cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells

Mitogen-activated protein kinases (MAPKs) are activated by a variety of extracellular stimuli, including agonists for G protein-coupled receptors. Using transient transfection of COS-7 cells, we have studied the stimulation of a hemagglutinin-tagged p44mapk (p44HA-mapk) by receptors coupled to Gs, Gq, and Gi. Agonists that act via all three G proteins stimulated p44HA-mapk activity. A constitutively activated alpha s mutant, forskolin, and a cAMP analog also increased p44HA-mapk activity, indicating that cAMP in COS-7 cells, in contrast to other cell types, activates the MAPK pathway. Similarly, a constitutively activated alpha q mutant, overexpression of phospholipase C-beta 2, and a phorbol ester also stimulated p44HA-mapk, suggesting that Gq-coupled receptors stimulate the MAPK pathway by increasing phosphatidylinositol turnover and probably stimulating protein kinase C. In COS-7 cells, in contrast to Rat-1 cells, mutationally activated alpha i did not stimulate the MAPK pathway. G protein beta and gamma subunits, overexpressed together, did activate p44HA-mapk; this finding suggests that in COS-7 cells Gi-coupled receptors may stimulate the MAPK pathway through beta gamma. These unexpected results in COS-7 cells show that G proteins and second messengers regulate the MAPK pathway differently in different cell types.     

Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli

We report the cloning of a novel human activator of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 7 (MKK7). The mRNA for MKK7 is widely expressed in humans and mice and encodes a 47-kDa protein (419 amino acids), as determined by immunoblotting endogenous MKK7 with an antibody raised against its N terminus. The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and was more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylated and activated JNK1 but failed to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 was activated by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 was also activated when cells were exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-alpha. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activated MKK7, suggesting that MKK7 will be involved in many physiological pathways.     

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.     

Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells

Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and beta-lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNKI or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicin-induced JNKI activity, but had no effect on JNKI activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a specific intracellular Ca2+ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no effect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The differential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of antimicrotubule drugs.     

Differential regulation of IkappaB kinase alpha and beta by two upstream kinases, NF-kappaB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1

NF-kappaB is activated by various stimuli including inflammatory cytokines and stresses. A key step in the activation of NF-kappaB is the phosphorylation of its inhibitors, IkappaBs, by an IkappaB kinase (IKK) complex. Recently, two closely related kinases, designated IKKalpha and IKKbeta, have been identified to be the components of the IKK complex that phosphorylate critical serine residues of IkappaBs for degradation. A previously identified NF-kappaB-inducing kinase (NIK), which mediates NF-kappaB activation by TNFalpha and IL-1, has been demonstrated to activate IKKalpha. Previous studies showed that mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-kappaB by an undefined mechanism. Here, we show that overexpression of MEKK1 preferentially stimulates the kinase activity of IKKbeta, which resulted in phosphorylation of IkappaBs. Moreover, a catalytically inactive mutant of IKKbeta blocked the MEKK1-induced NF-kappaB activation. By contrast, overexpression of NIK stimulates kinase activities of both IKKalpha and IKKbeta comparably, suggesting a qualitative difference between NIK- and MEKK1-mediated NF-kappaB activation pathways. Collectively, these results indicate that NIK and MEKK1 independently activate the IKK complex and that the kinase activities of IKKalpha and IKKbeta are differentially regulated by two upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli.     

Regulation of human neuronal calcium channels by G protein betagamma subunits expressed in human embryonic kidney 293 cells

Serotonin (5-HT) was found to inhibit steroid (17 alpha,20 beta-dihydroxy-4-pregnen-3-one; 17,20 beta P)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > alpha-methyl-5HT > 2-methyl-5HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20 beta P-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT1A, 5-HT2, and 5-HT3 receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs.     

Differential regulation of insulin receptor substrate-2 and mitogen-activated protein kinase tyrosine phosphorylation by phosphatidylinositol 3-kinase inhibitors in SH-SY5Y human neuroblastoma cells

Insulin-like growth factor I (IGF-I) is a potent neurotropic factor promoting the differentiation and survival of neuronal cells. SH-SY5Y human neuroblastoma cells are a well characterized in vitro model of nervous system growth. We report here that IGF-I stimulated the tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and insulin receptor substrate-2 (IRS-2) in a time- and concentration-dependent manner. These cells lacked IRS-1. After being tyrosine phosphorylated, IRS-2 associated transiently with downstream signaling molecules, including phosphatidylinositol 3-kinase (PI 3-K) and Grb2. Treatment of the cells with PI 3-K inhibitors (wortmannin and LY294002) increased IGF-I-induced tyrosine phosphorylation of IRS-2. We also observed a concomitant increase in the mobility of IRS-2, suggesting that PI 3-K mediates or is required for IRS-2 serine/threonine phosphorylation, and that this phosphorylation inhibits IRS-2 tyrosine phosphorylation. Treatment with PI 3-K inhibitors induced an increased association of IRS-2 with Grb2, probably as a result of the increased IRS-2 tyrosine phosphorylation. However, even though the PI 3-K inhibitors enhanced the association of Grb2 with IRS-2, these compounds suppressed IGF-I-induced mitogen-activated protein kinase activation and neurite outgrowth. Together, these results indicate that although PI 3-K participates in a negative regulation of IRS-2 tyrosine phosphorylation, its activity is required for IGF-IR-mediated mitogen-activated protein kinase activation and neurite outgrowth.     

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.     

Substance P-induced mitogenesis in human astrocytoma cells correlates with activation of the mitogen-activated protein kinase signaling pathway

The neuropeptide substance P (SP) regulates many biological processes through binding to and activating the SP receptor (NK-1 subtype). Activation of the SP receptor induces mitogenesis in several cell types. In this study, we characterized the mitogenic response induced by SP peptide in the U-373MG astrocytoma cell line and showed that activation of the SP receptor induces [3H]thymidine incorporation into DNA. We also found that SP potently induces c-myc mRNA and protein in the U-373MG cells. Tyrphostin A25, which blocks activity of tyrosine kinases, significantly inhibited SP-induced mitogenesis, suggesting that the mitogenic response induced by SP peptide involves phosphorylation by tyrosine kinases. Furthermore, stimulation of the SP receptor activates tyrosine phosphorylation and enzymatic activity of extracellular signal-regulated kinases (Erk1 and Erk2), also called the mitogen-activated protein kinases (MAPKs). This result suggests that MAPKs participate in the SP peptide-induced signaling pathway. The addition of CP 96,345 ([(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1 -azabicyclo[2.2.2]octan-3-amine]; an NK-1 receptor antagonist) or PD 098059 (MEK1 inhibitor) inhibited both DNA synthesis and activation of the MAPK pathway, substantiating that SP stimulates mitogenesis by activating the MAPK pathway through receptors of the NK-1 subtype. Our results demonstrate that SP peptide is a strong mitogen in the U-373MG astrocytoma cell line and establish a clear correlation between SP-induced mitogenesis and activation of MAPK signaling pathway.     

Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells

We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.     

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.     

Agonist-dependent internalization of the human melanocortin-4 receptors in human embryonic kidney 293 cells

A chimeric protein comprised of melanocortin-4 receptor (MC4R) and the green fluorescent protein (GFP) was created for studying receptor/ligand localization and trafficking. The ligand binding affinities and second messenger stimulation induced by MC4R-GFP closely resembled those of the wild-type receptor, suggesting functional integrity of the chimeric protein. As observed with a confocal microscope, in human embryonic kidney (HEK)-293 cells MC4R/GFP was distributed evenly along the cell membrane. Addition of [Nle4-d-Phe7]-alpha-melanocyte-stimulating hormone (NDP-MSH), a peptide MC4R agonist, induced receptor translocation into intracellular compartments in a time- and concentration-dependent manner. [Ac-Nle-c[Asp-His-d-Nal(2')-Arg-Trp-Lys]-NH2] (SHU9119), a potent MC4R antagonist, completely inhibited NDP-MSH-mediated internalization. MC4R-GFP internalization was unaffected by a protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), but was impaired by pretreatment with inhibitors of endocytosis through clathrin-coated pits, hypertonic sucrose, or concanavalin A. Time-dependent colocalization of MC4R-GFP with rhodamine-transferrin, an early endosome marker, and with LysoTraker, a lysosome marker, was observed after short-term (45 min) and prolonged (20 h) agonist exposure, respectively. Rhodamine-[AcNle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2] (MTII), a fluorescent derivative of an MC4R agonist, was found to cointernalize with MC4R-GFP into intracellular vesicles. No significant receptor recycling or segregation from the ligand was observed 60 min after removal of the agonist. In contrast, an antagonist rhodamine-Ac-Cys-Glu-His-(d-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2 (HS014) bound to and colocalized with MC4R-GFP on the cell surface and did not stimulate receptor internalization. In sum, these results suggest that MC4R is subject to agonist-dependent endocytosis via clathrin-coated pits. Prolonged agonist exposure directs MC4R into lysosomes, possibly for degradation. Receptor and ligand recycling is not efficient for MC4R in HEK-293 cells.     

Blockade of mitogen-activated protein kinase cascade signaling in interleukin 6-independent multiple myeloma cells

OBJECTIVE: There have been few studies concerning the clinical pathology of squamous cell carcinoma arising from ovarian mature cystic teratoma. Thus, the objective of this study is to determine clinicopathologic factors affecting survival in this rare tumor. METHODS: From September 1979 to June 1996, 37 patients with squamous cell carcinoma arising from ovarian mature cystic teratoma were treated by the Tokai Ovarian Tumor Study Group. A retrospective clinicopathologic and survival analysis of these patients was performed. The mode of infiltration was classified into expansive, moderately diffused, and diffused patterns. RESULTS: Although the 5-year survival rate was 94.7% and 80.0% for stage I and II patients, respectively, 12 of 13 patients with stage III died within 20 months (P = .0001). A significant difference was also observed between the survival of the groups with and without residual tumor at surgery (P = .0001). Pathologic features, grade, mode of infiltration, and vascular involvement were significant factors by univariate analysis. Multivariate analysis showed significant differences in survival related to grade (P = .0154) and mode of infiltration (P = .0053). The preoperative squamous cell carcinoma antigen level was significantly higher in the patients with squamous cell carcinoma arising from mature cystic teratoma than in patients with mature cystic teratoma (P < .0001). CONCLUSION: This study suggests that pathologic factors, grade, and mode of infiltration can provide valuable information for predicting the survival of patients with squamous cell carcinoma arising from mature cystic teratoma. In addition, squamous cell carcinoma antigen may be a useful marker to detect this disease preoperatively.     

Roles for protein kinase C and mitogen-activated protein kinase in nicotine-induced secretion from bovine adrenal chromaffin cells

Both the Ca2+/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca2+ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca2+ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC50 1-5 microM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC50 approximately 10 microM) than that induced by nicotine (IC50 approximately 30 microM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca2+ ionophore and the participation of both PKC and MEK/MAPK in optimal, secretion induced by nicotine.     

Leukotriene D4 activates mitogen-activated protein kinase through a protein kinase Calpha-Raf-1-dependent pathway in human monocytic leukemia THP-1 cells

Leukotriene D4 (LTD4) is a major lipid mediator involved in inflammatory and allergic disorders including bronchial asthma. Despite its potent biological activity, little is known about the receptor and intracellular signaling pathways. Here we analyzed the signal transduction mechanisms through LTD4 receptors using human monocytic leukemia THP-1 cells. When these cells were stimulated with LTD4, intracellular calcium concentration was increased and mitogen-activated protein kinase (MAP kinase) was activated severalfold. This activation was inhibited by staurosporine or GF109203X treatment or abolished by protein kinase C depletion. Cytosolic protein kinase Calpha was translocated to the membrane, and Raf-1 was activated by LTD4 treatment in a similar time course. LTD4-induced Raf-1 activation was diminished by protein kinase C depletion in the cells. A chemotactic response of THP-1 cells toward LTD4 was observed which was inhibited by pertussis toxin (PTX) pretreatment. Thus, LTD4 has at least two distinct signaling pathways in THP-1 cells, a PTX-insensitive mitogen-activated protein kinase activation through protein kinase Calpha and Raf-1 and a PTX-sensitive chemotactic response. This cellular signaling can explain in part the versatile activities of LTD4 in macrophages under inflammatory and allergic conditions.     

Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling

Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of beta2-adrenergic receptors (beta2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following beta2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits beta2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis.     

Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38

Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.     

Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells

Activation of stress-activated protein kinases, including the p38 and the c-Jun NH2-terminal kinases (JNK), have been associated with the onset of cardiac hypertrophy and cell death in response to hemodynamic overload and ischemia/reperfusion injury. Upon infection of cultured neonatal rat cardiac myocytes with recombinant adenoviral vectors expressing a wild type and a constitutively active mutant of MKK7 (or JNKK2), JNK was specifically activated without affecting other mitogen-activated protein kinases, including extracellular signal-regulated protein kinases and p38. Specific activation of the JNK pathway in cardiac myocytes induced characteristic features of hypertrophy, including an increase in cell size, elevated expression of atrial natriuretic factor, and induction of sarcomere organization. In contrast, co-activation of both JNK (by MKK7) and p38 (by MKK3 or MKK6) in cardiomyocytes led to an induction of cytopathic responses and suppression of hypertrophic responses. These data provide the first direct evidence that activation of JNK alone is sufficient to induce characteristic features of cardiac hypertrophy, thereby supporting an active role for the JNK pathway in the development of cardiac hypertrophy. The cytopathic response, as a result of co-activation of both JNK and p38, may contribute to the loss of contractile function and viability of cardiomyocytes following hemodynamic overload and cardiac ischemia/reperfusion injury.