Mitogen- Activated Protein Kinases

In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (ΔMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of t...

Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras. Oxidative Medicine and Cellular Longevity astrocytes, and that regulation of Ha-Ras critically involves MAPK activation.

EGFR overexpression and chromosome 3p deletion are two frequent events in head and neck cancers. We previously mapped the smallest region of recurrent copy-number loss at 3p12.2-p14.1. LRIG1, a negative regulator of EGFR, was found at 3p14, and its copynumber loss correlated with poor clinical outcome. Inducible expression of LRIG1 in head and neck cancer TW01 cells, a line with low LRIG1 levels, suppressed cell proliferation in vitro and tumor growth in vivo. Gene expression profiling, quantitative RT-PCR, chromatin immunoprecipitation, and western blot analysis demonstrated that LRIG1 modulated extracellular matrix (ECM) remodeling and EGFR-MAPK-SPHK1 transduction pathway by suppressing expression of EGFR ligands/activators, MMPs and SPHK1. In addition, LRIG1 induction triggered cell morphology changes and integrin inactivation, which coupled with reduced SNAI2 expression. By contrast, knockdown of endogenous LRIG1 in TW06 cells, a line with normal LRIG1 levels, significantly enhanced cell proliferation, migration and invasiveness. Such tumor-promoting effects could be abolished by specific MAPK or SPHK1 inhibitors. Our data suggest LRIG1 as a tumor suppressor for head and neck cancers; LRIG1 downregulation in cancer cells enhances EGFR-MAPK-SPHK1 signaling and ECM remodeling activity, leading to malignant phenotypes of head and neck cancers.

EGFR overexpression and chromosome 3p deletion are two frequent events in head and neck cancers. We previously mapped the smallest region of recurrent copy-number loss at 3p12.2-p14.1. LRIG1, a negative regulator of EGFR, was found at 3p14, and its copynumber loss correlated with poor clinical outcome. Inducible expression of LRIG1 in head and neck cancer TW01 cells, a line with low LRIG1 levels, suppressed cell proliferation in vitro and tumor growth in vivo. Gene expression profiling, quantitative RT-PCR, chromatin immunoprecipitation, and western blot analysis demonstrated that LRIG1 modulated extracellular matrix (ECM) remodeling and EGFR-MAPK-SPHK1 transduction pathway by suppressing expression of EGFR ligands/activators, MMPs and SPHK1. In addition, LRIG1 induction triggered cell morphology changes and integrin inactivation, which coupled with reduced SNAI2 expression. By contrast, knockdown of endogenous LRIG1 in TW06 cells, a line with normal LRIG1 levels, significantly enhanced cell proliferation, migration and invasiveness. Such tumor-promoting effects could be abolished by specific MAPK or SPHK1 inhibitors. Our data suggest LRIG1 as a tumor suppressor for head and neck cancers; LRIG1 downregulation in cancer cells enhances EGFR-MAPK-SPHK1 signaling and ECM remodeling activity, leading to malignant phenotypes of head and neck cancers.

In addition to increasing cGMP, the soluble guanylyl cyclase (sGC) activator 3-(5Ј-hydroxymethyl-2Ј-furyl)-1-benzylindazole (YC-1) can elevate intracellular cAMP levels. This response was assumed to be as a result of cGMP-dependent inhibition of cAMP phosphodiesterases; however, in this study, we show that YC-1-induced cAMP production in the rat pancreatic beta cell line INS-1E occurs independent of its function as a sGC activator and independent of its ability to inhibit phosphodiesterases. This YC-1-induced cAMP increase is dependent upon soluble adenylyl cyclase and not on transmembrane adenylyl cyclase activity. We previously showed that soluble adenylyl cyclase-generated cAMP can lead to extracellular signal-regulated kinase activation and that YC-1-stimulated cAMP production also stimulates extracellular signal-regulated kinase. Although YC-1 has been used as a tool for investigating sGC and cGMP-mediated pathways, this study reveals cGMP-independent pharmacological actions of this compound.

Background Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. Methods Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. Results MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER? subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER-subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER? and ER-cell lines. Conclusions MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathway.

MEKs, which operate within the ERK cascade, shuttle into the nucleus, but are rapidly exported from this location, forming an apparent cytosolic distribution both before and after stimulation. Two dierent mechanisms have been proposed for the nuclear translocation of MEKs. One of them involves a constant and nonregulated shuttling of MEKs into the nucleus operating both before and after mitogenic stimulation. The other mechanism seems to require the activity of MEKs and is facilitated in response to mitogenic stimulation. Here we show that these two mechanisms may coexist in the same cells. We found that leptomycin B (LMB), a potent inhibitor of nuclear export, induces a nuclear accumulation of MEKs, and this was signi®cantly facilitated by stimulation of LMB-treated cells with EGF, TPA and peroxovanadate. The EGF-stimulated, but not the LMBinduced translocation was attenuated by MEK inhibitors and by using inactive forms of MEK1. We also show that LMB slightly activates the ERK cascade, but this activity only partially induces the nuclear accumulation of MEKs in cells treated by LMB alone. Thus, MEKs translocate into the nucleus by a combination of nonregulated and stimulated processes that contribute to the nuclear translocation of MEKs either in resting cells or upon mitogenic stimulation. Oncogene (2001) 20, 7588 ± 7596.

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein–protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference ...

Platelet-derived growth factor (PDGF) stimulates transcription of an immediate-early gene set in Balb/c 3T3 cells. One cohort of these genes, typified by c-fos, is induced within minutes following activation of PDGF receptors. A second cohort responds to PDGF only after a significant time delay, although induction is still a primary response to receptor activation as shown by "superinduction" in the presence of the protein synthesis inhibitor cycloheximide. PDGF-receptor activated signaling pathways for the "slow" immediate-early genes are poorly resolved. Using gain-of-function mutations together with small molecule inhibitors of kinase activity, we show that activation of PI 3-kinase is both necessary and sufficient for the induction of the prototype slow immediate-early gene, monocyte chemoattractant-1 (MCP-1). Following activation of PDGF receptors, MCP-1 mRNA does not begin to accumulate for at least 90 min. However, only a brief (10 min) interval of PI 3-kinase activity is required to trigger this delayed response. The serine/threonine protein kinase, Akt/PKB, likely functions as a downstream affector of PI 3-kinase for this induction.

All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH2-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, ...

List of non-standard abbreviations (6)-[ 125 I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl ([ 125 I]DOI; G-protein coupled receptor (GPCR); G-protein coupled receptor kinase (GRK); human herpes virus 8 (HHV8); mitogen-activated protein kinase (MAPK); Multi-PDZ domain containing protein 1 (MUPP1); nuclear factor kappa B (NFkappa B); nitric oxide synthase (NOS); PSD-95, Dlg, ZO-1 (PDZ); serum response element (SRE) MOL#8268

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

Mitogen-activated protein kinases (MAPK) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPK: MPK3, MPK4, and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can, indeed, be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea, or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for em...

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1(high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined in...

Purpose: Oncogenic BRAF/Ras or NF1 loss can potentially trigger oncogene-induced senescence (OIS) through activation of the mitogen-activated protein kinase (MAPK) pathway. Somatic genetic abnormalities affecting this pathway occur in the majority of pilocytic astrocytomas (PA), the most prevalent brain neoplasm in children. We investigated whether OIS is induced in PA. Experimental Design: We tested expression of established senescence markers in three independent cohorts of sporadic PA. We also assessed for OIS in vitro, using forced expression of wild-type and V600E-mutant BRAF in two astrocytic cell lines: human telomerase reverse transcriptase (hTERT)-immortalized astrocytes and fetal astrocytes. Results: Our results indicate that PAs are senescent as evidenced by marked senescence-associated acidic β-galactosidase activity, low KI-67 index, and induction of p16INK4a but not p53 in the majority of 52 PA samples (46 of 52; 88.5%). Overexpression of a number of senescence-associa...

Las cianobacterias son organismos muy ubicuos que se encuentran ampliamente distribuidos por todo el planeta, ocupando ambientes muy diversos en los que deben enfrentarse a diferentes estreses abióticos. Uno de los principales mecanismos que permite a las cianobacterias adaptarse a estos estreses es la regulación de la expresión génica, que principalmente es llevada a cabo por reguladores transcripcionales y sistemas de dos componentes. En la cianobacteria Anabaena sp. PCC7120 una de las familias de reguladores transcripcionales más importantes es la familia FUR, compuesta por tres parálogos denominados FurA, FurB y FurC. Estos tres factores de transcripción son esenciales para orquestar las respuestas de esta cianobacteria a estreses abióticos y son reguladores globales que modulan la expresión de una gran número de genes en el genoma de Anabaena sp. PCC7120. Algunos de estos genes están regulados de forma directa pero muchos otros están regulados indirectamente a través de regulad...

In Hydra, head regeneration and bud formation appear to be very similar processes. The fact that there are genes whose expression is specific for one of the two processes suggests that they do not have identical molecular bases. We analyzed the signal transduction pathways regulating bud development using inhibitors of protein kinase C, Src, PI3K and ERK. The four inhibitors reversibly blocked bud formation in Hydra when applied before stage 1. Once the bud reached stage 3, three of them had no effect and the bud developed normally. The inhibitors blocked the expression of Budhead, an early head marker, and of CnOtx which are specific for bud formation. The results are in agreement with the central role of a signaling pathway mediated by Src on bud development.

All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH2-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, ...

c-Jun is an oncogenic transcription factor involved in the regulation of cell proliferation, apoptosis and transformation. We have previously reported that cell transformations induced by ornithine decarboxylase (ODC) and c-Ha-ras oncogene, commonly activated in various cancer cells, are associated with constitutively increased phosphorylation of c-Jun on Ser residues 63 and 73. In the present study, we examined the significance of c-Jun phosphorylation and activation on the phenotype of the ODC- and ras-transformants, by using specific inhibitors and dominant-negative (DN) mutants to c-Jun NH2-terminal kinase (JNK) and its upstream kinase, SEK1/MKK4 (mitogen-activated protein kinase kinase 4), and to c-Jun. The transformed morphology of both the ODC- and ras-expressing cells was reversed partially by JNK inhibitors and DN JNK1, more effectively by DN SEK1/MKK4 and phosphorylation-deficient c-Jun mutants (c-JunS63,73A, c-JunS63,73A,T91,93A) and most potently by a transactivation dom...

In the present study, we report that the RAS/BRAF/MAP kinase cascade plays a crucial role in the regulation of the Skp2/p27 pathway in thyroid cancer cells and that this is critical for cell proliferation. In vitro studies with cellular models of human thyroid carcinoma cells demonstrated that the adoptive expression of oncogenic RET/PTC1, Ha-RASV12 or BRAFV600E enhances Skp2 and reduces p27 protein expression in a MAP kinase-dependent manner; that RAS/BRAF/MAP kinase-dependent control of p27 expression in thyroid cancer cells occurs by regulating the stability of Skp2 and p27 protein; and that antisense oligonucleotides to p27 suppress growth arrest induced by MEK inhibitors. Finally, analysis of human thyroid carcinomas indicated that MAP kinasepositive thyroid tumors-as detected by immunostaining for p-ERK-presented high p27 degradative activity and low levels of p27 protein (n = 30; p < 0.05). In summary, our results indicate that constitutive signalling of the MAP kinase cascade contributes to the development of thyroid cancer promoted by activated RAS and BRAF oncogenes and that this occurs, at least in part, by compromising the inhibitory function of p27.

Background Like mammalian MAP kinases, the mating-specific Fus3 MAPK of yeast accumulates in the nuclei of stimulated cells. Because Fus3 does not appear to be subjected to active nucleo-cytoplasmic transport, it is not clear how its activation by mating pheromone effects the observed change in its localization. One possibility is that the activation of Fus3 changes its affinity for nuclear and cytoplasmic tethers. Results Dig1, Dig2, and Ste12 are nuclear proteins that interact with Fus3. We found that the pheromone-induced nuclear accumulation of a Fus3-GFP reporter is reduced in cells lacking Dig1 or Dig2, whereas Fus3T180AY182A-GFP localization was unaffected by the absence of these proteins. This suggests that Dig1 and Dig2 contribute to the retention of phosphorylated Fus3 in the nucleus. Moreover, overexpression of Ste12 caused the hyper-accumulation of Fus3-GFP (but not Fus3T180AY182A-GFP) in the nuclei of pheromone-treated cells, suggesting that Ste12 also plays a role in t...

CD40 engagement induces a variety of functional outcomes following association with adaptor molecules of the TNF receptor-associated factor (TRAF) family. Whereas TRAF2, -5, and -6 initiate NF-κB activation, the outcomes of TRAF3-initiated signaling are less characterized. To delineate CD40-induced TRAF3-dependent events, Ramos B cells stably transfected with a dominant negative TRAF3 were stimulated with membranes expressing recombinant CD154/CD40 ligand. In the absence of TRAF3 signaling, activation of p38 and control of Ig production were abrogated, whereas Jun N-terminal kinase activation and secretion of IL-10, lymphotoxin-α, and TNF-α were partially blocked. By contrast, induction of apoptosis, activation of NF-κB, generation of granulocyte-macrophage CSF, and up-regulation of CD54, MHC class II, and CD95 were unaffected by the TRAF3 dominant negative. Together, these results indicate that TRAF3 initiates independent signaling pathways via p38 and JNK that are associated with ...

To delineate the molecular mechanisms regulating Th2 cell differentiation, CD28-mediated generation of Th2 effectors was analyzed. In the absence of TCR ligation CD28 stimulation induced Th2 differentiation of memory but not of naive CD4+ T cells, whereas costimulation via CD28 and the TCR enhanced Th2 differentiation from naive T cells but suppressed it from memory T cells. Stimulation of T cells via the CD28 pathway, therefore, provided critical signals facilitating Th2 cell differentiation. By comparing the responses to CD28 stimulation in memory and naive T cells and by using specific inhibitors, signaling pathways were defined that contributed to Th2 differentiation. CD28-induced Th2 differentiation required IL-4 stimulation and the activation of the mitogen-activated protein kinases p38 and extracellular signal-regulated kinases 1/2. CD28 engagement directly initiated IL-4 gene transcription in memory T cells and induced activation of phosphatidylinositol 3-kinase, p38, and c-...

Tumour necrosis factor-K K (TNF) has a variety of cellular effects including apoptotic and necrotic cytotoxicity. TNF activates a range of kinases, but their role in cytotoxic mechanisms is unclear. HeLa cells expressing elevated type II 75 kDa TNF receptor (TNFR2) protein, analysed by flow cytometry and Western analysis, showed altered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK; but not MAPK) protein content and activation. There was greater JNK activation, but reduced p38MAPK activation in dying cells compared to those still to enter TNFinduced apoptosis. Moreover, cells displaying more rapid apoptosis possess higher levels of type I 55 kDa TNFR1 receptor isoform, but less TNFR2. These findings reveal differential kinase activation in TNF-induced apoptotic death.

TLRs play a crucial role in early host defense against invading pathogens. In the seminiferous epithelium, Sertoli cells are the somatic nurse cells that mechanically segregate germ cell autoantigens by means of the blood-tubular barrier and create a microenvironment that protects germ cells from both interstitial and ascending invading pathogens. The objective of this study was to examine TLR expression and their functional responses to specific agonists in mouse Sertoli cells. We measured the expression of TLR2, TLR4, TLR5, and TLR6 mRNAs and confirmed by FACS analysis the presence of proteins TLR2 and TLR5 on which we focused our study. Stimulation of Sertoli cells with macrophage-activating lipopeptide-2, agonist of TLR2/TLR6, and with flagellin, agonist of TLR5, induces augmented secretion of the chemokine MCP-1. To assess the functional significance of MCP-1 production following TLR stimulation, conditioned medium from either macrophage-activating lipopeptide-2 or flagellin-tr...

Toll-like receptors (TLRs) are known to play a key role in the innate immune system particularly in inflammatory response against invading pathogens. Recent reports strongly indicate that they play important roles in cancer cells. Prostate cancer represents one of the most common cancer for which no cure is available once metastatic and androgen refractory. Since TLR3 has been recently suggested as a possible therapeutic target in some cancer cell lines, we studied TLR3 expression and functionality in two human prostate cancer cell lines, LNCaP and PC3. We report that both cell lines express TLR3 and that the TLR3 agonist poly (I:C) activates mitogen-activated protein kinases and induces inhibition of proliferation as well as caspase-dependent apoptosis. By using pharmacological and genetic approaches, we demonstrate the involvement of TLR3 in poly (I:C)-induced effects. We also show that a novel interferon-independent pathway involving protein kinase C (PKC)-a activation, upstream of p38 and c-jun N-terminal kinase, is responsible for poly (I:C) proapoptotic effects on LNCaP cells. To our knowledge, this is the first report describing a role of PKC-a in poly (I:C)-mediated apoptosis. The comprehension of the mechanisms underlying TLR3-mediated apoptosis can contribute tools to develop new agonists useful for the treatment of prostate cancer.

Tolerogenic dendritic cells (tDCs) may offer an intervention therapy in autoimmune diseases or transplantation. Stable immaturity and tolerogenic function of tDCs after encountering inflammatory environment are prerequisite for positive outcome of immunotherapy. However, the signaling pathways regulating their stable tolerogenic properties are largely unknown. In this study, we demonstrated that human monocyte-derived tDCs established by using paricalcitol (analogue of vitamin D2), dexamethasone and monophosphoryl lipid A exposed for 24h to LPS, cytokine cocktail, polyI:C or CD40L preserved reduced expression of co-stimulatory molecules, increased levels of inhibitory molecules ILT-3, PDL-1 and TIM-3, increased TLR-2, increased secretion of IL-10 and TGF-β, reduced IL-12 and TNF-α secretion and reduced T cell stimulatory capacity. tDCs further induced IL-10-producing T regulatory cells that suppressed the proliferation of responder T cells. In the inflammatory environment, tDCs main...

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1b and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1b and IL-1a and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1b production in celiac PBMC show that PDWGF-induced de novo pro-IL-1b synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1b. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1b release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3 2/2 and ASC 2/2 knockout mice. Moreover, treatment of human PBMC as well as MyD88 2/ 2 and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-b (TRIF) 2/2 BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-kB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1b in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-kB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD.

Although several multiprotein complexes containing MAPKs (mitogen-activated protein kinases) have been identified using overexpression of kinases and scaffold proteins, the components of the complexes and their physical properties at endogenous expression levels have not been defined. We characterized a large protein complex containing a nerve-growth-factor-activated ERK (extracellular-signal-regulated kinase) and MEK (MAPK/ERK kinase) in rat pheochromocytoma (PC12) cells. This protein complex fractionated into a high-speed pellet and was resistant to non-ionic detergent treatments that solubilized membranes. Disruption of protein–protein interactions by treatment with high salt was required to facilitate immunoprecipitation of active ERK1 and co-precipitation of MEK1. Microtubule fragments were also present in the detergent-resistant high-speed pellet, and some kinases were bound to them, especially ERK1b (an alternatively spliced isoform of ERK1), which showed a strong preference ...

After a two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. One of the most rapidly and highly induced genes and proteins in regenerating liver is insulin-like growth factor binding protein 1 (IGFBP-1), a secreted protein that may modulate the activities of insulin-like growth factors (IGFs) or signal via IGF-independent mechanisms. To assess the functional role of IGFBP-1 in liver regeneration, mice with a targeted disruption of the IGFBP-1 gene were generated. Although IGFBP-1 −/− mice demonstrated normal development, they had abnormal liver regeneration after partial hepatectomy, characterized by liver necrosis and reduced and delayed hepatocyte DNA synthesis. The abnormal regenerative response was associated with blunted activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and a reduced induction of C/EBPβ protein expression posthepatectomy. ...

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1v/cek1v null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 Wl suspension containing 1U10 S , 1U10 T and 1U10 U blastospores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1v/cek1v null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10 ؊7 M) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2-5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-␣-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-␣ (TGF␣), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGF␣ release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPKmediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGF␣. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis. Substance P (SP), 1 an 11-amino acid neuropeptide belonging to the tachykinin family, is localized in the central nervous

Neurotensin (NT) is a highly expressed gastrointestinal (GI) neuropeptide, which modulates GI motility, secretion and cell growth as well as intestinal inflammation. Since EGF receptor is highly expressed in human colon cancer cells, we sought to examine whether NT stimulation contributes to the EGFR overexpression using nontransformed colonocyte NCM460 cells. The results show that NT treatment caused a significant increase in EGFR protein expression and gene transcription. Pretreatment with MAP kinase pathway inhibitor PD98059 blocked NT-induced EGFR expression. As the EGFR promoter has a functional Egr-1 site, previously shown to mediate its transcription in response to hypoxia, we examined the role of Egr-1 in the NT response. We first show that NT stimulated Egr-1 expression, which can be inhibited by PD98059. We also determined whether NT increases Egr-1 binding to its site within the EGFR promoter. The data indicate that NT enhanced the amount of Egr-1 binding to the EGFR Egr-1 site and that this binding was significantly decreased by PD98059. To verify that Egr-1 mediated NT-induced EGFR transcription, Egr-1 siRNA was used to knock down its expression. The data show that transfection of Egr-1 siRNA significantly inhibited NT-stimulated EGFR transcription. Together, our results suggest that NT can stimulate MAP kinase-mediated Egr-1 and EGFR gene expression in human colonocytes. Our results may be relevant to the mechanisms by which NT participates in the development of colon cancer. neurotensin; early growth response gene-1; EGF receptor Neurotensin (NT) is a 13 amino acid neuropeptide that was initially isolated from bovine hypothalamus by Carraway and Leeman. 1 Subsequently, it was found that its highest expression is localized in the gastrointestinal (GI) tract. 2 In the small intestine, NT is synthesized and secreted by specific mucosal endocrine cells in response to several different stimuli. NT modulates GI tract motility, 5-7 stimulates intestinal secretion, 8,9 stimulates

It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4 ؊ / CXCR4 ؉ /CCR5 ؉ T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 ␣ (SDF-1␣) (CXCL12) and macrophage inflammatory protein 1 ␤ (MIP-1␤) (CCL4) of their respective coreceptors. Thus, both p38 and SAPK/Jun N-terminal kinase mitogenactivated protein kinases (MAPKs) are activated on stimulation of these cells with either T-or M-tropic gp120, as well as with SDF-1␣ or MIP-1␤. In contrast, extracellular signal-related kinase 1 and 2 MAPKs are only activated by MIP-1␤ but not by M-tropic gp120. Importantly, T-and M-tropic gp120 are able to induce the secretion of matrix metalloproteinase 9 (MMP-9), an extracellular metalloproteinase present in cerebrospinal fluid of patients with HIV-1 by T cells or glioma cells. Specific inhibition of MAPK p38 activation resulted in a complete abrogation of the induction of the MMP-9 pathogenic factor expression by gp120 or chemokines in both cell types. Because neurodegenerative features in acquired immune deficiency syndrome dementia may involve demyelinization by MMP-9, the specific targeting of p38 could provide a novel means to control HIV-induced cytopathogenic effects and cell homing to viral replication sites. (Blood.

Hepatitis C virus (HCV) infection often leads to the development of hepatocellular carcinoma (HCC), but its molecular mechanism has not been clearly elucidated. Previously, transgenic mice constitutively expressing HCV core protein have been shown to develop HCC, suggesting a pivotal role of the core protein in hepatocarcinogenesis. Here, we analyzed the expression of cytokines associated with a variety of cellular processes, including cell proliferation, in the mouse model for HCV-associated HCC to define the molecular events prior to oncogenesis. The expression of tumor necrosis factor-␣ and interleukin-1␤ was increased at both protein and mRNA levels. In addition, the activities of c-Jun N-terminal kinase and activator protein-1 (AP-1), downstream effectors, were enhanced, while IB kinase or nuclear factor-B activities were not enhanced. Thus, the altered in vivo expression of cytokines with AP-1 activation in consequence to the core protein expression may contribute to hepatocarcinogenesis in persistent HCV infection.

Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH 2 -terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed.

Integrin-mediated signalling has been implicated in asbestos-induced carcinogenesis. In studies here, we examined signal transduction events associated with integrin-directed cell reactions triggered by crocidolite asbestos in the pleural mesothelial cell line 4/4 RM-4. Crocidolite fibres induced a significant time-and dosedependent activation of the extracellular-signal-regulated kinases ERK1 and ERK2. ERK activation was specifically inhibited by integrin-blocking agents, that are integrinbinding peptides containing the sequence arginine-glycineaspartic acid (RGD), and monoclonal antibodies against the integrin 1-chain. Integrin-dependent activation of ERK1/2 in response to asbestos appeared to be independent of focal adhesion kinase pp125 FAK (FAK) since FAK autophosphorylation remained unaffected in crocidoliteexposed mesothelial cells. Instead, we observed striking similarities in the kinetics of asbestos-induced ERK1/2 responses and phosphorylation of protein kinase B (AKT) at serine 473, a possible target residue for integrin-linked kinase. As with ERK activation, asbestos-induced AKT stimulation was significantly blocked by both the RGDpeptide and the 1-integrin antibodies. These studies are the first to establish that in mesothelial cells ERK1/2 and AKT are simultaneously phosphorylated upon asbestos exposure in a 1-integrin-dependent manner.

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)–targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS muta...

Introduction 3. RAS/RAF/MEK/ERK signaling pathway 3.1. Aberrant signaling through the RAS/RAF/MEK/ERK pathway in thyroid tumorigenesis 3.1.1. The role of RET/PTC rearrangements 3.1.2. The role of TRK rearrangements 3.1.3. The role of Ras protein and RAS mutation 3.1.4. The role of B-Raf kinase and BRAF mutation 3.2. RET/PTC, RAS, BRAF in RAS/RAF/MEK/ERK pathway and their influence on gene expression profiles in thyroid neoplasms 4.PI3K/Akt signaling pathway and its role in malignant transformation 4.1. Aberrant signaling of PI3K/Akt pathway in thyroid tumorigenesis 4.1.1. The role of PIK3CA alterations 4.1.2. The role of AKT alterations 4.1.3. The role of PTEN alterations 5. Interactions between RAS/RAF/MEK/ERK alterations and PI3K/Akt pathway activation 6. Acknowledgements 7. References

Introduction 3. RAS/RAF/MEK/ERK signaling pathway 3.1. Aberrant signaling through the RAS/RAF/MEK/ERK pathway in thyroid tumorigenesis 3.1.1. The role of RET/PTC rearrangements 3.1.2. The role of TRK rearrangements 3.1.3. The role of Ras protein and RAS mutation 3.1.4. The role of B-Raf kinase and BRAF mutation 3.2. RET/PTC, RAS, BRAF in RAS/RAF/MEK/ERK pathway and their influence on gene expression profiles in thyroid neoplasms 4.PI3K/Akt signaling pathway and its role in malignant transformation 4.1. Aberrant signaling of PI3K/Akt pathway in thyroid tumorigenesis 4.1.1. The role of PIK3CA alterations 4.1.2. The role of AKT alterations 4.1.3. The role of PTEN alterations 5. Interactions between RAS/RAF/MEK/ERK alterations and PI3K/Akt pathway activation 6. Acknowledgements 7. References

Melanoma is one of the most invasive skin cancers with the highest mortality risk. The PI3K/AKT/mTOR signaling pathways are a key regulatory point related to growth factors and involved in the cell's energy metabolism. They are responsible for cell life processes such as growth, proliferation, invasion, survival, apoptosis, autophagy, and angiogenesis. The studies undertaken concerned the effect of protein kinase inhibitors involved in the signaling pathways of AKT, MEK, and mTOR kinases on the expression of cytoskeletal and extracellular matrix proteins, invasion process, and activities of the matrix metalloproteinases (MMPs): MMP-2 and MMP-9 in melanoma cells. The study used mTOR kinase inhibitors: Everolimus and Torkinib; dual PI3K/mTOR inhibitors BEZ-235 and Omipalisib; and the mTORC1/2 inhibitor OSI-027. These compounds were used both as monotherapy and in combination with the MEK1/2 inhibitor AS-703026. mTOR kinase inhibitors, especially the third generation in combination with the MEK 1/2 kinase inhibitor AS-703026, significantly inhibited invasion and metalloproteinases (MMPs) activity in melanoma cell lines. The inhibition of the cell invasion process was accompanied by a significant change in the expression of proteins associated with EMT. The morphology of cells also changed significantly: their thickness, volume, roughness, convexity of shape, and irregularity, which may be a good diagnostic and prognostic factor for the response to treatment. Our studies to date on the effect of three generations of mTOR kinase inhibitors on the inhibition of the invasion process, the activation of apoptosis, and the reduction in cell proliferation suggest that they may be an important target for anticancer therapy.

A lternative splicing provides one of the major mechanisms by which eukaryotic genomes can increase the diversity of transcripts and protein products encoded by a limited amount of genes. The spliceosome is involved in recognizing and removing intronic sequences and it is guided in this activity by splicing regulatory factors that determine the particular splice sites that are to be recognized and used for intron removal. Thus, by modulating the spliceosome's activity, these factors can cause different mRNA transcripts to be generated from the same precursor mRNA. In a recent RNAi screen for factors modulating RAS/MAPK signaling, we identified the Drosophila exon junction complex (EJC) as one of the components regulating the splicing of mapk transcripts. We showed that removal of EJC components caused multiple consecutive mapk exons to be skipped. Moreover, we determined that other fly genes that, like mapk, had particularly large introns, were also sensitive to disruption of the EJC. The importance of intron length in determining sensitivity to EJC disruption suggests that the EJC is functioning in exon definition, a splicing mechanism that occurs for long introns in particular. ©2 0 1 1 L a n d e s B i o s c i e n c e . D o n o t d i s t r i b u t e .

An important mechanism of antitumoral targeted therapies is the induction of apoptosis in tumor cells. Tamoxifen and trastuzumab (Herceptin), respectively, are able to trigger apoptosis in human breast cancer cells. But, frequently altered apoptotic signal cascades, for instance through PTEN mutations, help tumor cells to escape antitumoral therapy. We studied to what extent the apoptotic effect of signal-transduction inhibitors is dependent on PTEN expression. PTEN expression was analysed by Western blot analysis in tumor cell lines of the breast (BT-474, MCF-7, MDA-MB-231), ovary (BG-1, SK-OV-3) and endometrium (Ishikawa, HEC-1A). Apoptotic effects of tamoxifen, trastuzumab, ZD1839 (Iressa) and different mitogen-activated protein kinase (MAP) inhibitors were measured after 24 h of treatment. Cellular apoptosis was determined by the detection of cytoplasmic histone-DNA complexes. The tested tumor cell lines exhibited a different PTEN expression, ranging from a high expression (ovarian cancer cell line BG-1 and BT-474 breast cancer cells) to a total absence of PTEN expression (endometrial Ishikawa cells). The apoptotic effect of receptor-targeting drugs (tamoxifen, trastuzumab, ZD1839) was dependent both on receptor expression and PTEN expression. When cells were treated with MAPK inhibitors, no correlation between PTEN expression and the apoptosis rate was observed. Our data underline the importance of PTEN expression regarding the induction of apoptosis through various targeted therapies.

Maternal obesity increases risk for childhood obesity, but molecular mechanisms are not well understood. We hypothesized that primary umbilical vein endothelial cells (HUVEC) from infants of overweight and obese mothers would harbor transcriptional patterns reflecting offspring obesity risk. In this observational cohort study, we recruited 13 lean (pre-pregnancy BMI &lt;25.0 kg/m(2)) and 24 overweight-obese (&#39;ov-ob&#39;, BMI ⩾25.0 kg/m(2)) women. We isolated primary HUVEC, and analyzed both gene expression (Primeview, Affymetrix) and cord blood levels of hormones and adipokines. 142 transcripts were differentially expressed in HUVEC from infants of overweight-obese mothers (false discovery rate, FDR&lt;0.05). Pathway analysis revealed that genes involved in mitochondrial and lipid metabolism were negatively correlated with maternal BMI…

Mitogen-activated protein kinase (MAPK) 3/MAPK1 (also known as ERK1/ERK2) plays an important role in the signal transduction pathways. To our knowledge, however, its role in the development of testicular ischemia-reperfusion injury has not yet been investigated. Therefore, we studied the pattern of MAPK3/MAPK1 activation in a experimental model of testicular ischemia-reperfusion injury. We also investigated MAPK8 to understand whether an association exists between these two MAPKs. Adult male Sprague-Dawley rats were subjected to 1 h of testicular ischemia followed by 24 h of reperfusion or to a sham testicular ischemia-reperfusion. Animals were randomized to receive PD98059, which is an inhibitor of MAPK3/MAPK1 (10 mg/kg i.p. administered immediately after detorsion), or its vehicle. The time course of MAPK3/MAPK1, MAPK8, and tumor necrosis factor (TNF; also known as TNF alpha) expression and a histological examination in both the ischemic-reperfused testis and the contralateral one were performed. In both testes, MAPK3/MAPK1 and MAPK8 expression appeared following 10 min of reperfusion and reached their highest activation after 30 min. The MAPK levels slowly decreased, and no significant expression of either kinase was observed following 2 h of reperfusion. Expression of TNF was evident after 1 h of reperfusion and reached its maximum increase after 3 h. PD98059 blunted MAPK3/MAPK1 and MAPK8, reduced TNF expression, and improved the testicular damage caused by ischemia-reperfusion injury in both testes. These data emphasize that MAPK3/MAPK1 has a role in testicular damage and that its blockade might have a future therapeutic role for the management of patients with unilateral testicular torsion.