Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the ¹⁰⁶PNTP¹⁰⁹ motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, ¹⁸³KKRKRKCLLL¹⁹² (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1''s role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.     

Phosphorylation of KIBRA by the extracellular signal-regulated kinase (ERK)–ribosomal S6 kinase (RSK) cascade modulates cell proliferation and migration

In mammals, KIBRA is defined as a memory performance-associated protein. The physiological function and regulation of KIBRA in non-neuronal cells are much less understood. Recent studies have identified KIBRA as a novel regulator of the Hippo signaling pathway, which plays a critical role in tumorigenesis by inhibiting cell proliferation and promoting apoptosis. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora and cyclin-dependent kinase 1 during mitosis. In this current study, we show that KIBRA is also phosphorylated by the ERK (extracellular signal-regulated kinases)–RSK (p90 ribosomal S6 kinases) cascade. We demonstrated that ERK1/2 phosphorylate KIBRA at Ser⁵⁴⁸ in cells as well as in vitro. Moreover, we found that RSK1/2 specifically phosphorylates KIBRA at two highly conserved sites (Thr⁹²⁹ and Ser⁹⁴⁷) in vitro and in cells. RSK-mediated phosphorylation is required for KIBRA binding to RSK1, but not RSK2. Surprisingly, KIBRA knockdown impaired cell migration and proliferation in breast cancer cells. By using inducible-expression cell lines, we further show that phospho-regulation of KIBRA by ERK1/2 and RSK1/2 is required for proper cell proliferation and RSK-mediated phosphorylation also modulates KIBRA's migratory activity in MDA-MB-231 breast cancer cells. Our findings uncover unexpected results and a new mechanism through which KIBRA regulates cell migration and proliferation.     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

Glial cell‐derived neurotrophic factor increases migration of human chondrosarcoma cells via ERK and NF‐κB pathways

Invasion of tumor cells is the primary cause of therapeutic failure in the treatment of malignant chondrosarcomas. Glial cell‐derived neurotrophic factor (GDNF) plays a crucial role in migration and metastasis of human cancer cells. Integrins are the major adhesive molecules in mammalian cells. Here we found that GDNF directed the migration and increased cell surface expression of αv and β3 integrin in human chondrosarcoma cells. Pretreated of JJ012 cells with MAPK kinase (MEK) inhibitors PD98059 or U0126 inhibited the GDNF‐mediated migration and integrin expression. Stimulation of cells with GDNF increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited GDNF‐mediated cells migration and integrin up‐regulation. Stimulation of cells with GDNF induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the GDNF‐mediated increasing of κB‐luciferase activity was inhibited by PD98059, U0126, PDTC and TPCK or MEK, ERK, IKKα, and IKKβ mutants. Taken together, these results suggest that the GDNF acts through MEK/ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrin and contributing the migration of human chondrosarcoma cells. J. Cell. Physiol. 220: 499–507, 2009. © 2009 Wiley‐Liss, Inc.     

Dieckol from Ecklonia cava suppresses the migration and invasion of HT1080 cells by inhibiting the focal adhesion kinase pathway downstream of Rac1-ROS signaling

We have previously isolated dieckol, a nutrient polyphenol compound, from the brown alga, Ecklonia cava (Lee et al., 2010a). Dieckol shows both antitumor and antioxidant activity and thus is of special interest for the development of chemopreventive and chemotherapeutic agents against cancer. However, the mechanism by which dieckol exerts its antitumor activity is poorly understood. Here, we show that dieckol, derived from E. cava, inhibits migration and invasion of HT1080 cells by scavenging intracellular reactive oxygen species (ROS). H₂O₂ or integrin signal-mediated ROS generation increases migration and invasion of HT1080 cells, which correlates with Rac1 activation and increased expression and phosphorylation of focal adhesion kinase (FAK). Rac1 activation is required for ROS generation. Depletion of FAK by siRNA suppresses Rac1-ROS-induced cell migration and invasion. Dieckol treatment attenuated intracellular ROS levels and activation of Rac1 as well as expression and phosphorylation of FAK. Dieckol treatment also decreases complex formation of FAK-Src-p130Cas and expression of MMP2, 9, and 13. These results suggest that the Rac1-ROS-linked cascade enhances migration and invasion of HT1080 cells by inducing expression of MMPs through activation of the FAK signaling pathway, whereas dieckol downregulates FAK signaling through scavenging intracellular ROS. This finding provides new insights into the mechanisms by which dieckol is able to suppress human cancer progresssion and metastasis. Therefore, we suggest that dieckol is a potential therapeutic agent for cancer treatment.     

ADP Signaling in Vascular Endothelial Cells: ADP-DEPENDENT ACTIVATION OF THE ENDOTHELIAL ISOFORM OF NITRIC-OXIDE SYNTHASE REQUIRES THE EXPRESSION BUT NOT THE KINASE ACTIVITY OF AMP-ACTIVATED PROTEIN KINASE*

ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser¹¹⁷⁹ and Ser⁶³⁵ and dephosphorylation at Ser¹¹⁶ in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y₁ receptor antagonist MRS 2179 or by knockdown of P2Y₁ using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser¹¹⁷⁹ and Ser⁶³⁵ phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-β (CaMKKβ) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser⁶³⁵ phosphorylation and eNOS activity but did not affect eNOS Ser¹¹⁷⁹ phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKβ knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKβ kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKβ is necessary for ADP signaling to eNOS.     

Down-regulation of Sprouty2 in non-small cell lung cancer contributes to tumor malignancy via extracellular signal-regulated kinase pathway-dependent and -independent mechanisms

Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase signaling mainly by interfering with the Ras/Raf/mitogen-activated protein kinase cascade, a pathway known to be frequently deregulated in human non-small cell lung cancer (NSCLC). In this study, we show a consistently lowered Spry2 expression in NSCLC when compared with the corresponding normal lung epithelium. Based on these findings, we investigated the influence of Spry2 expression on the malignant phenotype of NSCLC cells. Ectopic expression of Spry2 antagonized mitogen-activated protein kinase activity and inhibited cell migration in cell lines homozygous for K-Ras wild type, whereas in NSCLC cells expressing mutated K-Ras, Spry2 failed to diminish extracellular signal-regulated kinase (ERK) phosphorylation. Nonetheless, Spry2 significantly reduced cell proliferation in all investigated cell lines and blocked tumor formation in mice. Accordingly, a Spry2 mutant unable to inhibit ERK phosphorylation reduced cell proliferation significantly but less pronounced compared with the wild-type protein. Therefore, we conclude that Spry2 interferes with ERK phosphorylation and another yet unidentified pathway. Our results suggest that Spry2 plays a role as tumor suppressor in NSCLC by antagonizing receptor tyrosine kinase-induced signaling at different levels, indicating feasibility for the usage of Spry in targeted gene therapy of NSCLC.     

A novel domain of caveolin‐2 that controls nuclear targeting: regulation of insulin‐specific ERK activation and nuclear translocation by caveolin‐2

Herein, we report that insulin‐activated extracellular signal‐regulated kinase (ERK) is translocated to the nuclear envelope by caveolin‐2 (cav‐2) and associates with lamin A/C in the inner nuclear membrane in response to insulin. We identified that the Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain on the C‐terminal of cav‐2 is essential for insulin‐induced phosphorylation and nuclear targeting of ERK and cav‐2. In human embryonic kidney 293T cells, ERK was not activated and translocated to the nucleus by insulin in comparison to insulin‐like growth factor‐1 (IGF‐1). However, insulin‐stimulated activation of ERK was induced by exogenous addition of cav‐2. The activated ERK associated and translocated with the cav‐2 to the nucleus. In turn, cav‐2 promoted phospho‐ERK interaction with lamin A/C in the inner nuclear membrane. In contrast, ERK, but not cav‐2, was phosphorylated and translocated to the nucleus by IGF‐1. The nuclear targeted phospho‐ERK failed to localize in the nuclear envelope in response to IGF‐1. Together, our data demonstrate that translocation of phospho‐ERK to the nuclear envelope is mediated by Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain of cav‐2 and this event is an insulin‐specific action.     

Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc     

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF‐1 and CXCR4 (SDF‐1 receptor). Treatment of osteosarcoma cells with SDF‐1α increased the migration and cell surface expression of αvβ3 integrin. CXCR4‐neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF‐1α‐induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF‐1α‐mediated migration and integrin expression. Stimulation of cells with SDF‐1α increased the phosphorylation of MEK and extracellular signal‐regulating kinase (ERK). In addition, NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) also inhibited SDF‐1α‐mediated cell migration and integrin up‐regulation. Stimulation of cells with SDF‐1α induced IκB kinase (IKKα/β) phosphorylation, IκB phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. Furthermore, the SDF‐1α‐mediated increasing κB‐luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKα and IKKβ mutants. Taken together, these results suggest that the SDF‐1α acts through CXCR4 to activate MEK and ERK, which in turn activates IKKα/β and NF‐κB, resulting in the activations of αvβ3 integrins and contributing the migration of human osteosarcoma cells. J. Cell. Physiol. 221: 204–212, 2009. © 2009 Wiley‐Liss, Inc     

Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway

Curcumin, a natural and crystalline compound isolated from the plant Curcuma longa with low toxicity in normal cells, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about antimetastasis effects and mechanism of curcumin in lung cancer. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis. In this study, we examined the influence of curcumin on in vitro invasiveness of human lung cancer cells and the expressions of Rac1. The results indicate that curcumin at 10 μM slightly reduced the proliferation of 801D lung cancer cells but showed an obvious inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. Meanwhile, we demonstrated that the suppression of invasiveness correlated with inhibition of Rac1/PAK1 signaling pathways and matrix metalloproteinase (MMP) 2 and 9 protein expression by combining curcumin treatment with the methods of Rac1 gene silence and overexpression in lung cancer cells. Laser confocal microscope also showed that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. At last, through xenograft experiments, we confirmed the connection between Rac1 and the growth and metastasis inhibitory effect of curcumin in vivo. In summary, these data demonstrated that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signaling pathway and MMP-2 and MMP-9 expression, which provided a novel insight into the molecular mechanism of curcumin against lung cancer.     

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC‐1 cells

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c‐Jun N‐terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC‐1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC‐1 clones that express wild‐type (WT) or kinase‐dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone‐A (PonA). NT potently stimulated c‐Jun Ser⁶³ phosphorylation in both wild type and clonal derivatives of PANC‐1 cells. PonA‐induced expression of WT, but not K618N PKD1, rapidly blocked NT‐mediated c‐Jun Ser⁶³ phosphorylation either at the level of or upstream of MKK4, a dual‐specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT‐induced JNK/cJun activation in PANC‐1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT‐induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro‐mitogenicERK and pro‐apopotic JNK/c‐Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC‐1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC‐1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly‐(HEMA)]‐coated dishes to eliminate cell adhesion (anchorage‐independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1–10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC‐1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells. J. Cell. Physiol. 223: 309–316, 2010. © 2010 Wiley‐Liss, Inc.     

Targeting EP4 downstream c‐Jun through ERK1/2‐mediated reduction of DNMT1 reveals novel mechanism of solamargine‐inhibited growth of lung cancer cells

Lung cancer is the most common cancer and the leading cause of cancer deaths worldwide. We previously showed that solamargine, one natural phytochemicals from traditional plants, inhibited the growth of lung cancer cells through inhibition of prostaglandin E2 (PGE₂) receptor EP4. However, the potential downstream effectors of EP4 involving in the anti‐lung cancer effects of solamargine still remained to be determined. In this study, we further verified that solamargine inhibited growth of non‐small‐cell lung cancer (NSCLC) cells in multiple cell lines. Mechanistically, solamargine increased phosphorylation of ERK1/2. Moreover, solamargine inhibited the protein expression of DNA methyltransferase 1 (DNMT1) and c‐Jun, which were abrogated in cells treated with MEK/ERK1/2 inhibitor (PD98059) and transfected with exogenously expressed DNMT1 gene, respectively. Interestingly, overexpressed DNMT1 gene antagonized the effect of solamargine on c‐Jun protein expression. Intriguingly, overexpressed c‐Jun blocked solamargine‐inhibited lung cancer cell growth, and feedback resisted the solamargine‐induced phosphorylation of ERK1/2. A nude mouse xenograft model implanted with lung cancer cells in vivo confirmed the results in vitro. Collectively, our results show that solamargine inhibits the growth of human lung cancer cells through reduction of EP4 protein expression, followed by increasing ERK1/2 phosphorylation. This results in decrease in DNMT1 and c‐Jun protein expressions. The inter‐correlations between EP4, DNMT1 and c‐Jun and feedback regulation of ERK1/2 by c‐Jun contribute to the overall responses of solamargine in this process. This study uncovers an additional novel mechanism by which solamargine inhibits growth of human lung cancer cells.     

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells

Background

Methyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration.

Methods

MG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence.

Results

Treatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser⁸³ and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca^2 + and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor.

General significance

Our results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.     

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGFβ-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.     

Cross‐talk between MAP kinase pathways is involved in IGF‐independent,IGFBP‐6‐induced Rh30 rhabdomyosarcoma cell migration

Insulin‐like growth factor binding protein‐6 (IGFBP‐6) inhibits the tumorigenic properties of IGF‐II‐dependent cancer cells by directly inhibiting IGF‐II actions. However, in some cases, IGFBP‐6 is associated with increased cancer cell tumorigenicity, which is unlikely to be due to IGF‐II inhibition. The mechanisms underlying the contradictory actions of IGFBP‐6 remain unclear. We recently generated an IGFBP‐6 mutant that does not bind IGFs (mIGFBP‐6) to address this issue. Although RD rhabdomyosarcoma cells express IGF‐II, we previously showed that mIGFBP‐6 promoted migration through an IGF‐independent, p38‐dependent pathway. We further studied the role of MAP kinases in IGFBP‐6‐induced migration of Rh30 rhabdomyosarcoma cells, which also express IGF‐II. In these cells, mIGFBP‐6 induced chemotaxis rather than chemokinesis. Both wild‐type (wt) and mIGFBP‐6 transiently induced phosphorylation of ERK1/2 and JNK1, but not p38. Inhibition of ERK1/2 phosphorylation completely prevented mIGFBP‐6‐induced ERK1/2 activation and cell migration, whereas a JNK inhibitor partially prevented migration. Interestingly, p38 pathway inhibition completely prevented mIGFBP‐6‐induced ERK1/2 and JNK1 activation and migration despite mIGFBP‐6 not activating p38. Furthermore, blocking the ERK1/2 pathway also inhibited mIGFBP‐6‐induced JNK1 activation. In contrast, IGFBP‐6 had no effect on Akt phosphorylation and an Akt inhibitor had no effect on migration. These results indicate that IGFBP‐6 promotes Rh30 rhabdomyosarcoma chemotaxis in an IGF‐independent manner, and that MAPK signaling pathways and their cross‐talk play an important role in this process. Therefore, besides decreasing Rh30 cell proliferation by inhibiting IGF‐II, IGFBP‐6 promotes their migration via a distinct pathway. Understanding these disparate actions of IGFBP‐6 may lead to the development of novel cancer therapeutics. J. Cell. Physiol. 224: 636–643, 2010. © 2010 Wiley‐Liss, Inc.     

Anti-migratory and anti-invasive effect of somatostatin in human neuroblastoma cells: involvement of Rac and MAP kinase activity

Cell motility and invasion are crucial events for the spread of cancer and, consequently, the metastatic process. Platelet-derived growth factor (PDGF) is not only capable of stimulating the proliferation of SH-SY5Y human neuroblastoma cells, but also their migration and invasion through an extracellular matrix barrier. Experiments using wortmannin and PD98059, specific inhibitors of the phosphatidylinositol 3-kinase (PI3-K) and of the mitogen-activated protein kinases (ERK 1 and 2) signaling, respectively, show that the activation of both pathways is required for the PDGF-induced cell motility responses. We have previously shown that somatostatin inhibits cell division and ERK 1/2 and Ras activity in SH-SY5Y cells. We report here that it is also capable of potently and effectively inhibiting their PDGF-stimulated migration and invasion. The inhibitory effect of somatostatin is sensitive to pertussis toxin. Although somatostatin does not affect PI3-K, it inhibits ERK 1/2 and the small G-protein Rac activation and ruffle formation induced by PDGF. These results indicate that somatostatin can be considered an anti-migratory and anti-invasive agent that acts by inhibiting ERK 1/2 signaling and the PI3-K pathway via the inhibition of Rac in SHSY5Y cells.     

Phosphorylation of Cdc42 Effector Protein-4 (CEP4) by Protein Kinase C Promotes Motility of Human Breast Cells

Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser¹⁸, Ser⁷⁷, Ser⁸⁰, and Ser⁸⁶) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser¹⁸ and Ser⁸⁰ were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser¹⁸ and Ser⁸⁰ causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.     

Estrogen‐related receptor alpha triggers the proliferation and migration of human non‐small cell lung cancer via interleukin‐6

Human non‐small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths worldwide. Estrogenic signals have been suggested to be important for the growth and metastasis of NSCLC cells. Our present data showed that estrogen‐related receptor alpha (ERRα), while not ERRβ or ERRγ, was significantly elevated in NSCLC cell lines as compared with that in normal bronchial epithelial cell line BEAS‐2B. The expression of ERRα in clinical NSCLC tissues was significantly greater than that in their matched normal adjacent tissues. Over expression of ERRα can trigger the proliferation, migration, and invasion of NSCLC cells, while si‐ERRα or ERRα inhibitor showed opposite effects. ERRα can increase the mRNA and protein expression of IL‐6, while not IL‐8, IL‐10, IL‐22, VEGF, TGF‐β, or TNF‐α, in NSCLC cells. Silence of IL‐6 attenuated ERRα induced proliferation and cell invasion. Furthermore, our data revealed the inhibition of NF‐κB, while not ERK1/2 or PI3K/Akt, abolished ERRα induced production of IL‐6. This might be due to that overexpression of ERRα can increase the expression and nuclear translocation of p65 in NSCLC cells. Collectively, our data showed that activation of NF‐κB/IL‐6 is involved in ERRα induced migration and invasion of NSCLC cells. It suggested that ERRα might be a potential target for NSCLC treatment.