l‐NAME‐Induced Dispersion of Melanosomes in Melanophores Activates PKC,MEK and ERK1

Melanosome movement represents a good model of cytoskeleton‐mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME) induced dispersion in melanophores pre‐aggregated with melatonin. Activation of cyclic adenosine 3′,5′‐monophosphate (cAMP)‐dependent protein kinase (PKA) or calcium‐dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal‐regulated kinase (MEK)‐ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of l ‐NAME‐induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in l ‐NAME‐dispersed melanophores. l ‐NAME also caused dispersion in latrunculin‐B‐treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the l ‐NAME‐induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.     

Widdrol-induced lipolysis is mediated by PKC and MEK/ERK in 3T3-L1 adipocytes

Molecular and Cellular Biochemistry - Obesity is a serious medical condition causing various diseases such as heart disease, type-2 diabetes, and cancer. Fat cells (adipocytes) play an important...     

TPA induces the expression of EC-SOD in human monocytic THP-1 cells: Involvement of PKC,MEK/ERK and NOX-derived ROS

Abstract

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.     

TPA induces the expression of EC-SOD in human monocytic THP-1 cells: involvement of PKC, MEK/ERK and NOX-derived ROS

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.     

Growth and maturation of melanosomes in the melanophores of a teleost,Oryzias latipes

Summary Formation of melanosomes in melanophores of a teleost, Oryzias latipes, was studied by means of electron microscopy. Two distinct types of premelanosomes are observed in the same cell: (i) multivesicular premelanosomes, which later develop into melanosomes with electron-lucent hollows in the center, appear at early embryonic stages; (ii) premelanosomes with highly organized, fibrous internal structure are formed at later stages of development and give rise to melanosomes with a filamentous center. Melanosomes are generally ellipsoid in shape, and the difference in the dimensions of fibrillar premelanosomes, melanosomes in the cells at younger developmental stages and those developed fully in melanophores of adults indicates that these organelles grow during development. The growth is achieved by fusion of small unmelanized vesicles or fibrillar premelanosomes to preformed melanosome and by fusion of two or more premelanosomes to form a larger organelle. The addition of the matrix of fibrillar premelanosomes around preformed melanosomes, which are derived from either multivesicular or fibrillar premelanosomes, forms a concentric outer deposit, and the fusion of small vesicles produces electron-lucent pits which are scattered irregularly in mature melanosomes.     

Leptin Inhibits Glucose Intestinal Absorption via PKC,p38MAPK,PI3K and MEK/ERK

The role of leptin in controlling food intake and body weight is well recognized, but whether this is achieved by modulating nutrient absorption is still a controversial issue. The aim of this work was to investigate the direct effect of luminal leptin on glucose intestinal absorption and elucidate for the first time its signaling pathway. Fully differentiated Caco-2 cells grown on transwell filters were used for glucose transport studies. Leptin caused a significant reduction in glucose absorption. Individual and simultaneous inhibition of ERK, p38MAPK, PI3K or PKC abrogated completely the inhibitory effect of leptin. Activating PKC, lead to a stimulatory effect that appeared only when ERK, p38MAPK, or PI3K was inactive. Moreover, leptin increased the phosphorylation of ERK, Akt and p38MAPK. This increase changed into a decrease when p38MAPK and PKC were inactivated individually. Inhibiting ERK maintained the leptin-induced up-regulation of p-Akt and p-p38MAPK while inhibiting PI3K reduced the level of p-ERK and p-Akt but maintained the increase in p-p38MAPK. These results suggest that leptin reduces glucose absorption by activating PKC. Although the latter modulates glucose absorption via a stimulatory and an inhibitory pathway, only the latter is involved in leptin’s action. Active PKC leads to a sequential activation of p38MAPK, PI3K and ERK which exerts an inhibitory effect on glucose absorption. The results reveal a modulatory role of leptin in nutrient absorption in addition to its known satiety inducing effect.     

ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK

We identified a 46-kDa ERK, whose kinetics of activation was similar to that of ERK1 and ERK2 in most cell lines and conditions, but showed higher fold activation in response to osmotic shock and epidermal growth factor treatments of Ras-transformed cells. We purified and cloned this novel ERK (ERK1b), which is an alternatively spliced form of ERK1 with a 26-amino acid insertion between residues 340 and 341 of ERK1. When expressed in COS7 cells, ERK1b exhibited kinetics of activation and kinase activity similar to those of ERK1. Unlike the uniform pattern of expression of ERK1 and ERK2, ERK1b was detected only in some of the tissues examined and seems to be abundant in the rat and human heart. Interestingly, in Ras-transformed Rat1 cells, there was a 7-fold higher expression of ERK1b, which was also more responsive than ERK1 and ERK2 to various extracellular treatments. Unlike ERK1 and ERK2, ERK1b failed to interact with MEK1 as judged from its nuclear localization in resting cells overexpressing ERK1b together with MEK1 or by lack of coimmunoprecipitation of the two proteins. Thus, ERK1b is a novel 46-kDa ERK isoform, which seems to be the major ERK isoform that responds to exogenous stimulation in Ras-transformed cells probably due to its differential regulation by MEK.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Aggregation of melanosomes in melanophores is accompanied by the reorganization of microtubules and intermediate filaments

The structure of the cytoskeleton in cultured melanophores of the fish Gymnocorymbus ternetzi during aggregation of melanosomes was studied. It has been shown that the motion of pigment granules is accompanied by a reorganization of microtubules and intermediate filament systems. In melanophores with dispersed pigment granules, microtubules are wavy and form a loose network whilst intermediate filaments in such cells form a dense network around the dispersed melanosomes. During aggregation microtubules and intermediate filaments become radially oriented. It was also shown that the surface area of melanophores increased during aggregation.     

MEK/ERK regulates adherens junctions and migration through Rac1

Polyamine depletion with the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine (DFMO), prevents Rac1 activation causing the formation of a thick actin cortex at the cell periphery and inhibits migration of intestinal epithelial cells. In the present study, we demonstrate that MEK activation by EGF increased Rac1 activation, dissociation of intercellular contacts, and migration in both control and polyamine-depleted cells, while U0126, a specific inhibitor of MEK1, prevented disruption of junctions as well as EGF-induced Rac1 activation. Constitutively active MEK1 (CA-MEK) expression altered cell-cell contacts in control and polyamine depleted cells. The expression of constitutively active Rac1 (CA-Rac1) restored beta-catenin to the cell periphery and prevented the formation of actin cortex and caused the appearance of F-actin stress fibers in polyamine-depleted cells. Inhibition of Rac activation by NSC23766, a specific inhibitor of Tiam1, an upstream guanidine nucleotide exchange factor for Rac1, reproduced the beta-catenin localization and actin structure of polyamine-depleted cells. Tiam1 localized more extensively with beta-catenin at the cell periphery in CA-Rac1 cells compared to vector cells. Polyamine depletion decreased the expression of E-cadherin to a greater extent compared to beta-catenin. Subcellular fractionation further confirmed our immuno-localization and western blotting observations. These data suggest that EGF acting through MEK1/ERK to activate Rac1 regulates cell-cell contacts. Thus, decreased migration in polyamine depleted cells may be due to the inhibition of Tiam1 activation of Rac1 and the subsequent decreased expression of beta-catenin and E-cadherin leading to reduced cell-cell contacts.     

Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ∼5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission.     

The effect of arrestin conformation on the recruitment of c-Raf1, MEK1, and ERK1/2 activation

Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.     

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.     

Dual actions of nitric oxide on angiogenesis: possible roles of PKC, ERK, and AP-1

Regulation of angiogenesis by nitric oxide (NO) is controversial since NO has been shown to have both pro- and anti-angiogenic effects. In this study, we examined the effect of the NO donor, S-nitro-N-acetyl-penicillamine (SNAP), on in vitro angiogenesis, and the mechanisms involved: PKC activity, ERK and c-Jun phosphorylation, and AP-1 DNA binding activity, in microvascular endothelial cells. SNAP, at 0.5-4 mM, significantly and dose-dependently inhibited angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 80%, 83%, and 63% and 73%, respectively. SNAP at concentrations > 2mM also abolished AP-1 binding activity. Lower concentrations of SNAP (0.1-0.3 mM) significantly increased angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 46%, 60%, and 61% and 180%, respectively. These findings indicate that the dual pro- and anti-angiogenic actions of NO are dose-dependent and suggest that they are mediated by PKC and ERK acting on AP-1.     

The phosphorylation of phospholipase C-gamma1, Raf-1, MEK, and ERK1/2 induced by a conserved retroviral peptide

A synthetic 17-amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins has been found to exhibit suppressive properties for numerous immune functions. It has been shown that CKS-17 causes an imbalance of human types 1 and 2 cytokines and inhibition of the immune responses of lymphocytes, monocytes, and macrophages. CKS-17 induced increased intracellular levels of cAMP, which plays an important role in regulation of cytokine biosynthesis. In this study, using a Jurkat T-cell line and Western blot analysis, CKS-17 induced phosphorylation of PLC-gamma1, Raf-1, MEK and ERK1/2. Using a PLC selective inhibitor U73122 or PLC-gamma1-deficient Jurkat cell line, phosphorylation induced by CKS-17 of ERK1/2, PLC-gamma1, or Raf-1, respectively, were undetectable or significantly reduced. Reintroduction of PLC-gamma1 into the PLC-gamma1-deficient Jurkat cells restored the phosphorylation of ERK1/2 and PLC-gamma1 induced by CKS-17. Further, pretreatment of Jurkat cells with PKC inhibitors blocks the phosphorylation of Raf-1, MEK, and ERK1/2 induced by CKS-17. These results indicate that CKS-17 induces the PLC-gamma1-PKC-Raf-1-MEK-ERK1/2 signaling pathway.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.