Involvement of Cot activity in the proliferation of ALCL lymphoma cells

Anaplastic large-cell lymphoma (ALCL) cells overexpress CD30 on their cell surface, show increased levels of activated Erk1/2 and of JunB; participating JunB in the proliferative capacity of these lymphomas. Here, we show that ALCL lymphoma cells also present high expression levels of the proto-oncogenic Cot (MAP3K8). Using pharmacological drugs as well as the RNA interference technique we show that Cot protein is responsible for the constitutive Erk1/2 activation in the ALCL lymphoma cells, SUDHL-1. Besides, inhibition of Cot activity reduces the number of cell divisions which is achieved, at least in part, by the control that Cot exercises on the activation state of p70 S6K and on the expression levels of JunB. Since Cot represents an alternative mode, independently of RAF, to activate Erk1/2, all these data strongly suggest that molecular targeting of Cot may be a potential new specific strategy for ALCL lymphomas therapy, without the fully disturbance of the Erk1/2 function.     

Sterile Inflammation in Acetaminophen-induced Liver Injury Is Mediated by Cot/tpl2

Cot/tpl2 (MAP3K8) activates MKK1/2-Erk1/2 following stimulation of the Toll-like/IL-1 receptor superfamily. Here, we investigated the role of Cot/tpl2 in sterile inflammation and drug-induced liver toxicity. Cot/tpl2 KO mice exhibited reduced hepatic injury after acetaminophen challenge, as evidenced by decreased serum levels of both alanine and aspartate aminotransferases, decreased hepatic necrosis, and increased survival relative to Wt mice. Serum levels of both alanine and aspartate aminotransferases were also lower after intraperitoneal injection of acetaminophen in mice expressing an inactive form of Cot/tpl2 compared with Wt mice, suggesting that Cot/tpl2 activity contributes to acetaminophen-induced liver injury. Furthermore, Cot/tpl2 deficiency reduced neutrophil and macrophage infiltration in the liver of mice treated with acetaminophen, as well as their hepatic and systemic levels of IL-1α. Intraperitoneal injection of damage-associated molecular patterns from necrotic hepatocytes also impaired the recruitment of leukocytes and decreased the levels of several cytokines in the peritoneal cavity in Cot/tpl2 KO mice compared with Wt counterparts. Moreover, similar activation profiles of intracellular pathways were observed in Wt macrophages stimulated with Wt or Cot/tpl2 KO damage-associated molecular patterns. However, upon stimulation with damage-associated molecular patterns, the activation of Erk1/2 and JNK was deficient in Cot/tpl2 KO macrophages compared with their Wt counterparts; an effect accompanied by weaker release of several cytokines, including IL-1α, an important component in the development of sterile inflammation. Taken together, these findings indicate that Cot/tpl2 contributes to acetaminophen-induced liver injury, providing some insight into the underlying molecular mechanisms.     

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.     

Oncoprotein Cot1 represses kinase suppressors of Ras1/2 and 1,25-dihydroxyvitamin D3-induced differentiation of human acute myeloid leukemia cells

Metabolites and derivatives of vitamin D are well-known inducers of monocytic differentiation, but the mechanistic basis for their action is not fully elucidated. Here we show that the product of protooncogene Cot1 represses the monocytic phenotype in human acute myeloid leukemia (AML) cells induced to differentiate by 1,25-dihydroxyvitamin D(3) (1,25D), even though the expression of cellular Cot1 increases early in the process of 1,25D-induced differentiation. Interestingly, the expression of the two members of the Kinase Suppressor of Ras (KSR) family of molecular scaffolds, known to be positive regulators of Ras signaling and of 1,25D-induced differentiation, increases in parallel with Cot1 in 1,25D-treated cells. However, KSR1/2 are negatively regulated by Cot1, as determined by transfection of siCot1, and confirmed by a reverse effect of ectopic expression of Cot1. The effect of Cot1 in AML cells appears to be cell-type specific, as previous reports in other cell types found KSR-2 to be a negative regulator of Cot1, a reverse relationship. Also in contrast to findings in other cells, in AML cells Cot1 exerts negative control on the MAP kinase pathways, since siCot1 increases the levels of activated Raf1, p90RSK, JNK1, c-jun, and p38, though not of MEK/ERK. These findings have implications for therapy of AML, since in AML cells active MAPKs hasten cell differentiation, and specific pharmacological inhibitors of Cot1 kinase activity have recently became available, thus making Cot1 a "druggable" target.     

Lysophosphatidic acid down-regulates stress fibers and up-regulates pro-matrix metalloproteinase-2 activation in ovarian cancer cells

Epithelial ovarian cancer (EOC) is asymptomatic at early stages and is often diagnosed late when tumor cells are highly metastatic. Lysophosphatidic acid (LPA) has been implicated in ovarian oncogenesis as levels of this lipid are elevated in patient ascites and plasma. Because the underlying mechanism governing LPA regulation of matrix metalloproteinase-2 (MMP-2) activation remains undefined, we investigated the relationship between LPA-induced changes in actin microfilament organization and MMP-2 enzymatic activity. We report that when cells were cultured at a high density, LPA mediated stress fiber and focal adhesion disassembly and significantly repressed RhoA activity in EOC cells. Inhibition of Rho-kinase/ROCK enhanced both LPA-stimulated loss of stress fibers and pro-MMP-2 activation. In contrast, expression of the constitutively active RhoA(G14V) mutant diminished LPA-induced pro-MMP-2 activation. LPA had no effects on membrane type 1-MMP or tissue inhibitor of metalloproteinase-2 expression, but up-regulated MMP-2 levels, contributing to the induction of MMP-2 activation. Interestingly, when cells were cultured at a low density, stress fibers were present after LPA stimulation, and ROCK activity was required for EOC cell migration. Collectively, these results were consistent with a model in which LPA stimulates the metastatic dissemination of EOC cells by initiating loss of adhesion and metalloproteinase activation.     

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.     

Live imaging of glioblastoma cells in brain tissue shows requirement of actin bundles for migration

Live-cell imaging of glioblastoma U373 and U87 cells transfected with actin cytoskeleton markers has been used to study there-arrangements that are associated with migration in two- and three-dimensional matrices and in brain tissue. In collagen gels and in brain slices, both cell types developed neuronal-like processes with ruffling membranes and filopodia. Blebbing cells were also observed, but these were mainly immobile. The retraction of trailing cell processes in a tissue environment was associated with the transient development and contraction of bundles of axial stress fibers. The inhibition of Rho-kinase caused glioblastoma cells in brain slices to become immobile and develop neurite-like processes at random, which indicates the requirement of Rho signaling and contractility for migration. Actin stress fibers were also observed in glioblastoma cells injected into the brains of living mice. Thus, invading glioblastoma cells use neurite-like extensions to penetrate between neuronal fibers and contractile actin bundles for traction of the cell body.     

The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin-dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg-/- fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg-/- fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain-containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg-/- cells, the increased contractility of arg-/- cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.     

Vascular endothelial growth factor (VEGF)-driven actin-based motility is mediated by VEGFR2 and requires concerted activation of stress-activated protein kinase 2 (SAPK2/p38) and geldanamycin-sensitive phosphorylation of focal adhesion kinase

In endothelial cells, vascular endothelial growth factor (VEGF) induces an accumulation of stress fibers associated with new actin polymerization and rapid formation of focal adhesions at the ventral surface of the cells. This cytoskeletal reorganization results in an intense motogenic activity. Using porcine endothelial cells expressing one or the other type of the VEGF receptors, VEGFR1 or VEGFR2, or human umbilical vein endothelial cells pretreated with a VEGFR2 neutralizing antibody, we show that VEGFR2 is responsible for VEGF-induced activation of the stress-activated protein kinase-2/p38 (SAPK2/p38), phosphorylation of focal adhesion kinase (FAK), and enhanced migratory activity. Activation of SAPK2/p38 triggered actin polymerization whereas FAK, which was phosphorylated independently of SAPK2/p38, initiated assembly of focal adhesions. Both processes contributed to the formation of stress fibers. Geldanamycin, an inhibitor of HSP90 blocked tyrosine phosphorylation of FAK, assembly of focal adhesions, actin reorganization, and cell migration, all of which were reversed by overexpressing HSP90. We conclude that VEGFR2 mediates the physiological effect of VEGF on cell migration and that two independent pathways downstream of VEGFR2 regulate actin-based motility. One pathway involves SAPK2/p38 and leads to enhanced actin polymerization activity. The other involves HSP90 as a permissive signal transduction factor implicated in FAK phosphorylation and assembly of focal adhesions.     

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor–activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow–derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP–eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene–encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene–encoding mRNA translation in Toll-like receptor–activated macrophages.     

Prostaglandin E2-mediated migration of human trophoblast requires RAC1 and CDC42

The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E(2) produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE(2)-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE(2) or 17-phenyl trinor PGE(2) (PGE receptor PTGER1 agonist) or PGE(1) Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE(2)-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE(2), PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE(2)-mediated migration of human EVTs.     

Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.     

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.     

B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.     

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.     

Grape seed proanthocyanidins inhibit melanoma cell invasiveness by reduction of PGE2 synthesis and reversal of epithelial-to-mesenchymal transition

Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of grape seed proanthocyanidins (GSPs) on melanoma cancer cell migration and the molecular mechanisms underlying these effects using highly metastasis-specific human melanoma cell lines, A375 and Hs294t. Using in vitro cell invasion assays, we observed that treatment of A375 and Hs294t cells with GSPs resulted in a concentration-dependent inhibition of invasion or cell migration of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2 expression and prostaglandin (PG) E(2) production. Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of melanoma cells with COX-2 small interfering RNA, also inhibited melanoma cell migration. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, an inducer of COX-2, enhanced the phosphorylation of ERK1/2, a protein of mitogen-activated protein kinase family, and subsequently cell migration whereas both GSPs and celecoxib significantly inhibited 12-O-tetradecanoylphorbol-13-acetate-promoted cell migration as well as phosphorylation of ERK1/2. Treatment of cells with UO126, an inhibitor of MEK, also inhibited the migration of melanoma cells. Further, GSPs inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Additionally, inhibition of melanoma cell migration by GSPs was associated with reversal of epithelial-mesenchymal transition process, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin and cytokeratins) while loss of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in melanoma cells. Together, these results indicate that GSPs have the ability to inhibit melanoma cell invasion/migration by targeting the endogenous expression of COX-2 and reversing the process of epithelial-to-mesenchymal transition.