Insulin stimulates nitric oxide production in rat adipocytes

In adipocytes, insulin regulates the activity of different protein kinases (PI3K/Akt, MAPK, PKC) and protein phosphatases (PP-1, PP-2A). Since these enzymes are implicated in the regulation of NOS activity which is present in adipose tissue, we tested the effects of insulin on white adipocyte NOS activity. Exposure of adipocytes to insulin resulted simultaneously in NOS activity stimulation and Akt activation with maximal effect observed at 1 nM. Higher concentrations of insulin induced a progressive decline of NOS activity. In the presence of wortmannin, a PI3K inhibitor, 1 nM insulin failed to stimulate NOS activity. Insulin (1 nM)-stimulated NOS activity was also abolished by U0126, an inhibitor of p42/p44 MAPK activation, and by 1 microM okadaic acid (OA), which inhibits both PP-1 and PP-2A but not by 1 nM OA which inhibits only PP-2A. Moreover, inhibition of cPKC allowed a high (1 microM) insulin concentration to stimulate NOS activity. These results (i) demonstrate that insulin activates NO production in adipocytes through both PI3K/Akt and MAPK/PP-1 activation and (ii) suggest that PP-1 activation protects NOS against the inhibitory effect of cPKC activation.     

Negative regulation of the protein kinase C activator-induced ICAM-1 expression in the human bronchial epithelial cell line NCI-H292 by p44/42 mitogen-activated protein kinase

The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one.     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Proadrenomedullin N-terminal 20 peptide (PAMP) enhances proliferation of rat zona glomerulosa cells by activating MAPK cascade.

The effect of proadrenomedullin N-terminal 20 peptide (PAMP) on the proliferative activity of rat zona glomerulosa (ZG) cells has been investigated. Dispersed rat ZG cells were cultured in vitro for 24 h and then exposed to PAMP for an additional 24 h, and the proliferation rate was assessed by the 5-bromo-2'-deoxyuridine (BrdU) incorporation technique. PAMP dose-dependently increased the percentage of BrdU-positive cells, with a maximal effective concentration observed at 10(-8) M. The tyrosine kinase (TK) inhibitor, tyrphostin-23, and the p42/p44 MAPK inhibitor, PD-98059, abolished the proliferogenic effect of PAMP, while the protein kinase (PK) A inhibitor, H-89, and the PKC inhibitor, calphostin-C, were ineffective in blocking the response to PAMP. PAMP (10(-8) M) enhanced TK and MAPK activity of dispersed rat ZG cells. The stimulatory action of PAMP on TK activity was annulled by tyrphostin-23, while that on MAPK activity was abolished by either tyrphostin-23 or PD-98059. Taken together, these data indicate that PAMP enhances proliferation of cultured rat ZG cells, through the TK-dependent activation of p42/p44 MAPK cascade.     

PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK

Brain natriuretic peptide (BNP) produced by cardiac myocytes has antifibrotic and antigrowth properties and is a marker of cardiac hypertrophy. We previously showed that prostaglandin E2 (PGE2) is the main prostaglandin produced in myocytes treated with proinflammatory stimuli and stimulates protein synthesis by binding to its EP4 receptor. We hypothesized that PGE2, acting through EP4, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE2 increased hBNP promoter activity 3.5-fold. An EP4 antagonist reduced the stimulatory effect of PGE2 but not an EP1 antagonist. Because EP4 signaling can involve adenylate cyclase, cAMP, and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE2 stimulation of the hBNP promoter. H-89 at 5 muM decreased PGE2 stimulation of BNP promoter activity by 100%. Because p42/44 MAPK mediates the effect of PGE2 on protein synthesis, we also examined the role of MAPKs in the regulation of BNP promoter activity. PGE2 stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effect of PGE2. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE2 stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP4, PKA, Rap, and p42/44 MAPK.     

Sustained phosphorylation of mitogen-activated protein kinase is required for basic fibroblast growth factor-mediated axonal branch formation in cultured rat hippocampal neurons

Basic fibroblast growth factor (bFGF) has been reported to promote the formation of axonal branches in cultured brain neurons. In the present study, we investigated whether the mitogen-activated protein kinase (MAPK) cascade was involved in this action of bFGF in cultured rat hippocampal neurons. Recombinant human bFGF (0.1-10 ng/ml) induced phosphorylation of p44/42 MAPK in a concentration and time-dependent manner. The phosphorylation of p44/42 MAPK occurred rapidly within 5 min after addition of bFGF, and lasted for 48 h. The bFGF-induced phosphorylation of p44/42 MAPK and axonal branch formation were both blocked by simultaneous addition of U0126 and PD98059, specific inhibitors of MAPK kinases. Furthermore, when U0126 and PD98059 were added 24 h after bFGF, phosphorylation of p44/42 m MAPK was decreased, and axonal branch formation was stopped. These results suggest that sustained activation of the MAPK cascade is required for bFGF-mediated axonal branch formation.     

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.     

Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model

Mitogen-activated protein kinase (MAPK) signaling cascade plays a pivotal role in the activation of inflammatory cells. Recent findings revealed that the activity of p42/44 MAPK (also known as extracellular signal-regulated kinase (ERK)) in the lungs was significantly higher in asthmatic mice than in normal controls. We hypothesized that inhibition of ERK activity may have anti-inflammatory effects in allergic asthma. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of VCAM-1 expression, and airway hyperresponsiveness. Intraperitoneal administration of U0126, a specific MAPK/ERK kinase inhibitor, significantly (p < 0.05) inhibited OVA-induced increases in total cell counts, eosinophil counts, and IL-4, IL-5, IL-13, and eotaxin levels recovered in bronchoalveolar lavage fluid in a dose-dependent manner. U0126 also substantially (p < 0.05) reduced the serum levels of total IgE and OVA-specific IgE and IgG1. Histological studies show that U0126 dramatically inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and expression of VCAM-1 in lung tissues. In addition, U0126 significantly (p < 0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine in a dose-dependent manner. Western blot analysis of whole lung lysates shows that U0126 markedly attenuated OVA-induced tyrosine phosphorylation of ERK1/2. Taken together, our findings implicate that inhibition of ERK signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.     

Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.     

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

OxLDL induces mitogen-activated protein kinase activation mediated via PI3-kinase/Akt in vascular smooth muscle cells

Oxidized low-density lipoprotein (OxLDL) is a risk factor in atherosclerosis and stimulates multiple signaling pathways, including activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK), which are involved in mitogenesis of vascular smooth muscle cells (VSMCs). We therefore investigated the relationship between PI3-K/Akt and p42/p44 MAPK activation and cell proliferation induced by OxLDL. OxLDL stimulated Akt phosphorylation in a time- and concentration-dependent manner, as determined by Western blot analysis. Phosphorylation of Akt stimulated by OxLDL and epidermal growth factor (EGF) was attenuated by inhibitors of PI3-K (wortmannin and LY294002) and intracellular Ca2+ chelator (BAPTA/AM) plus EDTA. Pretreatment of VSMCs with pertussis toxin, cholera toxin, and forskolin for 24 h also attenuated the OxLDL-stimulated Akt phosphorylation. In addition, pretreatment of VSMCs with wortmannin or LY294002 inhibited OxLDL-stimulated p42/p44 MAPK phosphorylation and [3H]thymidine incorporation. Furthermore, treatment with U0126, an inhibitor of MAPK kinase (MEK)1/2, attenuated the p42/p44 MAPK phosphorylation, but had no effect on Akt activation in response to OxLDL and EGF. Overexpression of p85-DN or Akt-DN mutants attenuated MEK1/2 and p42/p44 MAPK phosphorylation stimulated by OxLDL and EGF. These results suggest that the mitogenic effect of OxLDL is, at least in part, mediated through activation of PI3-K/Akt/MEK/MAPK pathway in VSMCs.     

Leptin and insulin induce mutual resistance for nitric oxide synthase III activation in adipocytes

Obesity‐induced hyperleptinemia is frequently associated with insulin resistance suggesting a crosstalk between leptin and insulin signaling pathways. Our aim was to determine whether insulin and leptin together interfere on NOS activation in adipocytes. We examined insulin and leptin‐induced nitric oxide synthase (NOS) activity, protein amount and NOS III phosphorylation at Ser¹¹⁷⁹ in isolated epididymal adipocytes from rat, in the presence or not of inhibitors of kinases implicated in insulin or leptin signaling pathways. Insulin or leptin induced NOS III phosphorylation at Ser¹¹⁷⁹ leading to increased NO production in rat adipocytes, in agreement with our previous observations. When insulin and leptin at a concentration found in obese rats (10 ng/ml) were combined, NOS activity was not increased, suggesting a negative crosstalk between insulin and leptin signaling mechanisms. Chemical inhibitors of kinases implicated in signaling pathways of insulin, such as PI‐3 kinase, or of leptin, such as JAK‐2, did not prevent this negative interaction. When leptin signaling was blocked by PKA inhibitors, insulin‐induced NOS activity and NOS III phosphorylation at Ser¹¹⁷⁹ was observed. In the presence of leptin and insulin, (i) IRS‐1 was phosphorylated on Ser³⁰⁷ and this effect was prevented by PKA inhibitors, (ii) JAK‐2 was dephosphorylated, an effect prevented by SHP‐1 inhibitor. A mutual resistance occurs with leptin and insulin. Leptin phosphorylates IRS‐1 to induce insulin resistance while insulin dephosphorylates JAK‐2 to favor leptin resistance. This interference between insulin and leptin signaling could play a crucial role in insulin‐ and leptin‐resistance correlated with obesity. J. Cell. Biochem. 108: 982–988, 2009. © 2009 Wiley‐Liss, Inc.     

Evidence that inhibition of p44/42 mitogen-activated protein kinase signaling is a factor in proteasome inhibitor-mediated apoptosis

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-myc human mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.     

Cyclic adenosine 3',5'monophosphate/protein kinase A and mitogen-activated protein kinase 3/1 pathways are involved in adenylate cyclase-activating polypeptide 1-induced common alpha-glycoprotein subunit gene (Cga) expression in mouse pituitary gonadotroph LbetaT2 cells

Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.