Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism

The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.     

A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways.

A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway.     

Inhibition of phosphatidylinositol-3-kinase enhances insulin stimulation of insulin receptor substrate 1 tyrosine phosphorylation and extracellular signal-regulated kinases in mouse R- fibroblasts

Insulin stimulates phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases (ERK) in various mammalian cells. To study the role of PI3K in insulin stimulation of ERK, we employed PI3K inhibitor LY294002 and mouse embryonic R- fibroblasts lacking IGF-1 receptors. In these R- cells, PI3K inhibition by LY294002 enhanced insulin stimulation of ERK phosphorylation whereas LY294002 inhibited insulin stimulation of Akt phosphorylation. The enhanced insulin stimulation of ERK phosphorylation was accompanied by increased IRS-1 tyrosine phosphorylation. Insulin stimulation of insulin receptor tyrosine phosphorylation was not altered. PI3K inhibition increased IRS-1-Grb2 complex formation and ras activity following insulin treatment of cells. Increased insulin stimulation of ERK by PI3K inhibition was mediated by the MEK/ERK pathway, but did not involve inhibitory Ser259 phosphorylation of raf that was reported to be mediated by Akt. In summary, PI3K inhibition in R- cells enhanced insulin stimulation of ERK phosphorylation by mechanisms involving enhancement of IRS-1 tyrosine phosphorylation, IRS-1-Grb2 complex formation and the ras/MEK/ERK pathway.     

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Persistence was established after most of the SARS-CoV-infected Vero E6 cells died. RNA of the defective interfering virus was not observed in the persistently infected cells by Northern blot analysis. SARS-CoV diluted to 2 PFU failed to establish persistence, suggesting that some particular viruses in the seed virus did not induce persistent infection. Interestingly, a viral receptor, angiotensin converting enzyme (ACE)-2, was down-regulated in persistently infected cells. G418-selected clones established from parent Vero E6 cells, which were transfected with a plasmid containing the neomycin resistance gene, were infected with SARS-CoV, resulting in a potential cell population capable of persistence in Vero E6 cells. Our previous studies demonstrated that signaling pathways of extracellular signal-related kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3'-kinase (PI3K)/Akt were activated in SARS-CoV-infected Vero E6 cells. Previous studies also showed that the activation of p38 MAPK by viral infection-induced apoptosis, and a weak activation of Akt was not sufficient to protect from apoptosis. In the present study, we showed that the inhibitors of JNK and PI3K/Akt inhibited the establishment of persistence, but those of MAPK/ERK kinase (MEK; as an inhibitor for ERK1/2) and p38 MAPK did not. These results indicated that two signaling pathways of JNK and PI3K/Akt were important for the establishment of persistence in Vero E6 cells.     

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.     

Insulin targets the Na(+)/K(+) ATPase in enterocytes via PI3K, PKC, and MAPKS

The effect of insulin on intestinal Na(+)/K(+) ATPase is till now undetermined, and it is still unclear whether insulin exerts any modulatory effect on glucose absorption by targeting the ATPase. This work attempted to address this question and to unravel the signaling pathway involved using Caco-2 cells as a model. After an overnight starvation, cells were treated with insulin in presence and absence of specific inhibitors of some known mediators. The activity of the pump was assayed by measuring the ouabain-inhibitable inorganic phosphate (P(i)) released, whereas changes in its abundance were determined by western blot analysis. Insulin decreased the activity and abundance of the ATPase in a crude membrane homogenate. This effect disappeared completely upon inhibition of either phosphotidylinositol-3 kinase (PI3K) or protein kinase C (PKC), but was partially abolished when p38MAPK or MEK/ERK were inhibited separately. Activation of PKC with phorbol-12-myristate-13-acetate (PMA) imitated the effect of insulin and was not affected by inhibition of PI3K. The data suggest that PI3K and PKC are along the same pathway that branches into two separate ones involving each either p38MAP kinase or MEK/ERK. This hypothesis was confirmed by the data obtained from the treatment of Caco-2 cells with PMA, when p38MAPK and MEK/ERK were inhibited simultaneously. Concomitant inhibition of p38MAPK and MEK/ERK abrogated fully the effect of insulin, indicating that no other pathways are present in addition to the ones proposed above.     

Inhibition of Phosphatidylinositol-3-kinase Enhances Insulin Stimulation of Insulin Receptor Substrate 1 Tyrosine Phosphorylation and Extracellular Signal-Regulated Kinases in Mouse R− Fibroblasts

Insulin stimulates phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases (ERK) in various mammalian cells. To study the role of PI3K in insulin stimulation of ERK, we employed PI3K inhibitor LY294002 and mouse embryonic R^? fibroblasts lacking IGF-1 receptors. In these R^? cells, PI3K inhibition by LY294002 enhanced insulin stimulation of ERK phosphorylation whereas LY294002 inhibited insulin stimulation of Akt phosphorylation. The enhanced insulin stimulation of ERK phosphorylation was accompanied by increased IRS-1 tyrosine phosphorylation. Insulin stimulation of insulin receptor tyrosine phosphorylation was not altered. PI3K inhibition increased IRS-1–Grb2 complex formation and ras activity following insulin treatment of cells. Increased insulin stimulation of ERK by PI3K inhibition was mediated by the MEK/ERK pathway, but did not involve inhibitory Ser²⁵⁹ phosphorylation of raf that was reported to be mediated by Akt. In summary, PI3K inhibition in R^? cells enhanced insulin stimulation of ERK phosphorylation by mechanisms involving enhancement of IRS-1 tyrosine phosphorylation, IRS-1–Grb2 complex formation and the ras/MEK/ERK pathway.     

MEK kinase 3 directly activates MKK6 and MKK7, specific activators of the p38 and c-Jun NH2-terminal kinases.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) activates the c-Jun NH2-terminal kinase (JNK) pathway, although no substrates for MEKK3 have been identified. We have examined the regulation by MEKK3 of MAPK kinase 7 (MKK7) and MKK6, two novel MAPK kinases specific for JNK and p38, respectively. Coexpression of MKK7 with MEKK3 in COS-7 cells enhanced MKK7 autophosphorylation and its ability to activate recombinant JNK1 in vitro. MKK6 autophosphorylation and in vitro activation of p38alpha were also observed following coexpression of MKK6 with MEKK3. MEKK2, a closely related homologue of MEKK3, also activated MKK7 and MKK6 in COS-7 cells. Importantly, immunoprecipitates of either MEKK3 or MEKK2 directly activated recombinant MKK7 and MKK6 in vitro. These data identify MEKK3 as a MAPK kinase kinase specific for MKK7 and MKK6 in the JNK and p38 pathways. We have also examined whether MEKK3 or MEKK2 activates p38 in intact cells using MAPK-activated protein kinase-2 (MAPKAPK2) as an affinity ligand and substrate. Anisomycin, sorbitol, or the expression of MEKK3 in HEK293 cells enhanced MAPKAPK2 phosphorylation, whereas MEKK2 was less effective. Furthermore, MAPKAPK2 phosphorylation induced by MEKK3 or cellular stress was abolished by the p38 inhibitor SB-203580, suggesting that MEKK3 is coupled to p38 activation in intact cells.     

Inhibition of mitochondrial respiratory chain in the brain of rats after hepatic failure induced by acetaminophen

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.     

The p38 pathway provides negative feedback for Ras proliferative signaling

Ras activates three mitogen-activated protein kinases (MAPKs) including ERK, JNK, and p38. Whereas the essential roles of ERK and JNK in Ras signaling has been established, the contribution of p38 remains unclear. Here we demonstrate that the p38 pathway functions as a negative regulator of Ras proliferative signaling via a feedback mechanism. Oncogenic Ras activated p38 and two p38-activated protein kinases, MAPK-activated protein kinase 2 (MK2) and p38-related/activated protein kinase (PRAK). MK2 and PRAK in turn suppressed Ras-induced gene expression and cell proliferation, whereas two mutant PRAKs, unresponsive to Ras, had little effect. Moreover, the constitutive p38 activator MKK6 also suppressed Ras activity in a p38-dependent manner whereas arsenite, a potent chemical inducer of p38, inhibited proliferation only in a tumor cell line that required Ras activity. MEK was required for Ras stimulation of the p38 pathway. The p38 pathway inhibited Ras activity by blocking activation of JNK, without effect upon ERK, as evidenced by the fact that PRAK-mediated suppression of Ras-induced cell proliferation was reversed by coexpression of JNKK2 or JNK1. These studies thus establish a negative feedback mechanism by which Ras proliferative activity is regulated via signaling integrations of MAPK pathways.     

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.     

Hyperosmotic stress activates the insulin receptor in CHO cells

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.     

Changes in the balance of phosphoinositide 3-kinase/protein kinase B (Akt) and the mitogen-activated protein kinases (ERK/p38MAPK) determine a phenotype of visceral and vascular smooth muscle cells.

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860-28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I-triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.     

Enhanced insulin action on glucose transport and insulin signaling in 7-day unweighted rat soleus muscle.

Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly (P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser(473) phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89-92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.     

MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.