A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection

The MAP kinase pathway has been shown to be active in many growth factor signaling systems, including that of prolactin (PRL). In our studies, the main objective was to examine the possible involvement of MEK kinases (Map/Erk kinase kinases) in PRL-stimulated mitogenic and lactogenic processes. We used the MEK kinase inhibitor PD 098059 to block MEK kinase activation in the Nb2 cell line and mammary gland explants derived from 12- to 14-day pregnancy mice. PD 098059 attenuated PRL-induced Nb2 cell mitogenesis at 10 microM and a maximum inhibition was observed at 100 microM. In cultured mammary tissues, PD 098059 at 100 microM had no effect on the PRL stimulation of lipid, casein and lactose synthesis and iodide uptake. Further, the growth-inhibitory effect of PD 098059 on Nb2 cells was ameliorated when the drug was removed from the culture medium, indicating that PD 098059 acts in a reversible manner. When MEK1 was immunoprecipitated from PD 098059 and/or PRL treated Nb2 cells, PRL-stimulated MEK1 kinase activity was directly inhibited by PD 098059 at concentrations employed in the culture experiments. PRL has no effect on the tyrosyl phosphorylation of MAP kinases in cultured mammary tissues derived from pregnant mice, whereas earlier we found that PRL stimulates the tyrosyl phosphorylation of all four MAP kinases in Nb2 cells. The results suggest that the MAP kinase pathway plays an important role in the PRL stimulation of Nb2 cell mitogenesis but is not involved in the PRL stimulation of milk product synthesis.     

Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation

Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In the present study, we examined the signaling pathway underlying IGF-I-mediated neurite outgrowth. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induced concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation was observed within 1 min, maximal phosphorylation of ERKs was not reached for 30 min. Both IGF-IR and ERK phosphorylation were maintained for at least 24 h. Also, the concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation paralleled that of IGF-I-mediated neurite outgrowth. We further examined the role of mitogen-activated protein kinase activation in IGF-I-stimulated neuronal differentiation using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059. Whereas PD98059 had no effect on IGF-IR phosphorylation, PD98059 reduced IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produced a parallel reduction of IGF-I-stimulated neurite outgrowth. Finally, consistent with its ability to block neuronal differentiation, PD98059 inhibited IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation.     

Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase

p38 is a member of the mitogen-activated protein (MAP) kinase superfamily activated by stress signals and implicated in cellular processes involving inflammation and apoptosis. Unlike the extracellular signal-regulated kinases (p42 and p44 MAP kinases), which are stimulated by insulin in many cell types, p38 activity is inhibited by insulin in postmitotic fetal neurons for which insulin is a potent survival factor (Heidenreich, K. A., and Kummer, J. L. (1996) J. Biol. Chem. 271, 9891-9894). These data suggested that insulin's effects on neuronal survival are mediated by inhibition of a p38-mediated apoptotic pathway. To better understand the relationship between p38 activity and cell survival, we induced apoptosis in two cell lines and examined the ability of insulin or a specific p38 inhibitor (a pyridinyl imidazole compound PD169316) to block p38 activity and cell death. In Rat-1 fibroblasts grown in the presence of serum, p38 activity was undetectable by immune complex assays, and the number of apoptotic cells was very low (<0.5%). After the removal of serum for 16 h, p38 activity was markedly elevated, and apoptosis increased by 14-15-fold. Insulin (50 ng/ml) inhibited p38 activity by approximately 70% and blocked apoptosis by at least 80%. PD169316 also blocked p38 enzyme activity and apoptosis by approximately 80%. Similar results were obtained in differentiated PC12 cells that were deprived of nerve growth factor (NGF) for 16 h. In the presence of NGF, p38 activity and the number of apoptotic cells was very low (approximately 1.0%). After NGF withdrawal, p38 activity was selectively elevated and apoptosis increased to 15%. Both insulin and PD169316 markedly blocked the increase in p38 activity and apoptosis. The MAP kinase kinase inhibitor, PD98059, had no effect on apoptosis in Rat-1 fibroblasts and only partially blocked apoptosis in PC12 cells. PD98059 did not influence insulin's ability to block apoptosis, indicating that the extracellular signal-regulated kinase pathway does not mediate insulin's survival effects. These data further support the role of p38 in cellular apoptosis and support the hypothesis that insulin promotes cell survival, at least in part, by inhibiting the p38 pathway.     

Human metabotropic glutamate receptor 2 couples to the MAP kinase cascade in chinese hamster ovary cells

We have examined the functional coupling of the human metabotropic glutamate receptor type 2 (mGluR2) with the regulation of the mitogen activated protein kinase (MAP kinase) signal transduction cascade. We demonstrated that L-glutamate stimulation of the human mGluR2 receptor transiently expressed in chinese hamster ovary (CHO) cells leads to a rapid increase in the activity of p42/p44 MAP kinase (also known as the extracellular signal regulated kinases, ERK1 and ERK2). Activation of p42/p44 MAP kinase has been demonstrated in a peptide phosphorylation assay and through the demonstration of a shift in electrophoretic mobility of p42 MAP kinase following activation. In both assay systems L-glutamate stimulation of MAP kinase was inhibited by pertussis toxin and by the MEK (MAP/ERK activating kinase) inhibitor PD 98059. We conclude that L-glutamate stimulation of the mGluR2 receptor in CHO cells mediated regulation of p42/p44 MAP kinase following the activation of pertussis toxin-sensitive G alpha(i) G-proteins via a distinct protein kinase signalling pathway that utilizes MEK.     

An inhibitory fragment derived from protein kinase Cepsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters

We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKCdelta and PKCepsilon, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKCepsilon (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKCepsilon in cardiac myocytes. Phorbol ester-stimulated translocation of PKCepsilon was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKCdelta. These results demonstrate that PKCepsilon mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.     

Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization

In SH-SY5Y human neuroblastoma cells, insulin-like growth factor (IGF)-I mediates membrane ruffling and growth cone extension. We have previously shown that IGF-I activates the tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) 2. In the current study, we examined which signaling pathway underlies IGF-I-mediated FAK phosphorylation and cytoskeletal changes and determined if an intact cytoskeleton was required for IGF-I signaling. Treatment of SH-SY5Y cells with cytochalasin D disrupted the actin cytoskeleton and prevented any morphological changes induced by IGF-I. Inhibitors of phosphatidylinositol 3-kinase (PI 3-K) blocked IGF-I-mediated changes in the actin cytoskeleton as measured by membrane ruffling. In contrast, PD98059, a selective inhibitor of ERK kinase, had no effect on IGF-I-induced membrane ruffling. In parallel with effects on the actin cytoskeleton, cytochalasin D and PI 3-K inhibitors blocked IGF-I-induced FAK tyrosine phosphorylation, whereas PD98059 had no effect. It is interesting that cytochalasin D did not block IGF-I-induced ERK2 tyrosine phosphorylation. Therefore, it is likely that FAK and ERK2 tyrosine phosphorylations are regulated by separate pathways during IGF-I signaling. Our study suggests that integrity as well as dynamic motility of the actin cytoskeleton mediated by PI 3-K is required for IGF-I-induced FAK tyrosine phosphorylation, but not for ERK2 activation.     

Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cgamma1 (PLCgamma1). We used recombinant glutathione-S-transferase (GST)-PLCgamma1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCgamma1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCgamma1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway

PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359-2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.     

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.     

Interleukin-8 and chemotactic activity of middle ear effusions

Chemotactic factors, i.e., an N-formyl peptide, C5a, interleukin-8, and leukotriene B4, induced neutrophils to activate mitogen-activated protein (MAP) kinases, as defined by the tyrosine phosphorylation and decrease in electrophoretic mobility of immunodetected 44-, 42-, and 40-kDa proteins. PD 98059, an inhibitor of MAP kinase kinase activation, blocked these changes. The drug likewise blocked neutrophil chemotaxis but did not alter superoxide anion production and paradoxically enhanced degranulation responses to the stimuli. The MAP kinase pathway appears to have a highly selective role in mediating motility but not other cellular responses.     

Sustained activation of the mitogen-activated protein kinase pathway. A mechanism underlying receptor tyrosine kinase specificity for matrix metalloproteinase-9 induction and cell migration

Activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is required for ligand-dependent regulation of numerous cellular functions by receptor tyrosine kinases. We have shown previously that although many receptor tyrosine kinase ligands are mitogens for keratinocytes, cell migration and induction of the 92-kilodalton gelatinase/matrix metalloproteinase (MMP)-9 are selectively regulated by the epidermal growth factor and scatter factor/hepatocyte growth factor receptors. In this report we present evidence of an underlying mechanism to account for these observed differences in receptor tyrosine kinase-mediated response. Ligands that are mitogenic, but do not induce MMP-9 or colony dispersion, transiently activate the p42/p44 ERK/MAP kinases. In contrast, ligands that stimulate MMP-9 induction and colony dispersion induced sustained activation of these kinases. The functional significance of sustained MAPK activation was demonstrated by inhibition of the MAP kinase kinase MEK1. Disruption of the prolonged signal by addition of the MEK1 inhibitor PD 98059 up to 4 h after growth factor stimulation substantially impaired ligand-dependent colony dispersion and MMP-9 induction. These findings support the conclusion that duration of MAPK activation is an important determinant for certain growth factor-mediated functions in keratinocytes.     

Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen-activated protein kinase (MAPK) cascades in relation to oxidant-induced cell death in an oligodendrocyte cell line, central glia-4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H2O2) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet-derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK). Dose-response studies showed differential sensitivities of PDGF receptor phosphorylation (>1 mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1 mM) activation by H2O2. The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H2O2 also activated MAPK-activated protein kinase-2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant-induced cell death was indicated by morphological changes, decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose-dependently by the MEK inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant-mediated cytotoxicity.     

Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current

Astrocytes swell during neuronal activity as they accumulate K+ to buffer the increase in external K+ released from neurons. This swelling activates volume-sensitive Cl- channels, which are thought to be important in regulatory volume decrease and in the response of the CNS to trauma and excitotoxicity. Mitogen-activated protein (MAP) kinases also are activated by cell volume changes, but their roles in volume regulation are unknown. We have investigated the role of tyrosine and MAP kinases in the activation of volume-activated Cl- channels in cultured astrocytes, using whole-cell patch-clamp recording and Western immunoblots. As previously described, hypo-osmotic solution induced an outwardly rectifying Cl- current, which was blocked by NPPB and SITS. This Cl- current did not depend on [Ca2+ ]i because it was still observed when 20 mM BAPTA was included in the pipette, but it did exhibit rundown when ATP was omitted. Inhibition of tyrosine kinases with genistein or tyrphostin A23 (but not the inactive agents daidzein and tyrphostin A1) blocked the Cl- current. The MAP kinase kinase (MEK) inhibitor PD 98059 reversibly inhibited activation of the Cl- current by hypo-osmotic solution. Western immunoblots showed that genistein or PD 98059 blocked activation of Erk-1 and Erk-2 by hypo-osmotic solution in astrocytes. Therefore, activation of tyrosine and MAP kinases by swelling is a critical step in the opening of volume-sensitive Cl- channels.     

Regulation of mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle cells

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.