Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1579-1589 ◽  
Author(s):  
M. Reyes-Reyes ◽  
N. Mora ◽  
A. Zentella ◽  
C. Rosales
Keyword(s):  
Signaling Pathways ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Monocytic Leukemia ◽  
Monocytic Cells ◽  
Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

scholarly journals β-Arrestin2 Is Critically Involved in CXCR4-mediated Chemotaxis, and This Is Mediated by Its Enhancement of p38 MAPK Activation

2002 ◽  
Vol 277 (51) ◽  
pp. 49212-49219 ◽  
Author(s):  
Yue Sun ◽  
Zhijie Cheng ◽  
Lan Ma ◽  
Gang Pei
Keyword(s):  
P38 Mapk ◽  
Stromal Cell ◽  
Cancer Metastasis ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mapk Activation ◽  
Endogenous Expression ◽  
Stromal Cell Derived Factor ◽  
Negative Mutant

Chemotaxis mediated by chemokine receptors such as CXCR4 plays a key role in lymphocyte homing and hematopoiesis as well as in breast cancer metastasis. We have demonstrated previously that β-arrestin2 functions to attenuate CXCR4-mediated G protein activation and to enhance CXCR4 internalization. Here we show further that the expression of β-arrestin2 in both HeLa and human embryonic kidney 293 cells significantly enhances the chemotactic efficacy of stromal cell-derived factor 1α, the specific agonist of CXCR4, whereas the suppression of β-arrestin2 endogenous expression by antisense or RNA-mediated interference technology considerably attenuates stromal cell-derived factor 1α-induced cell migration. Expression of β-arrestin2 also augmented chemokine receptor CCR5-mediated but not epidermal growth factor receptor-mediated chemotaxis, indicating the specific effect of β-arrestin2. Further analysis reveals that expression of β-arrestin2 strengthened CXCR4-mediated activation of both p38 MAPK and ERK, and the suppression of β-arrestin2 expression blocked the activation of two kinases. Interestingly, inhibition of p38 MAPK activation (but not ERK activation) by its inhibitors or by expression of a dominant-negative mutant of p38 MAPK effectively blocked the chemotactic effect of β-arrestin2. Expression of a dominant-negative mutant of ASK1 also exerted the similar blocking effect. The results of our study suggest that β-arrestin2 can function not only as a regulator of CXCR4 signaling but also as a mediator of stromal cell-derived factor 1α-induced chemotaxis and that this activity probably occurs via the ASK1/p38 MAPK pathway.

Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Development ◽  
2000 ◽  
Vol 127 (14) ◽  
pp. 3091-3100 ◽  
Author(s):  
A. Djiane ◽  
J. Riou ◽  
M. Umbhauer ◽  
J. Boucaut ◽  
D. Shi
Keyword(s):  
Equatorial Region ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Treated Animal ◽  
Dependent Manner ◽  
Convergent Extension ◽  
Loss Of Function ◽  
Opposing Effects

Wnt signalling plays a crucial role in the control of morphogenetic movements. We describe the expression and functional analyses of frizzled 7 (Xfz7) during gastrulation in Xenopus. Low levels of Xfz7 transcripts are expressed maternally during cleavage stages; its zygotic expression strongly increases at the beginning of gastrulation and is predominantly localized to the presumptive neuroectoderm and deep cells of the involuting mesoderm. Overexpression of Xfz7 in the dorsal equatorial region affects the movements of convergent extension and delays mesodermal involution. It alters the correct localization, but not the expression, of mesodermal and neural markers. These effects can be rescued by extra-Xfz7, which is a secreted form of the receptor that also weakly inhibits convergent extension when overexpressed. This suggests that the wild-type and truncated receptors have opposing effects when coexpressed and that overexpression of Xfz7 causes an increased signalling activity. Consistent with this, Xfz7 biochemically and functionally interacts with Xwnt11. In addition, Dishevelled, but not (β)-catenin, synergizes with Xfz7 to affect convergent extension. Furthermore, overexpression of Xfz7 and Xwnt11 also affects convergent extension in activin-treated animal caps, and this can be efficiently reversed by coexpression of Cdc42(T17N), a dominant negative mutant of the small GTPase Cdc42 known as a key regulator of actin cytoskeleton. Conversely, Cdc42(G12V), a constitutively active mutant, rescues the effects of extra-Xfz7 on convergent extension in a dose-dependent manner. That both gain-of-function and loss-of-function of both frizzled and dishevelled produce the same phenotype has been well described in Drosophila tissue polarity. Therefore, our results suggest an endogenous role of Xfz7 in the regulation of convergent extension during gastrulation.

PKCε modulates NF-κB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes

2000 ◽  
Vol 279 (4) ◽  
pp. H1679-H1689 ◽  
Author(s):  
Richard C. X. Li ◽  
Peipei Ping ◽  
Jun Zhang ◽  
William B. Wead ◽  
Xinan Cao ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Protein Kinases ◽  
Mapk Pathway ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adult Rabbit ◽  
Jnk Pathway ◽  
Mitogen Activated Protein Kinases ◽  
Mitogen Activated Protein

We have previously shown that protein kinase C (PKC)-ε, nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPKs) are essential signaling elements in ischemic preconditioning. In the present study, we examined whether activation of PKCε affects the activation of NF-κB in cardiac myocytes and whether MAPKs are mediators of this signaling event. Activation of PKCε (+108% above control) in adult rabbit cardiomyocytes to a degree that has been previously shown to protect myocytes against hypoxic injury increased the DNA-binding activity of NF-κB (+164%) and activator protein (AP)-1 (+127%) but not that of Elk-1. Activation of PKCη did not have an effect on these transcription factors. Activation of PKCε also enhanced the phosphorylation activities of the p44/p42 MAPKs and the p54/p46 c-Jun NH2-terminal kinases (JNKs). PKCε-induced activation of NF-κB and AP-1 was completely abolished by inhibition of the p44/p42 MAPK pathway with PD98059 and by inhibition of the p54/p46 JNK pathway with a dominant negative mutant of MAPK kinase-4, indicating that both signaling pathways are necessary. Taken together, these data identify NF-κB and AP-1 as downstream targets of PKCε, thereby establishing a molecular link between activation of PKCε and activation of NF-κB and AP-1 in cardiomyocytes. The results further demonstrate that both the p44/p42 MAPK and the p54/p46 JNK signaling pathways are essential mediators of this event.

scholarly journals The Crossroads between RAS and RHO Signaling Pathways in Cellular Transformation, Motility and Contraction

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 819
Author(s):  
Olga Soriano ◽  
Marta Alcón-Pérez ◽  
Miguel Vicente-Manzanares ◽  
Esther Castellano
Keyword(s):  
Signaling Pathways ◽  
Actin Polymerization ◽  
Mapk Pathway ◽  
Human Cancer ◽  
Cellular Transformation ◽  
Dependent Manner ◽  
Rho Proteins ◽  
Filament Assembly ◽  
Multiple Signaling ◽  
Mechanical Feedback

Ras and Rho proteins are GTP-regulated molecular switches that control multiple signaling pathways in eukaryotic cells. Ras was among the first identified oncogenes, and it appears mutated in many forms of human cancer. It mainly promotes proliferation and survival through the MAPK pathway and the PI3K/AKT pathways, respectively. However, the myriad proteins close to the plasma membrane that activate or inhibit Ras make it a major regulator of many apparently unrelated pathways. On the other hand, Rho is weakly oncogenic by itself, but it critically regulates microfilament dynamics; that is, actin polymerization, disassembly and contraction. Polymerization is driven mainly by the Arp2/3 complex and formins, whereas contraction depends on myosin mini-filament assembly and activity. These two pathways intersect at numerous points: from Ras-dependent triggering of Rho activators, some of which act through PI3K, to mechanical feedback driven by actomyosin action. Here, we describe the main points of connection between the Ras and Rho pathways as they coordinately drive oncogenic transformation. We emphasize the biochemical crosstalk that drives actomyosin contraction driven by Ras in a Rho-dependent manner. We also describe possible routes of mechanical feedback through which myosin II activation may control Ras/Rho activation.

Ras induces NBT-II epithelial cell scattering through the coordinate activities of Rac and MAPK pathways

2002 ◽  
Vol 115 (12) ◽  
pp. 2591-2601 ◽  
Author(s):  
Natacha Edme ◽  
Julian Downward ◽  
Jean-Paul Thiery ◽  
Brigitte Boyer
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Mapk Pathway ◽  
Small Gtpase ◽  
Dominant Negative Mutant ◽  
Cell Periphery ◽  
Cell Dissociation ◽  
Downstream Targets ◽  
Cell Dispersion ◽  
Cell Scattering

Cell dissociation and cell migration are the two main components of epithelium-mesenchyme transitions (EMT). We previously demonstrated that Ras is required for the accomplishment of both of these processes during the EGF-induced EMT of the NBT-II rat carcinoma cell line in vitro. In this study,we examined the downstream targets of Ras that are responsible for the dissociation and motility of NBT-II cells. Overexpression of activated forms of c-Raf and MEK1 (a component of the mitogen-activated protein kinase pathway, MAPK) led to cell dissociation, as inferred by the loss of desmosomes from the cell periphery. By contrast, active PI3K, RalA and RalB did not induce desmosome breakdown. The MEK1 inhibitor PD098059 inhibited EGF- and Ras-induced cell dispersion, whereas the PI3K inhibitor LY294002 had no effect. Accordingly, among the partial loss-of-function mutants of Ras(RasV12) that were used to distinguish between downstream targets of Ras, we found that the Raf-specific Ras mutants RasV12S35 and RasV12E38 induced cell dissociation. The PI3K- and RalGDS-activating Ras mutants had, in contrast, no effect on cell dispersion. However, MEK1 was unable to promote cell motility,whereas RasV12S35 and RasV12E38 induced cell migration, suggesting that another Ras effector was responsible for cell motility. We found that the small GTPase Rac is necessary for EGF-mediated cell dispersion since overexpression of a dominant-negative mutant of Rac1 (Rac1N17) inhibited EGF-induced NBT-II cell migration. All stimuli that promoted cell migration also induced Rac activation. Finally, coexpression of active Rac1 and active MEK1 induced the motility of NBT-II cells, suggesting that Ras mediates NBT-II cell scattering through the coordinate activation of Rac and the Raf/MAPK pathway.

Stimulation of pro-α1(I) collagen by TGF-β1 in mesangial cells: role of the p38 MAPK pathway

2001 ◽  
Vol 280 (3) ◽  
pp. F495-F504 ◽  
Author(s):  
Beek Yoke Chin ◽  
Amir Mohsenin ◽  
Su Xia Li ◽  
Augustine M. K. Choi ◽  
Mary E. Choi
Keyword(s):  
P38 Mapk ◽  
Intracellular Signaling ◽  
Mesangial Cells ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Glomerular Mesangial Cells ◽  
Mapk Activation ◽  
Mapk Phosphorylation ◽  
Collagen Mrna

Transforming growth factor-β1(TGF-β1) is a potent inducer of extracellular matrix protein synthesis and a key mediator of renal fibrosis. However, the intracellular signaling mechanisms by which TGF-β1stimulates this process remain incompletely understood. In this report, we examined the role of a major stress-activated intracellular signaling cascade, belonging to the mitogen-activated protein kinase (MAPK) superfamily, in mediating TGF-β1 responses in rat glomerular mesangial cells, using dominant-negative inhibition of TGF-β1 signaling receptors. We first stably transfected rat glomerular mesangial cells with a kinase-deleted mutant TGF-β type II receptor (TβR-IIM) designed to inhibit TGF-β1 signaling in a dominant-negative fashion. Next, expression of TβR-IIM mRNA was confirmed by Northern analysis. Cell surface expression and ligand binding of TβR-IIM protein were demonstrated by affinity cross-linking with 125I-labeled-TGF-β1. TGF-β1 rapidly induced p38 MAPK phosphorylation in wild-type and empty vector (pcDNA3)-transfected control mesangial cells. Interestingly, transfection with dominant-negative TβR-IIM failed to block TGF-β1-induced p38 MAPK phosphorylation. Moreover, dominant-negative TβR-IIMfailed to block TGF-β1-stimulated pro-α1(I) collagen mRNA expression and cellular protein synthesis, whereas TGF-β1-induced extracellular signal-regulated kinase (ERK) 1/ERK2 activation and antiproliferative responses were blocked by TβR-IIM. In the presence of a specific inhibitor of p38 MAPK, SB-203580, TGF-β1 was unable to stimulate pro-α1(I) collagen mRNA expression in the control and TβR-IIM-transfected mesangial cells. Finally, we confirmed that both p38 MAPK activation and pro-α1(I) collagen stimulation were TGF-β1 effects that were abrogated by dominant-negative inhibition of TGF-β type I receptor. Thus we show first demonstration of p38 MAPK activation by TGF-β1 in mesangial cells, and, given the rapid kinetics, this TGF-β1 effect is likely a direct one. Furthermore, our findings suggest that the p38 MAPK pathway functions as a component in the signaling of pro-α1(I) collagen induction by TGF-β1 in mesangial cells.

Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction

1999 ◽  
Vol 112 (24) ◽  
pp. 4485-4489 ◽  
Author(s):  
C. Wu
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Focal Adhesion ◽  
Adaptor Protein ◽  
Small Gtpase ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Mesenchymal Transition ◽  
Integrin Linked Kinase

Integrin-linked kinase (ILK) is a focal adhesion serine/threonine protein kinase that is emerging as a key signaling protein functioning at one of the early convergence points of integrin- and growth factor-signaling pathways. ILK binds to PINCH through the N-terminal ankyrin (ANK) repeat domain and the PINCH binding is crucial for focal adhesion localization of ILK. The ILK-PINCH interaction also connects ILK to Nck-2, an SH2-SH3-containing adaptor protein that interacts with components of growth factor and small GTPase signaling pathways. The kinase activity of ILK is regulated by both cell adhesion and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. ILK phosphorylates downstream targets such as protein kinase B (PKB, also known as Akt) and glycogen synthase kinase 3 (GSK-3) and regulates their activities. Overexpression of ILK in epithelial cells leads to striking morphological changes mimicking epithelial-mesenchymal transition, including upregulation of integrin-mediated fibronectin matrix assembly and downregulation of cell-cell adhesions. Furthermore, ILK regulates nuclear translocation of (beta)-catenin and gene expression, and promotes cell cycle progression and tumor formation. Recent genetic studies in Drosophila melanogaster and Caenorhabditis elegans have shown that lack of expression of ILK or PINCH results in phenotypes resembling those of integrin-null mutants, which demonstrates that ILK and PINCH are indispensable for integrin function during embryonic development.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

2021 ◽  
Author(s):  
Toshihiro Kobayashi ◽  
Hitomi Imachi ◽  
Kensaku Fukunaga ◽  
Jingya Lyu ◽  
Seisuke Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Promoter Activity ◽  
Active Form ◽  
Specific Inhibitor ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Activation Mechanism ◽  
Dominant Negative Mutant ◽  
Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

scholarly journals The multi-site docking protein Grb2-associated binder 1 (Gab1) enhances interleukin-6-induced MAPK-pathway activation in an SHP2-, Grb2-, and time-dependent manner

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Hannes Bongartz ◽  
Karen Gille ◽  
Wiebke Hessenkemper ◽  
Katharina Mandel ◽  
Marc Lewitzky ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Molecular Mechanisms ◽  
Mapk Pathway ◽  
Sh2 Domain ◽  
Time Dependent ◽  
Signalling Pathways ◽  
Dependent Manner ◽  
Mapk Activation ◽  
Pathway Activation ◽  
Mapk Signalling

Abstract Background Cytokine-dependent activation of signalling pathways is tightly orchestrated. The spatiotemporal activation of signalling pathways dictates the specific physiological responses to cytokines. Dysregulated signalling accounts for neoplastic, developmental, and inflammatory diseases. Grb2-associated binder (Gab) family proteins are multi-site docking proteins, which expand cytokine-induced signal transduction in a spatial- and time-dependent manner by coordinating the recruitment of proteins involved in mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) and phosphatidyl-inositol-3-kinase (PI3K) signalling. Interaction of Gab family proteins with these signalling proteins determines strength, duration and localization of active signalling cascades. However, the underlying molecular mechanisms of signal orchestration by Gab family proteins in IL-6-induced signalling are only scarcely understood. Methods We performed kinetic analyses of interleukin-6 (IL-6)-induced MAPK activation and analysed downstream responses. We compared signalling in wild-type cells, Gab1 knock-out cells, those reconstituted to express Gab1 mutants, and cells expressing gp130 receptors or receptor mutants. Results Interleukin-6-induced MAPK pathway activation can be sub-divided into an early Gab1-independent and a subsequent Gab1-dependent phase. Early Gab1-independent MAPK activation is critical for the subsequent initiation of Gab1-dependent amplification of MAPK pathway activation and requires binding of SH2 domain-containing phosphatase 2 (SHP2) to the interleukin-6 receptor complex. Subsequent and coordinated recruitment of Grb2 and SHP2 to Gab1 is essential for Gab1-dependent amplification of IL-6-induced late MAPK pathway activation and subsequent gene expression. Conclusions Overall, we elaborated the molecular requirements for Gab1-dependent, spatiotemporal orchestration of interleukin-6-dependent MAPK signalling. We discriminated IL-6-induced Gab1-independent, early activation of MAPK signalling and Gab1-dependent, sustained activation of MAPK signalling.

scholarly journals pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line.

1995 ◽  
Vol 131 (3) ◽  
pp. 761-773 ◽  
Author(s):  
J M Rodier ◽  
A M Vallés ◽  
M Denoyelle ◽  
J P Thiery ◽  
B Boyer
Keyword(s):  
Growth Factor ◽  
Signaling Pathways ◽  
Cell Line ◽  
Intracellular Signaling ◽  
Carcinoma Cell ◽  
Biological Activities ◽  
Dominant Negative ◽  
Carcinoma Cell Line ◽  
Dominant Negative Mutant ◽  
Cell Scattering

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.

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