While the IGF-I-induced activation of PKB/Akt was inhibited by PI3-K inhibitor LY294002 but not by MEK inhibitor PD98059, the activation of both MEK and ERK by IGF-I was inhibited by both. These findings suggest that the IGF-I signal that leads to stimulation of DNA synthesis of MCF-7 cells is transduced to ERK through PI3-K, only when they are anchorage-deficient.     

Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.