Inhibition of angiogenesis by a mouse sprouty protein

Sprouty negatively modulates branching morphogenesis in the Drosophila tracheal system. To address the role of mammalian Sprouty homologues in angiogenesis, another form of branching morphogenesis, a recombinant adenovirus engineered to express murine Sprouty-4 selectively in endothelial cells, was injected into the sinus venosus of embryonic day 9.0 cultured mouse embryos. Sprouty-4 expression inhibited branching and sprouting of small vessels, resulting in abnormal embryonic development. In vitro, Sprouty-4 inhibited fibroblast growth factor and vascular endothelial cell growth factor-mediated cell proliferation and migration and prevented basic fibroblast growth factor and vascular endothelial cell growth factor-induced MAPK phosphorylation in endothelial cells, indicating inhibition of tyrosine kinase-mediated signaling pathways. The ability of constitutively activated mutant Ras(L61) to rescue Sprouty-4 inhibition of MAPK phosphorylation suggests that Sprouty inhibits receptor tyrosine kinase signaling upstream of Ras. Thus, Sprouty may regulate angiogenesis in normal and disease processes by modulating signaling by endothelial tyrosine kinases.     

Differential Regulation of MAPK Phosphorylation in the Dorsal Hippocampus in Response to Prolonged Morphine Withdrawal-Induced Depressive-Like Symptoms in Mice

Depression is one of the most frequent neuropsychiatric comorbidities associated with opiate addiction. Mitogen activated protein kinase (MAPK) and MAPK phosphatase (MKP) are involved in drug addiction and depression. However, the potential role of MAPK and MKP in depression caused by morphine withdrawal remains unclear. We utilized a mouse model of repeated morphine administration to examine the molecular mechanisms that contribute to prolonged withdrawal induced depressive-like behaviors. Depressive-like behaviors were significant at 1 week after withdrawal and worsened over time. Phospho-ERK (extracellular signal-regulated protein kinase) was decreased and MKP-1 was elevated in the hippocampus, and JNK (c-Jun N-terminal protein kinase), p38 (p38 protein kinase) and MKP-3 were unaffected. A pharmacological blockade of MKP-1 by intra-hippocampal sanguinarine (SA) infusion prevented the development of depressive-like behaviors and resulted in relatively normal levels of MKP-1 and phospho-ERK after withdrawal. Our findings support the association between hippocampal MAPK phosphorylation and prolonged morphine withdrawal-induced depression, and emphasize the MKP-1 as an negative regulator of the ERK phosphorylation that contributes to depression.     

cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.     

MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. Here we show that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI3K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.     

Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression

Degradation of collagenous extracellular matrix by collagenase 1 (also known as matrix metalloproteinase 1 [MMP-1]) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, chronic ulcers, and tumor invasion and metastasis. Here, we have investigated the role of distinct mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-1 gene expression. The activation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (designated ERK1,2) pathway by oncogenic Ras, constitutively active Raf-1, or phorbol ester resulted in potent stimulation of MMP-1 promoter activity and mRNA expression. In contrast, activation of stress-activated c-Jun N-terminal kinase and p38 pathways by expression of constitutively active mutants of Rac, transforming growth factor beta-activated kinase 1 (TAK1), MAPK kinase 3 (MKK3), or MKK6 or by treatment with arsenite or anisomycin did not alone markedly enhance MMP-1 promoter activity. Constitutively active MKK6 augmented Raf-1-mediated activation of the MMP-1 promoter, whereas active mutants of TAK1 and MKK3b potently inhibited the stimulatory effect of Raf-1. Activation of p38 MAPK by arsenite also potently abrogated stimulation of MMP-1 gene expression by constitutively active Ras and Raf-1 and by phorbol ester. Specific activation of p38alpha by adenovirus-delivered constitutively active MKK3b resulted in potent inhibition of the activity of ERK1,2 and its upstream activator MEK1,2. Furthermore, arsenite prevented phorbol ester-induced phosphorylation of ERK1,2 kinase-MEK1,2, and this effect was dependent on p38-mediated activation of protein phosphatase 1 (PP1) and PP2A. These results provide evidence that activation of signaling cascade MKK3-MKK3b-->p38alpha blocks the ERK1,2 pathway at the level of MEK1,2 via PP1-PP2A and inhibits the activation of MMP-1 gene expression.     

ERK pathway positively regulates the expression of Sprouty genes

Sprouty was originally identified as an inhibitor of Drosophila development-associated receptor tyrosine kinase (RTK) signaling. Although RTK signaling has been shown to induce Sprouty gene expression, the precise induction pathway downstream of RTK remains unclear. As RTK signaling pathway includes activation of extracellular signal-regulated kinases (ERKs), we have examined a correlation between activation of ERKs and induction of Sprouty gene expression. All reagents which induce the activation of ERKs induce Sprouty gene expression; these agents include not only growth factors which bind to RTK but also phorbol 12-myristate-13-acetate and active Raf-1 kinase. Furthermore, the Sprouty gene expression induced by all those agents is totally suppressed when the cells are pretreated with specific inhibitors of ERK kinase (MEK). Human tumor cells which exhibit constitutive activation of ERKs show elevated expression of Sprouty genes, which is abolished by treatment of these cells with MEK inhibitors. All these findings clearly indicate that Sprouty gene expression is positively regulated by the ERK pathway downstream of RTK.     

Rap1A Regulates Osteoblastic Differentiation via the ERK and p38 Mediated Signaling

Rap1A is a member of small G proteins belonging to the Ras family. Recently, an integration of human genome-wide association studies (GWAS) and gene expression profiling study revealed that single-nucleotide polymorphisms (SNPs) within human Rap1A were strongly associated with narrow neck width in women. However, the regulatory role of Rap1A in osteoblasts remains to be elucidated. Here we report that Rap1A is a key regulator in osteoblast differentiation. Rap1A expression and activity were gradually enhanced during the induced differentiation of multipotent mesenchymal progenitor cells (C2C12) and preosteoblastic cells (MC3T3-E1). Knockdown of endogenous Rap1A significantly inhibited the osteogenic marker gene expression and matrix mineralization in cells with osteogenesis. In addition, knockdown of endogenous Rap1A suppressed the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), while overexpression of Rap1A accelerated osteoblast differentiation and enhanced the phosphorylation of ERK and p38. Taken together, our study suggests that Rap1A regulates osteoblast differentiation through modulating the ERK/p38 signaling.     

Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases.

Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocked NGF- and TPA-induced tyrosine phosphorylation of two proteins of approximately 42 and 44 kd. Conversely, expression of an oncogenic variant of Ras induced tyrosine phosphorylation of the same 42 and 44 kd proteins. The 44 kd protein was immunoprecipitated with an antibody directed against extracellular signal-regulated kinase 1/mitogen-activated protein kinase (MAPK) and the 42 kd protein comigrated with a 42 kd MAPK, indicating that at least one and probably both Ras-regulated phosphoproteins are MAPKs. In addition, MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, was also regulated by Ras. Ras was not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-gamma 1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs.     

Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation

Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. In this study, we provide a functional characterization of mammalian Sprouty1 and Sprouty2. Sprouty1 and Sprouty2 inhibited events downstream of multiple receptor tyrosine kinases and regulated both cell proliferation and differentiation. Using NIH3T3 cell lines conditionally expressing Sprouty1 or Sprouty2, we found that these proteins specifically inhibit the Ras/Raf/MAP kinase pathway by preventing Ras activation. In contrast, activation of the phosphatidylinositol 3-kinase pathway was not affected by Sprouty1 or Sprouty2. We further showed that Sprouty1 and Sprouty2 do no prevent the formation of a SNT.Grb2.Sos complex upon fibroblast growth factor stimulation, yet block Ras activation. Taken together, these results establish mammalian Sprouty proteins as important negative regulators of growth factor signaling and suggest that Sprouty proteins act downstream of the Grb2.Sos complex to selectively uncouple growth factor signals from Ras activation and the MAP Kinase pathway.     

Tyrosine phosphorylation of Sprouty proteins regulates their ability to inhibit growth factor signaling: a dual feedback loop

Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling.     

The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF--but not TSP-1--stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.     

Activation of calcium-dependent kinases and epidermal growth factor receptor regulate muscarinic acetylcholine receptor-mediated MAPK/ERK activation in thyroid epithelial cells

We previously showed that stimulation of muscarinic acetylcholine receptors (mAChR) by carbachol (Cch) caused a time- and dose-dependent increase of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) phosphorylation in thyroid epithelial cells. In this study, we demonstrated that mAChR stimulation also induced a time-dependent increase in the tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), which was prevented by pretreatment of thyroid epithelial cells with the specific Src-family tyrosine kinase inhibitor PP2. Besides, phosphorylation of Pyk2 was attenuated by chelation of extracellular Ca(2+) or inhibition of phospholipase C (PLC), and was evoked by thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor. Incorporation of Pyk2 antisense oligonucleotides in thyroid epithelial cells to down-regulated Pyk2 expression or pretreatment of cells with the Ca(2+)/calmodulin protein kinase II (CaM kinase II) inhibitor KN-62 significantly reduced Cch-induced MAPK/ERK phosphorylation. In addition, Cch-induced MAPK/ERK phosphorylation was partially inhibited by LY294002 and wortmannin, two selective inhibitors of phosphatidylinositol 3-kinase (PI3K), tyrphostin AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, and (-)-perillic acid, a post-translational inhibitor of small G-proteins isoprenylation. Taken together, our data suggest that Pyk2, CaM kinase II and Src-family tyrosine kinases are key molecules for the activation of MAPK/ERK cascade through the EGFR/Ras/Raf pathway in thyroid epithelial cells in response to mAChR stimulation.     

Glu-256 is a main structural determinant for oligomerisation of human arginase I

The role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2-SH3-SH2 domains of RasGAP suppressed Ras activity, extracellular signal-regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2-SH3-SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet-derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF-BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes involves RasGAP as a co-activator of Src to stimulate the Ras/mitogen-activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction.