Inhibition Of MAP Kinase Activity Research Articles

Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription

The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor Nɷ-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50μM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10μM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5μM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10μM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO.

Steroid-induced oocyte maturation in Indian shad Tenualosa ilisha (Hamilton, 1822) is dependent on phosphatidylinositol 3 kinase but not MAP kinase activation

Fully grown fish and amphibian oocytes exposed to a maturation-inducing steroid (MIS) activates multiple signal transduction pathways, leading to formation and activation of maturation-promoting factor (MPF) and induction of germinal vesicle breakdown (GVBD). The present study was to investigate if phosphatidylinositol 3 kinase (PI3 kinase) and mitogen-activated protein kinase (MAP kinase) activation are required for naturally occurring MIS, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced cdc2 activation and oocyte maturation (OM) in Tenualosa ilisha. We observed that17,20β-P-induced OM was significantly inhibited by PI3 kinase inhibitors Wortmannin and LY29400. 17,20β-P was shown to activate PI3 kinase maximally at 90min and cdc2 kinase at 16h of treatment. Relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in 17,20β-P-induced OM was examined. MAP kinase was rapidly phosphorylated and activated (60–120min) after MIS treatment and this response preceded the activation of cdc2 kinase by several hours. A selective inhibitor of MAP kinase (MEK), PD98059, sufficiently blocked the phosphorylation and activation of MAP kinase. Inhibition of MAP kinase activity using PD98059 however, had no effect on MIS-induced cdc2 kinase activation and GVBD. These results demonstrate that activation of the PI3 kinase is required for 17,20β-P-induced cdc2 kinase activation and OM in T. ilisha. MAP kinase although was activated in response to 17,20β-P and PI3 kinase activation, it is not necessary for cdc2 activation and OM in this species.

PKC‐dependent inhibition of MAP kinase activity and caldesmon phosphorylation in rabbit bladder smooth muscle (865.14)

Smooth muscle contraction is primarily regulated by myosin light chain (MLC) phosphorylation although thin filament proteins, specifically caldesmon, may also be involved. Our previous studies have shown that in vascular smooth muscle, p42 mitogen activated protein kinase (MAPK) but not p44 MAPK activity was enhanced in the presence of an inhibitor of PKC, bisindolylmaleimide‐1 (Bis). The goal of this study was to determine if this is a vascular‐specific pathway or a pathway common to all smooth muscles. Experiments were performed using carbachol‐stimulated rabbit bladder smooth muscle in the presence and absence of Bis; p42 and p44 MAPK activity and caldesmon phosphorylation were measured at different time points during the contraction. Our results showed that carbachol‐dependent p42 MAPK activity is enhanced in the presence of Bis with a concomitant increase in caldesmon phosphorylation in bladder smooth muscle. Carbachol dependent force generation increased concurrently with p42 MAPK phosphorylation which was reduced by inhibition of PKC during the entire contraction; a result not found in vascular smooth muscle. Together with our previous results, this novel signaling pathway appears to be general to both vascular and non‐vascular smooth muscles, however the effects on force are different. Future studies will be aimed at determining the mechanism by which inhibition of PKC increases p42 MAPK activity.Grant Funding Source: NIH DK 85734

Novel findings in membrane depolarization‐induced force development in vascular smooth muscle (865.11)

A unique phenomenon of vascular smooth muscle (VSM) is its ability to maintain force for a prolonged period of time. The exact mechanism responsible is not known but studies suggest a critical role for L‐type Ca2+ channels (LTCC). The purpose of this study was to investigate the consequences of prolonged LTCC opening with an agonist, Bay K8644, to elucidate the intracellular mechanisms responsible for maintaining VSM tone. Smooth muscle tissue isolated from swine carotid arteries was mounted for isometric force recording. Tissues were stimulated with Bay K8644 alone or following pretreatment with one of the following inhibitors: nifedipine (LTCC), ML‐7 (MLC kinase), bisindolylmaleimide (PKC), U0126 (MAP kinase), or H‐1152 (Rho kinase). The following results were obtained: • Bay K8644 increased MLC phosphorylation, PKC activity, MAP kinase activity, and force development but partially inhibited ROCK activity; CPI‐17 phosphorylation was not changed.• Bay K8644‐dependent activation of PKC inhibited MAP kinase activity; the Bay K8644 partial inhibition of ROCK activity was reversed by inhibition of LTCC with nifedipine .• Inhibition of MAP kinase activity significantly increased MLC kinase activity and inhibition of MLC kinase activity greatly increased MAP kinase activity.These results indicate that the maintenance of force in VSM involves an intricate signaling network with multiple modulatory steps.

The p21-activated kinase PAK 5 is involved in formalin-induced nociception through regulation of MAP-kinase signaling and formalin-specific receptors

p21-activated kinases (PAKs) are involved in signal cascades relevant for nociceptive processing and neuropathic pain. Particularly, the recently described group B PAKs 4, 5 and 6 regulate MAP-kinases and the rearrangement of the actin cytoskeleton, both of which have been linked to pain processing. However, a specific role of these PAKs in nociception has not yet been demonstrated. We found PAK 4, 5 and 6 expression in pain-relevant tissues in peripheral and CNS. Since viable knock-out mice only exist for the PAK isoform 5, we further assessed the impact of this PAK on acute and chronic pain using different behavioral models in mice. PAK 5 knock-out mice showed normal acute nociception and did not differ from wild type mice in their neuropathic pain behavior. However, the nociceptive response in formalin-induced paw inflammation was significantly reduced in knock-out mice associated with inhibition of MAP-kinase activation and a decreased number of formalin-induced c-Fos positive neurons in the spinal cord. Furthermore, in isolated neurons, we found a significantly reduced calcium response after stimulation of TRPA1-channels in PAK 5−/−- compared to PAK 5+/+-cells.Our results indicate that PAK 5 is involved in formalin-induced inflammatory nociception through regulation of MAPK-induced c-Fos-activation and formalin-specific TRP-channels.

Abstract A66: Phase IIa placebo controlled trial of sulindac in individuals at increased risk for melanoma: Preliminary findings

Abstract Some epidemiological studies and several experimental publications suggest that regular use of NSAIDs may reduce the risk of melanoma. Proposed mechanisms of action include: inhibition of MAP kinase activity and nuclear translocation, reduction in the concentration of prostaglandins associated with the inhibition of VEGF and bFGF production and HIF-1α stabilization, reduction of the activation state of the VEGF and Ftl-1 genes, and inhibition of PI-3 kinase/Akt signaling pathway. However, specific information on the ability of NSAIDs to reach melanocytes in vivo and to modulate key biomarkers in pre-neoplastic lesions, such as dysplastic nevi, have not been evaluated to date. Methods: A phase IIa placebo controlled clinical intervention trial was designed with the primary end point to determine whether oral sulindac and related metabolites were present in benign nevi (BN) after an 8 week course of oral sulindac; secondary end points were included to determine whether a short exposure to sulindac had an effect on apoptosis and VEGF expression in atypical nevi (AN), as well as measurement of sulindac and metabolite levels in plasma. Exploratory objectives will evaluate the feasibility of reverse phase protein microarray to identify intervention related changes in cell signaling proteins, focusing initially on the pAKT/mTOR pathway and COX expression. Fifty healthy subjects with ≥ 4 large (≥5 mm and < 15 mm) AN and one BN were enrolled between 2/09 and 7/10. Subjects underwent clinical and dermoscopic evaluation of their melanocytic lesions with 2 AN randomized to be excised at baseline for biomarker evaluation and 2 AN to post-intervention evaluation. Subsequently, participants were randomized to receive sulindac (150 mg BID) or placebo for 8 weeks. Upon completion of the intervention the residual 2 AN and the BN were surgically removed. Results: To date, accrual has been completed and study intervention in all subjects is expected to be concluded in 09/10. The study intervention was well tolerated with some mild or moderate gastrointestinal adverse events that are possibly or probably related to the intervention. Sulindac, sulindac sulfide, sulindac sulfone concentrations were 0 — 1.4, 0 −9.07, and 0 — 6.1 µg/ml, respectively, in 41 post-intervention plasma samples analyzed to date. Sulindac, sulindac sulfide, sulindac sulfone concentrations were 0 — 5.2, 0 — 0.25, and 0 — 14.27 µg/g, respectively, in 40 postintervention BN samples. Conclusion: Current results suggest that NSAIDs, in particular sulindac and the active sulindac metabolites, can reach measurable levels in the skin, and specifically in melanocytic nevi, further supporting the need to assess the effect of this group of drugs as chemopreventive agents for melanoma. Supported by N01CN35158 from the National Cancer Institute, Division of Cancer Prevention. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A66.

Homocysteine stimulates antioxidant response element-mediated expression of glutamate-cysteine ligase in mouse macrophages

Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Uptake of homocysteine induces oxidative stress in macrophages. Antioxidant response elements (AREs) are regulatory elements within promoters of genes, which protect cells against oxidative stress. The current study investigated whether homocysteine induces transcription of glutamate-cysteine ligase (Gcl), via ARE driven gene expression in mouse macrophages. Gcl is the rate-limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Gcl is heterodimeric and the genes encoding the subunits of Gcl contain several AREs within their 5′-promoter regions. Treatment of mouse macrophages with d-/l-homocysteine (50μM) induced depletion of intracellular glutathione and a compensatory increase in Gcl activity. Electro mobiliy shift assays demonstrated increased binding of nuclear proteins to ARE-containing oligonucleotides. Real-time RT-PCR revealed increased mRNA-expression of the catalytic subunit of Gcl (Gclc) after treatment with homocysteine, and this occurred via increased transcription as demonstrated with luciferase promoter reporter constructs for Gclc. Additional site directed mutagenesis demonstrated that ARE4 plays a direct role in mediating induction of Gclc by homocysteine. Supershift analysis and Western blotting revealed that Nrf2 signalling is critical in homocysteine-induced activation of ARE4. Inhibition of MAP kinase activity reduced binding of nuclear proteins to the AREs, nuclear expression of Nrf2 and mRNA expression of Gclc. Western blotting demonstrated phosporylation of ERK1/2 in homocysteine treated macrophages. These data suggest that ARE-driven gene expression of Gclc via a MEK/Nrf2 pathway could help to protect macrophages from oxidative stress due to hyperhomocysteinemia.

TCR-induced downregulation of protein tyrosine phosphatase PEST augments secondary T cell responses

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4 + and CD8 + T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4 + and CD8 + primary human T cells upon T cell activation. This was also true in mouse CD4 + T cells, but less striking in mouse CD8 + T cells. PTP-PEST reintroduced into Jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-κB elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells.

The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology.

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.

Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation.

Hyaluronan Regulates Transforming Growth Factor-β1 Receptor Compartmentalization

Transforming growth factor-beta1 (TGF-beta1) is a key cytokine involved in the pathogenesis of fibrosis in many organs. We previously demonstrated in renal proximal tubular cells that the engagement of the extracellular polysaccharide hyaluronan with its receptor CD44 attenuated TGF-beta1 signaling. In the current study we examined the potential mechanism by which the interaction between hyaluronan (HA) and CD44 regulates TGF-beta receptor function. Affinity labeling of TGF-beta receptors demonstrated that in the unstimulated cells the majority of the receptor partitioned into EEA-1-associated non-lipid raft-associated membrane pools. In the presence of exogenous HA, the majority of the receptors partitioned into caveolin-1 lipid raft-associated pools. TGF-beta1 increased the association of activated/phosphorylated Smad proteins with EEA-1, consistent with activation of TGF-beta1 signaling following endosomal internalization. Following addition of HA, caveolin-1 associated with the inhibitory Smad protein Smad7, consistent with the raft pools mediating receptor turnover, which was facilitated by HA. Antagonism of TGF-beta1-dependent Smad signaling and the effect of HA on TGF-beta receptor associations were inhibited by depletion of membrane cholesterol using nystatin and augmented by inhibition of endocytosis. The effect of HA on TGF-beta receptor trafficking was inhibited by inhibition of HA-CD44 interactions, using blocking antibody to CD44 or inhibition of MAP kinase activation. In conclusion, we have proposed a model by which HA engagement of CD44 leads to MAP kinase-dependent increased trafficking of TGF-beta receptors to lipid raft-associated pools, which facilitates increased receptor turnover and attenuation of TGF-beta1-dependent alteration in proximal tubular cell function.

Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes.

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.

Characterization of a variant of PAC-1 in large granular lymphocyte leukemia

Phosphatase in activated T cells ( PAC-1) is a mitogen-induced early responsive gene. It encodes a 32 kDa tyrosine–threonine dual specificity phosphatase. Constitutive expression of PAC-1 leads to an inhibition of MAP kinase activity in vivo. Such constitutive expression was reported in HTLV-1 infected cell lines. In the present study, we observed the constitutive over-expression of two transcripts related to PAC-1 in large granular lymphocyte (LGL) leukemia. By screening a LGL leukemia cDNA library using the 3 ′ end of a PAC-1 probe, we obtained a clone (clone 8) which retains one and one half introns, excludes two exons, and matches one hundred percent with a DNA sequence on chromosome 2. The deduced amino acid sequence of the predicted protein contains 170 amino acids and is 144 amino acids shorter than PAC-1. When we expressed this protein in Escherichia coli as a GST-fusion protein, a 45 kDa (19 kDa PAC-1 variant + 26 kDa GST protein) protein was obtained. The expressed protein was purified to near homogeneity by using a glutathione affinity column. The purified protein did not have any intrinsic phosphatase activity when assayed in vitro. But when this purified protein was added to a phosphatase assay system in combination with a recombinant dual specificity phosphatase, CL100, enhanced phosphatase activity was observed. The significance of the constitutive over-expression and its physiological role of this protein remain to be established in leukemic LGL.

A(2b) receptors mediate the antimitogenic effects of adenosine in cardiac fibroblasts.

Adenosine inhibits growth of cardiac fibroblasts; however, the adenosine receptor subtype that mediates this antimitogenic effect remains undefined. Therefore, the goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat left ventricular cardiac fibroblasts, PDGF-BB (25 ng/mL) stimulated DNA synthesis ((3)H-thymidine incorporation), cellular proliferation (cell number), collagen synthesis ((3)H-proline incorporation), and MAP kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, but not N(6)-cyclopentyladenosine, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine, or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A(2B) receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5'-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A(2) receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A(2B) receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and collagen synthesis. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5'-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A(2B) receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of CF growth by activating A(2B) receptors coupled to inhibition of MAP kinase activity. Thus, A(2B) receptors may play a critical role in regulating cardiac remodeling associated with CF proliferation. Pharmacologic or molecular biological activation of A(2B) receptors may prevent cardiac remodeling associated with hypertension, myocardial infarction, and myocardial reperfusion injury after ischemia.

Chronic Angiotensin II Augments Integrin-Dependent Cell Functions Via PKC and Map-Kinase Activation in Rat Vsmcs

P78 The activation of local and systemic renin-angiotensin-system is directly and indirectly involved in the mechanisms of vascular remodeling during chronic hypertension. The following study investigated the effect of chronic angiotensin II (AII) admininstration on cell- matrix interaction of VSMCs in vitro. Adhesion to vitronectin (VN) and collagen I (ColI) as well as migration of rat vascular smooth muscle cells (VSMCs) were significantly augmented by chronic treatment with AII for 48 hours and 72 hours. These changes were not due to an increase of the integrins on the cells surface known to be capable in binding vitronectin and collagen I: alpha1, alpha5, beta1 and beta5, as demonstrated by flow cytometry. Also the phosporylation of the focal adhesion kinase (FAK), known to play an important role in adhesion and spreading processes, was not affected after chronic stimulation with Ang II. Downregulation of PKC activity by phorbol-myristate acetate (PMA, 0.1μmol/L), or treatment with the PKC inhibitor calphostin C before treatment with AII significantly reduced the effect of AII on adhesion to VN and ColI (each p<0.05) and also inhibited the effect on VSMC migration (p<0.01). Inhibition of MAP-kinase activation with PD 98059 before treatment with AII demonstrated comparable effects. PD 98059 significantly reduced the increase in adhesion on both, VN and ColI and diminished the effect on PDGF-directed migration of rVSMCs. Theses data indicate that regulation of these functional alterations in integrin-dependent cellular behaviour after chronic AII treatment involved protein kinase C (PKC) and mitogen-activated protein kinase (MAPK)-activation. These data demonstrate that Ang II plays a critical role in augmenting cellular functions in VSMCs, which are important molecular events for the development of vascular hyperplasia .

The roles of integrin-linked kinase in the regulation of myogenic differentiation.

Myogenic differentiation is a highly orchestrated, multistep process that is coordinately regulated by growth factors and cell adhesion. We show here that integrin-linked kinase (ILK), an intracellular integrin- and PINCH-binding serine/threonine protein kinase, is an important regulator of myogenic differentiation. ILK is abundantly expressed in C2C12 myoblasts, both before and after induction of terminal myogenic differentiation. However, a noticeable amount of ILK in the Triton X-100-soluble cellular fractions is significantly reduced during terminal myogenic differentiation, suggesting that ILK is involved in cellular control of myogenic differentiation. To further investigate this, we have overexpressed the wild-type and mutant forms of ILK in C2C12 myoblasts. Overexpression of ILK in the myoblasts inhibited the expression of myogenic proteins (myogenin, MyoD, and myosin heavy chain) and the subsequent formation of multinucleated myotubes. Furthermore, mutations that eliminate either the PINCH-binding or the kinase activity of ILK abolished its ability to inhibit myogenic protein expression and allowed myotube formation. Although overexpression of the ILK mutants is permissive for the initiation of terminal myogenic differentiation, the myotubes derived from myoblasts overexpressing the ILK mutants frequently exhibited an abnormal morphology (giant myotubes containing clustered nuclei), suggesting that ILK functions not only in the initial decision making process, but also in later stages (fusion or maintaining myotube integrity) of myogenic differentiation. Additionally, we show that overexpression of ILK, but not that of the PINCH-binding defective or the kinase-deficient ILK mutants, prevents inactivation of MAP kinase, which is obligatory for the initiation of myogenic differentiation. Finally, inhibition of MAP kinase activation reversed the ILK-induced suppression of myogenic protein expression. Thus, ILK likely influences the initial decision making process of myogenic differentiation by regulation of MAP kinase activation.

Insulin-like growth factor-1 causes a switch-like reduction of endogenous growth hormone mRNA in rat MtT/S somatotroph cells.

Reduction of mRNA expression from the endogenous GH gene by insulin-like growth factor 1 (IGF-1) in somatotroph-like rat MtT/S cells was measured. GH mRNA levels were reduced by 65 nM IGF-1 treatment in a time-dependent manner over 5 d of culture with a calculated GH mRNA half-life of 50 h, in line with previous values from primary cultures. Inhibition of inositol 3-phosphate kinase by wortmannin or LY-294,002 treatment was ineffective in blocking IGF-1 decreases in GH mRNA, as was inhibition of MAP kinase activity by PD 098059. The inhibition by IGF-1 also did not regulate Pit-1 (GHF-1) mRNA levels, which were constant during 65 nM IGF-1 treatment. MtT/S cells were shown to have both IGF-1 and insulin receptors as detected by Western blotting. There was also shown to be the suggestion of "hybrid" receptors containing different beta chains from each of these related heterotetrameric receptors. Analysis of the effects of IGF-1 and insulin on MtT/S cells showed that each reduced GH mRNA in a dose-dependent manner gave a calculated EC50 of 15.5 nM for IGF-1 and 0.6 nM for insulin, suggesting that the respective receptors for each hormone were activated. However, GH mRNA response to IGF-1 treatment was "ultrasensitive," exhibiting a switch-like effect; below 10 nM IGF-1, there was no decline in GH mRNA, but then maximal reduction occurred at IGF-1 concentrations above 20 nM. The degree of this ultrasensitive effect was calculated from the Hill equation for cooperativity, with a Hill coefficient of -4.1, greater than the classic cooperativity exhibited by hemoglobin binding to oxygen. The ultrasensitive response was specific for IGF-1, as insulin did not display this effect. These results suggest that the response evoked by the IGF-1 receptor could act as a binary molecular switch controlling GH mRNA expression in somatotrophs.

Mitogen-activated protein kinase inhibits 1,25-dihydroxyvitamin D3-dependent signal transduction by phosphorylating human retinoid X receptor alpha.

Human retinoid X receptor alpha (hRXR alpha) is a member of the nuclear receptor family of transcriptional regulators. It regulates transcription through its association with several heterodimeric partners, including the vitamin D3 receptor (VDR). Signaling through the VDR is essential for normal calcium homeostasis and has been shown to inhibit the proliferation of cancer cells derived from a number of tissues. Here we show that phosphorylation of hRXR alpha in ras-transformed human keratinocytes through the activated Ras-Raf-mitogen-activated protein kinase (Ras-Raf-MAP kinase) pathway results in attenuated transactivation by the VDR and resistance to the growth inhibitory action of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and RXR-specific agonist LG1069 (4-[1-(5,6,7, 8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) ethenyl]-benzoic acid). Phosphorylation of hRXR alpha occurs at serine 260, a consensus MAP kinase site. Inhibition of MAP kinase activity or point mutagenesis of serine 260 of hRXR alpha reverses the observed resistance to 1,25(OH)2D3 and LG1069. Thus, hRXR alpha is a downstream target of MAP kinase, and its phosphorylation may play an important role in malignant transformation.

C-reactive Protein Inhibits Chemotactic Peptide-induced p38 Mitogen-activated Protein Kinase Activity and Human Neutrophil Movement

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.

Inhibition of p42/p44 mitogen-activated protein kinase by oxysterols in rat astrocyte primary cultures and C6 glioma cell lines.

We previously demonstrated that oxysterols inhibit the growth of experimental glioblastoma induced in the rat brain cortex. Mechanism of action of these compounds remains obscure. In this study, we investigated the effect of 7beta-hydroxycholesterol (7beta-OHCH) and 7ketocholesterol (7k-CH) on the growth and MAP kinase activity in three in vitro biological models: rat astrocyte primary cultures, primary cultures treated by dibutyryl-cAMP (reactive cells), and the C6 glioma cell line. The oxysterols are not lethal to primary astrocytes, even if MAP kinase activity is decreased, particularly when cells were treated with 7k-CH. Both oxysterols are toxic to reactive astrocytes, and as compared with untreated primary cultures, they amplified the MAP kinase activity decrease. However, the mechanism of action of oxysterols on reactive astrocytes seems not to be linked to the MAP kinase pathway. In highly proliferating C6 cell lines, only 7beta-OHCH has an antiproliferative effect and is cytotoxic. The inhibition of MAP kinase activity is a function of 7beta-OHCH concentration. PD098059, a MAP kinase pathway inhibitor, has only a time-limited antiproliferative effect on C6 cell growth. We conclude that in C6 cells, the MAP kinase activity decrease is correlated with the toxic effect of 7beta-OHCH and occurs at first stages of 7beta-OHCH action.