Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway

Expression ofendothelial nitric oxide synthase (eNOS) in transfected U-937 cellsupregulates phorbol 12-myristate 13-acetate (PMA)-induced tumornecrosis factor- (TNF-) production through a superoxide(O)-dependent mechanism. Because mitogen-activatedprotein kinases (MAPK) have been shown to participate in both reactiveoxygen species signaling and TNF- regulation, their possible role ineNOS-derived O signal transduction was examined. Aredox-cycling agent, phenazine methosulfate, was found to bothupregulate TNF- (5.8 ± 1.0 fold; P = 0.01) andincrease the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. AlthoughS-nitroso-N-acetylpenicillamine, a nitric oxide(NO) donor, also increased TNF- production, NO exposure led tophosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-production by eNOS transfection was associated with increases inactivated p42/44 MAPK (P = 0.001), whereas levels ofphosphorylated p38 MAPK were unaffected. Furthermore, cotransfectionwith Cu/Zn superoxide dismutase, which blocks TNF- upregulation byeNOS, also abolished the effects on p42/44 MAPK. Expression ofGln³⁶¹eNOS, a mutant that produces O but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, twoNADPH binding site deletion mutants of eNOS that lack oxidase activityhad no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathwayinhibitor, blocked TNF- upregulation by eNOS (P = 0.02).Thus O produced by eNOS increases TNF- productionvia a mechanism that involves p42/44 MAPK activation.

     

Role of cAMP inhibition of p44/p42 mitogen-activated protein kinase in potentiation of protein secretion in rat lacrimal gland

We previously found that addition of cAMP and a Ca(2+)/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined whether cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity in the lacrimal gland. Since we know that activation of MAPK attenuates protein secretion stimulated by Ca(2+)- and PKC-dependent agonists, we also determined whether this activation causes potentiation of secretion. Freshly prepared rat lacrimal gland acinar cells were incubated with dibutyryl cAMP (DBcAMP), carbachol (a cholinergic agonist), phenylephrine (an alpha(1)-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of G(s)-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca(2+) and PKC were increased by agonists.     

Activation of p44/p42 mitogen-activated protein kinase limits triiodothyronine-stimulated alkaline phosphatase activity in osteoblasts

It has been shown that thyroid hormone stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated whether p44/p42 mitogen-activated protein (MAP) kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. Triiodothyronine (T(3)) markedly induced the phosphorylation of p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly enhanced the T(3)-induced alkaline phosphatase activity in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by T(3) was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase takes part in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblasts and that p44/p42 MAP kinase plays an inhibitory role in the thyroid hormone-effect.     

Distinct region of the granulocyte colony-stimulating factor receptor mediates proliferative signaling through activation of Janus kinase 2 and p44/42 mitogen-activated protein kinase.

The granulocyte colony-stimulating factor receptor (G-CSFR) regulates the proliferation, differentiation and survival of neutrophilic progenitor cells. In these studies, we introduced mutant G-CSFRs with cytoplasmic domains truncated approximately every 30 amino acids from the C-terminus into interleukin-3 (IL-3)-dependent myeloid LGM-1 cells. The G-CSFR membrane proximal region containing the Box 2 homology sequence was determined to be critical for proliferative signaling, as well as for activation of Janus kinase (JAK2) and p44/42 mitogen-activated protein kinase (MAPK) following G-CSF stimulation. In the presence of increasing concentrations of JAK2 or p44/42 MAPK inhibitors, LGM-1 cells expressing the full-length G-CSFR exhibited a decreased capacity to proliferate in response to G-CSF. These results demonstrate that JAK2 and p44/42 MAPK activation is involved in proliferative signaling through the G-CSFR membrane proximal region containing the Box 2 homology sequence.     

Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.     

mTORC1 signaling can regulate growth factor activation of p44/42 mitogen-activated protein kinases through protein phosphatase 2A

The mTORC1 complex (mammalian target of rapamycin (mTOR)-raptor) is modulated by mitogen-activated protein (p44/42 MAP) kinases (p44/42) through phosphorylation and inactivation of the tuberous sclerosis complex. However, a role for mTORC1 signaling in modulating activation of p44/42 has not been reported. We show that in two cancer cell lines regulation of the p44/42 MAPKs is mTORC1-dependent. In Rh1 cells rapamycin inhibited insulin-like growth factor-I (IGF-I)-stimulated phosphorylation of Thr(202) but not Tyr(204) and suppressed activation of p44/42 kinase activity. Down-regulation of raptor, which inhibits mTORC1 signaling, had a similar effect to rapamycin in blocking IGF-I-stimulated Tyr(204) phosphorylation. Rapamycin did not block maximal phosphorylation of Tyr(204) but retarded the rate of dephosphorylation of Tyr(204) following IGF-I stimulation. IGF-I stimulation of MEK1 phosphorylation (Ser(217/221)) was not inhibited by rapamycin. Higher concentrations of rapamycin (> or =100 ng/ml) were required to inhibit epidermal growth factor (EGF)-induced phosphorylation of p44/42 (Thr(202)). Rapamycin-induced inhibition of p44/42 (Thr(202)) phosphorylation by IGF-I was reversed by low concentrations of okadaic acid, suggesting involvement of protein phosphatase 2A (PP2A). Both IGF-I and EGF caused dissociation of PP2A catalytic subunit (PP2Ac) from p42. Whereas low concentrations of rapamycin (1 ng/ml) inhibited dissociation of PP2Ac after IGF-I stimulation, it required higher concentrations (> or =100 ng/ml) to block EGF-induced dissociation, consistent with the ability for rapamycin to attenuate growth factor-induced activation of p44/42. The effect of rapamycin on IGF-I or insulin activation of p44/42 was recapitulated by amino acid deprivation. Rapamycin effects altering the kinetics of p44/42 phosphorylation were completely abrogated in Rh1mTORrr cells that express a rapamycin-resistant mTOR, whereas the effects of amino acid deprivation were similar in Rh1 and Rh1mTORrr cells. These results indicate complex regulation of p44/42 by phosphatases downstream of mTORC1. This suggests a model in which mTORC1 modulates the phosphorylation of Thr(202) on p44/42 MAPKs through direct or indirect regulation of PP2Ac.     

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Delayed and sustained activation of p42/p44 mitogen-activated protein kinase induced by proteasome inhibitors through p21(ras) in PC12 cells

Proteolysis by the ubiquitin/proteasome pathway regulates the intracellular level of several proteins, some of which control cell proliferation and cell cycle progression. To determine what kinds of signaling cascades are activated or inhibited by proteasome inhibition, we treated PC12 cells with specific proteasome inhibitors and subsequently performed in-gel kinase assays. N-Acetyl-Leu-Leu-norleucinal and lactacystin, which inhibit the activity of the proteasome, induced the activation of p42/p44 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases (ERKs) 1 and 2]. In contrast, N-acetyl-Leu-Leu-methional, which inhibits the activity of calpains, but not of the proteasome, failed to induce ERK activation. Uniquely, the kinetics of MAP kinase activation induced by proteasome inhibitors are very slow compared with those resulting from activation by nerve growth factor; ERK activation is detectable only after a 5-h treatment with the inhibitors, and its activity remained unchanged for at least until 27 h. Proteasome inhibitor-initiated ERK activation is inhibited by pretreatment with the ERK kinase inhibitor PD 98059, as well as by overexpression of a dominant-negative form of Ras. Thus, proteasome inhibitors induce sustained ERK activation in a Ras-dependent manner. Proteasome inhibitor-induced neurite outgrowth, however, is not inhibited by PD 98059, indicating that sustained activation of ERKs is not the factor responsible for proteasome inhibitor-induced morphological differentiation. Our data suggest the presence of a novel mechanism for activation of the MAP kinase cascade that involves proteasome activity.     

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFα in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFα, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFα, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFα release and caspase expression.     

Factor VIIa-induced p44/42 mitogen-activated protein kinase activation requires the proteolytic activity of factor VIIa and is independent of the tissue factor cytoplasmic domain.

Signal transduction induced by activated factor VII (FVIIa) was studied with baby hamster kidney (BHK) cells transfected with human tissue factor (TF). FVIIa induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in cells expressing TF, BHK(+TF), but not in wild-type BHK(-TF) cells. BHK(+TF) cells responded to FVIIa in a dose-dependent manner, with detectable phosphorylation above 10-20 nM FVIIa. BHK cells transfected with a cytoplasmic domain-deleted version of TF, (des248-263)TF, or a C245S substitution variant of TF also supported FVIIa-induced MAPK activation. Experiments with active site-inhibited FVIIa, thrombin, factor Xa, and hirudin confirmed that the catalytic activity of FVIIa was mandatory for p44/42 MAPK activation. Furthermore, a high concentration of FVIIa in complex with soluble TF induced p44/42 MAPK phosphorylation in BHK(-TF) cells. These data suggest that TF was not directly involved in FVIIa-induced p44/42 MAPK phosphorylation but rather served to localize the action of FVIIa to the cell surface, potentially to cleave a cell surface receptor. Desensitization experiments with sequential addition of proteases suggested that the p44/42 MAPK response induced by FVIIa was distinctly different from the thrombin response, possibly involving a novel member of the protease-activated receptor family.     

Confluence of vascular endothelial cells induces cell cycle exit by inhibiting p42/p44 mitogen-activated protein kinase activity

Like other cellular models, endothelial cells in cultures stop growing when they reach confluence, even in the presence of growth factors. In this work, we have studied the effect of cellular contact on the activation of p42/p44 mitogen-activated protein kinase (MAPK) by growth factors in mouse vascular endothelial cells. p42/p44 MAPK activation by fetal calf serum or fibroblast growth factor was restrained in confluent cells in comparison with the activity found in sparse cells. Consequently, the induction of c-fos, MAPK phosphatases 1 and 2 (MKP1/2), and cyclin D1 was also restrained in confluent cells. In contrast, the activation of Ras and MEK-1, two upstream activators of the p42/p44 MAPK cascade, was not impaired when cells attained confluence. Sodium orthovanadate, but not okadaic acid, restored p42/p44 MAPK activity in confluent cells. Moreover, lysates from confluent 1G11 cells more effectively inactivated a dually phosphorylated active p42 MAPK than lysates from sparse cells. These results, together with the fact that vanadate-sensitive phosphatase activity was higher in confluent cells, suggest that phosphatases play a role in the down-regulation of p42/p44 MAPK activity. Enforced long-term activation of p42/p44 MAPK by expression of the chimera DeltaRaf-1:ER, which activates the p42/p44 MAPK cascade at the level of Raf, enhanced the expression of MKP1/2 and cyclin D1 and, more importantly, restored the reentry of confluent cells into the cell cycle. Therefore, inhibition of p42/p44 MAPK activation by cell-cell contact is a critical step initiating cell cycle exit in vascular endothelial cells.     

p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor.

Mitogen-activated protein (MAP) kinase is a 42-kDa serine/threonine-specific protein kinase that requires phosphorylation on both tyrosine and threonine residues for activity. This enzyme is rapidly and transiently activated in quiescent cells after addition of various agonists, including insulin, epidermal growth factor, platelet-derived growth factor, and phorbol esters. We show here that addition of the growth factors thrombin or basic fibroblast growth factor to CCL39 fibroblasts rapidly induces tyrosine phosphorylation of the p42 MAP kinase protein and concomitantly stimulates MAP kinase enzymatic activity. To elucidate the signaling pathways utilized in this activation, we took advantage of the sensitivity of CCL39 cells to the toxin of bordetella pertussis, which ADP-ribosylates two Gi proteins in this cell system. We show that pretreatment of cells with the toxin inhibited thrombin stimulation of MAP kinase by greater than 75% but had no detectable effect on the stimulation induced by basic fibroblast growth factor. We also demonstrate that these two growth factors that synergize for mitogenicity are able to cooperate in activation of MAP kinase and that this synergism is partially sensitive to pertussis toxin. Finally, we describe a 44-kDa protein, the tyrosine phosphorylation of which appears to be coregulated with p42 MAP kinase. We conclude that p42 MAP kinase (and the pp44 protein) are at or are downstream from a point of convergence of two different receptor-induced signaling pathways and might well play a key role in integrating those signals.     

Protein kinase C zeta plays a central role in activation of the p42/44 mitogen-activated protein kinase by endotoxin in alveolar macrophages

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC zeta, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC zeta, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC zeta partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC zeta to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC zeta. PKC zeta has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC zeta/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC zeta activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC zeta.     

The inhibitory gamma subunit of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase is a novel intermediate regulating p42/p44 mitogen-activated protein kinase signaling in human embryonic kidney 293 cells

The inhibitory gamma subunits of the retinal rod and cone photoreceptor type 6 retinal cyclic guanosine monophosphate phosphodiesterase (PDEgamma) are expressed in non-retinal tissues. Here, we show that PDEgamma interacts with the G-protein-coupled receptor kinase 2 signaling system to regulate the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. This is based upon several lines of evidence. First, the transfection of cells with an antisense rod PDEgamma plasmid construct, which reduced endogenous rod PDEgamma expression, ablated the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase. Second, the transfection of cells with recombinant rod or cone PDEgamma and/or G-protein-coupled receptor kinase 2 increased the stimulation of p42/p44 mitogen-activated protein kinase by epidermal growth factor or thrombin. In contrast, a G-protein-coupled receptor kinase 2 phosphorylation-resistant rod PDEgamma mutant failed to increase the epidermal growth factor- or thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase and, in fact, functioned as a dominant negative. Thrombin also stimulated the association of endogenous rod PDEgamma with dynamin II, which was increased in cells transfected with rod PDEgamma or G-protein-coupled receptor kinase 2. Dynamin II plays a critical role in regulating endocytosis of receptor signal complexes required for activation of p42/p44 mitogen-activated protein kinase. Therefore, PDEgamma may have an important role in promoting endocytosis of receptor signal complexes leading to the activation of p42/p44 mitogen-activated protein kinase. We conclude that PDEgamma is an entirely novel intermediate regulating mitogenic signaling from both receptor tyrosine kinase and G-protein-coupled receptors in human embryonic kidney 293 cells.