Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation

Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.     

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.     

AZU-1: a candidate breast tumor suppressor and biomarker for tumor progression

To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.     

Macrophage-colony-stimulating factor-induced activation of extracellular-regulated kinase involves phosphatidylinositol 3-kinase and reactive oxygen species in human monocytes

We previously reported that activation of the phosphatidylinositol (PI) 3-kinase pathway was important in M-CSF-induced monocyte survival. Because M-CSF also induces activation of the mitogen-activated protein (MAP) kinase extracellular-regulated kinase (Erk), we focused on dissecting the mechanism used by M-CSF to induce Erk activation in human monocytes. We found that, in addition to the MAP/Erk kinase inhibitor PD098059, the PI 3-kinase inhibitors LY294002 and wortmannin both suppressed Erk activation in M-CSF-treated monocytes, suggesting that 3-phosphorylated products of PI 3-kinase played a role in Erk activation. Investigating the biochemical pathways regulated by PI 3-kinase to activate Erk, we found that, in response to M-CSF, normal human monocytes induced reactive oxygen species (ROS), which were suppressed by the PI 3-kinase inhibitor wortmannin but not by the solvent control DMSO or the MAP/Erk kinase inhibitor PD098059. We next found that, in the absence of M-CSF, ROS could induce Erk activation in human monocytes. Exogenous H(2)O(2) induced Erk activation in human monocytes, which was suppressed by exogenous catalase. To determine whether ROS induced by M-CSF played a role in Erk activation, we found that N-acetylcysteine and diphenyleneiodonium both suppressed Erk activation in M-CSF-treated monocytes. Erk activation by M-CSF also seemed to play a role in cellular survival in monocytes. These data suggest that, in M-CSF-stimulated human monocytes, PI 3-kinase products and ROS production play a role in Erk activation and monocyte survival.     

MKP3 negatively modulates PDGF-induced Akt and Erk5 phosphorylation as well as chemotaxis

MAP kinase phosphatase-3 (MKP3), also known as DUSP6 or Pyst1, is a dual specificity phosphatase considered to selectively dephosphorylate extracellular-signal-regulated kinase 1/2 (Erk1/2). Here, we report that in NIH3T3 cells, MKP3 is induced in response to platelet-derived growth factor (PDGF)-BB treatment in an Erk1/2- and phosphatidylinositol 3-kinase (PI3K)-dependent manner, but independently of Erk5 expression. Silencing of MKP3 expression did not affect PDGF-BB-induced Erk1/2 or p38 phosphorylation; however, their basal level of phosphorylation was elevated. Furthermore, we found that PDGF-BB-mediated activation of Erk5 and Akt was enhanced when the MKP3 expression was reduced. Interfering with Mek1/2 or PI3K using the inhibitors CI-1040 and LY-294002, respectively, inhibited PDGF-BB-induced MKP3 expression. Functionally, we found that MKP3 silencing did not affect cell proliferation, but enhanced the chemotactic response toward PDGF-BB. Although both Akt and Erk5 have been linked to increased cell survival, downregulation of MKP3 did not alter the ability of PDGF-BB to protect NIH3T3 cells from starvation-induced apoptosis. However, we observed an increased apoptosis in untreated cells with reduced MKP3 expression. In summary, our data indicate that there is negative cross-talk between Erk1/2 and Erk5 that involves regulation of MKP3 expression, and that PI3K in addition to promoting Akt phosphorylation also negatively modulates Akt, through MKP3 expression.     

Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G_i-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.     

Modulation of Src Homology 3 Proteins by the Proline-Rich Adaptor Protein Shb

In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.     

Analysis of protein expression during oxidative stress in breast epithelial cells using a stable isotope labeled proteome internal standard

Normal cells undergo a variety of molecular and physiological changes upon malignant transformation, including their responses to environmental factors that induce oxidative stress. Understanding the molecular pathways regulating these changes would facilitate the development of novel cancer treatments and chemoprevention strategies. Differences in the oxidative stress response were investigated between nonmalignant (S-1) and malignant (T4-2) cell lines (both derived from the HMT-3522 breast epithelial cells) using proteomic approaches. A modification of the stable isotope labeling of amino acids in cell culture (SILAC) approach was employed in which a [(13)C,(15)N]-labeled proteome was prepared from both cells. Relative quantification of the proteome derived from the S-1 cells and the T4-2 cells was then conducted using a [(13)C,(15)N]-labeled proteome as the internal standard. Differentially expressed proteins that changed in a similar manner in both cell lines were mainly stress response proteins, including heat shock proteins, peroxiredoxins, and redox proteins. Proteins that showed significant change in expression level in only one the cell lines included cytoskeleton proteins and proteins implicated in cell cycle and apoptosis regulation. Fortilin was found to be significantly up regulated in the transformed T4-2 cells after H(2)O(2) treatment but not in the parental S-1 cells. However, Ran/TC4 was up regulated by H(2)O(2) in the nonmalignant breast epithelial cells but not in the malignant cells. These results suggest that the malignant T4-2 cells have acquired more resistance to H(2)O(2)-induced apoptosis than the nonmalignant S-1 cells.     

Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts.

Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have developed a sensitive method to measure PI 3-kinase activity in diluted, detergent-containing whole cell extracts and used this method to determine total, soluble, and membrane-associated PI 3-kinase activity in PDGF-stimulated NIH 3T3 fibroblasts. PDGF stimulation induced a 1.4-fold increase in total Nonidet P-40-extractable PI 3-kinase activity, which occurred within 1 min and was maintained above basal levels at 10 min. At the same time, PI 3-kinase activity in the soluble fraction decreased 30-50%. However, membrane-bound PI 3-kinase activity increased 2.4-fold at 1 min and 3.1-fold at 5 min. Translocation of the p85 PI 3-kinase subunit to the membrane was maximal at 10 min. These results suggest that PDGF-mediated activation of PI 3-kinase in membrane fraction results from initial intrinsic enzymatic activation followed by translocation from the cytosol.     

Role of Phosphoinositide 3-Kinase in Activation of Ras and Mitogen-Activated Protein Kinase by Epidermal Growth Factor

The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances. Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2. However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110alpha is by itself never sufficient to activate Ras or ERK2. PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation. The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase. The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system.     

Activation of host cell phosphatidylinositol 3-kinases by Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas' disease in humans, is an intracellular protozoan parasite with the ability to invade a wide variety of mammalian cells by a unique and remarkable process in cell biology that is poorly understood. Here we present evidence suggesting a role for the host phosphatidylinositol (PI) 3-kinases during T. cruzi invasion. The PI 3-kinase inhibitor wortmannin marked inhibited T. cruzi infection when macrophages were pretreated for 20 min at 37 degrees C before inoculation. Infection of macrophages with T. cruzi markedly stimulated the formation of the lipid products of the phosphatidylinositol (PI) 3-kinases, PI 3-phospate, PI 3,4-biphosphate, and PI 3,4,5-triphosphate, but not PI 4-phosphate or PI 4,5-biphosphate. This activation was inhibited by wortmannin. Infection with T. cruzi also stimulated a marked increase in the in vitro lipid kinase activities that are present in the immunoprecipitates of anti-p85 subunit of class I PI 3-kinase and anti-phosphotyrosine. In addition, T. cruzi invasion also activated lipid kinase activity found in immunoprecipitates of class II and class III PI 3-kinases. These data demonstrate that T. cruzi invasion into macrophages strongly activates separated PI 3-kinase isoforms. Furthermore, the inhibition of the class I and class III PI 3-kinase activities abolishes the parasite entry into macrophages. These findings suggest a prominent role for the host PI 3-kinase activities during the T. cruzi infection process.     

Interleukin-1β and cyclic AMP mediate the invasion of sheared chondrosarcoma cells via a matrix metalloproteinase-1-dependent mechanism

Matrix metalloproteinase-1 (MMP-1) is a potential biomarker for chondrosarcoma that is overexpressed at the invading edges of articular cartilage, and its expression correlates with poor survival rates. However, the molecular mechanisms of MMP-1 regulation and its potential contribution to chondrosarcoma cell invasion have yet to be elucidated, especially in shear-activated cells. Using molecular biology tools and an in vitro fluid shear model, we report that shear stress upregulates cyclic AMP (cAMP) and interleukin-1β (IL-1β) release, which in turn promotes the invasion of chondrosarcoma cells via the induction of MMP-1 in a phosphoinositide 3-kinase (PI3-K)- and ERK1/2-dependent manner. Activated PI3-K and ERK1/2 signaling pathways phosphorylate c-Jun, which in turn transactivates MMP-1 in human chondrosarcoma cells. Collectively, fluid shear stress upregulates matrix MMP-1 expression, which is responsible for the enhanced invasion of human chondrosarcoma cells.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS

In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE(1) in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE(1) induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE(1)-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE(1)-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the G(s) protein.     

Novel role of Giα2 in cell migration: Downstream of PI3-kinase–AKT and Rac1 in prostate cancer cells

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor β1 (TGFβ1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFβ1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase–AKT–Rac1 axis.     

The Actin Binding Domain of the Epidermal Growth Factor Receptor Is Required for EGF-Stimulated Tissue Invasion

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.     

EGF promotes invasion by PANC-1 cells through Rac1/ROS-dependent secretion and activation of MMP-2

Cancer metastasis involves tumor cells invading the surrounding tissue. Remodeling of tissue barriers depends on the ability of tumor cells to degrade the surrounding collagen matrix and then migrate through the matrix defects. Epidermal growth factor (EGF) has been shown to regulate tumor cell invasion through activation of matrix metalloproteinase-2 (MMP-2) in various tumor cell types. In the present study, we investigated the role of MMP-2 and the signaling pathway involved in EGF-promoted invasion by human pancreatic cancer cells PANC-1. Using specific inhibitors, we found that EGF stimulation of these tumor cells induced secretion and activation of the collagenase MMP-2, which was required for EGF-stimulated basement membrane degradation and cell invasion. Our results also indicate that signaling events downstream of EGF receptor involved PI3K- and Src-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-2 secretion and activation.