Epidermal growth factor directs sex-specific steroid signaling through Src activation

Estrogens and androgens exert many biological effects that do not require interactions of their receptors with chromosomal DNA. However, it has been a long-standing question how the sex steroid receptors provoke signal transduction outside the nucleus. Here we have shown that epidermal growth factor (EGF) directs sex-specific steroid signaling through Src activation. We have revealed that estrogen (E2)-induced Src activation takes place in, not only plasma, but also endomembranes. This was found ascribed to the existence of EGF and the occurrence of EGF receptor (EGFR)-involved endocytosis of estrogen receptor together with Src. EGFR, estrogen receptor, and Src were found to form a complex upon E2 stimulation. The cell growth of breast cancer-derived MCF-7 cells was found to remarkably increase through the above EGF-involved estrogen-signaling process. In contrast, the androgen 5alpha-dihydrotestosterone-induced Src activation occurs only in the plasma membrane free from the interaction of EGFR with androgen receptor, irrespective of EGF. The cell growth occurred only moderately as a result. The spatial difference in Src activation between E2 and 5alpha-dihydrotestosterone may be responsible for the different extent of observed cell growth.     

Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase

In this study, we examined the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85alpha oligonucleotide or LY294002, a selective inhibitor of PI3-kinase, independently inhibited the formation of erythropoietin-dependent colonies. In K562 cells, Src associated with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin-induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3-kinase in F-36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine-phosphorylated erythropoietin receptor, and associated with PI3-kinase. In vitro binding experiments proved that glutathione S-transferase-p85alpha N- or C-terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine-phosphorylated by Src. Taken together, Src transduces the erythropoietin-induced erythroid differentiation signals by regulating PI3-kinase activity.     

Naturally occurring ether-linked phosphatidylcholine activates phosphatidylinositol 3-kinase and stimulates cell growth

Phosphatidylcholine (PC) from marine invertebrates is enriched in ether-linked forms. PCs from ray fish, Dasyatis sp., and bivalve, Macoma birmanica, used in the present study, contain 65% and 75% (w/w of total PC) of ether-linked forms, respectively. Ether-linked PCs also occur in mammalian membranes. Agonist-mediated hydrolysis of PC generates second messengers which participate in cellular responses. In this study, we tested whether PCs from marine invertebrates directly affect mammalian cell growth and activity of phosphatidylinositol (PI-3-kinase). PI-3-kinase participates in mitogenesis initiated by a variety of growth factors. PI-3-kinase converts polyphosphoinositides to 3′ phosphorylated isomers and these products accumulate in response to mitogenic stimuli. Whether cell membrane lipids regulate PI-3-kinase activity is not known. The marine animal–derived PCs and dioleoyl DAG (dioleoylglycerol) stimulated growth of murine pre-B lymphocytes, whereas chicken PC (egg lecithin) inhibited growth of these cells. Egg lecithin is also a potent inhibitor of PI-3-kinase activity in vitro. We studied the effect of PCs and DAG on PI-3-kinase activity. Unlike egg lecithin, marine animal PCs enhanced PI-3-kinase activity. We investigated the effect of lipids on PI-3-kinase substrate utilization. PCs enriched in ether-linked species increased utilization of substrates by PI-3-kinase. PCs purified from marine organisms also contain a substantially higher percentage of the cis-unsaturated fatty acids, especially of the ? ω3 series (25% and 30% of total fatty acids for Dasyatis sp. and Macoma birmanica, respectively), as compared to vertebrate sources. In spite of differences in fatty acid composition, marine PCs and dioleoyl DAG showed similar effects on cell growth and PI-3-kinase activity. These findings indicate that ether-linked phospholipids activate PI-3-kinase and may participate in mitogenic responses. © 1994 Wiley-Liss, Inc.     

Hepatocyte growth factor/scatter factor stimulates migration of rat mammary fibroblasts through both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways.

Hepatocyte growth factor/scatter factor (HGF/SF) is considered to be a mesenchymal-derived factor that acts via a dual system receptor, consisting of the MET receptor and proteoglycans present on adjacent epithelial cells. Surprisingly, HGS/SF stimulated the migration of rat mammary (Rama) 27 fibroblasts, although it failed to stimulate their proliferation. HGF/SF stimulated a transient activation of mitogen-activated protein kinases p44 and p42 (p42/44(MAPK)), with a maximum level of dual phosphorylation of p42/44(MAPK) occurring 10-15 min after the addition of the growth factor, which was followed by a rapid decrease to near basal levels after 20 min. Interestingly, a second phase of p42/44(MAPK) dual phosphorylation was observed at later times (3 h to 10 h). PD098059, a specific inhibitor of MEK-1, prevented the dual phosphorylation of p42/44(MAPK) and also the phosphorylation of p90(RSK) (ribosomal subunit S6 kinase), which mirrored the kinetics of p42/44(MAPK) phosphorylation. Moreover, PD098059 prevented the HGF/SF-induced migration of Rama 27 cells. HGF/SF also induced an early increase in the phosphorylation of protein kinase B/Akt. Akt phosphorylation was elevated 15 min after the addition of HGF/SF and then declined to basal levels by 30 min. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), prevented the increase in Akt phosphorylation and abolished HGF/SF-induced migration of fibroblasts. PD098059 also inhibited the stimulation of Akt phosphorylation by HGF/SF and wortmannin similarly inhibited the stimulation of p42/44(MAPK) dual phosphorylation. These results suggest that HGF/SF-induced motility depends on both the transient dual phosphorylation of p42/44(MAPK) and the activation of PtdIns3K in Rama 27 fibroblasts and that these pathways are mutually dependent.     

Hepatoma-derived growth factor stimulates podosome rosettes formation in NIH/3T3 cells through the activation of phosphatidylinositol 3-kinase/Akt pathway

Hepatoma-derived growth factor (HDGF) stimulates the migration, invasion and metastasis in several types of cancer cells. However, the mechanism underlying HDGF-stimulated migration remains unclear. In this study, we investigated the influence of HDGF on cytoskeleton remodeling and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in non-transformed NIH/3T3 cells. Exogenous HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression and Akt phosphorylation in dose- and time-dependent manners. Application of LY294002, a PI3K inhibitor, attenuated the HDGF-induced migration, dorsal ruffles and podosome rosettes formation. Consistently, the HDGF-overexpressing NIH/3T3 transfectants exhibited significantly increased motility and elevated PI3K/Akt activities, which were repressed by LY294002 or adenovirus-mediated overexpression of endogenous PI3K antagonist, PTEN. In summary, HDGF elicits the activation of PI3K/Akt signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration.     

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.     

Formyl peptide receptor-1 activation enhances intestinal epithelial cell restitution through phosphatidylinositol 3-kinase-dependent activation of Rac1 and Cdc42

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.     

Epidermal growth factor stimulates phosphatidylinositol turnover in human foreskin fibroblasts without activation of protein kinase C

Epidermal growth factor stimulates phosphatidylinositol turnover in human foreskin fibroblasts. This is a primary cell culture with normal numbers of epidermal growth factor receptors that is stimulated to divide by epidermal growth factor. Increases are seen in the inositol phospholipids and inositol phosphates. Despite this activation of phosphatidylinositol turnover, there is no detectable activation of protein kinase C.     

Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling

Members of the epidermal growth factor (EGF) family of ligands and their receptors regulate migration and growth of intestinal epithelial cells. However, our understanding of the signal transduction pathways determining these responses is incomplete. In this study we tested the hypothesis that p38 is required for EGF-stimulated intestinal epithelial monolayer restitution. EGF-stimulated migration in a wound closure model required continuous presence of ligand for several hours for maximal response, suggesting a requirement for sustained signal transduction pathway activation. In this regard, prolonged exposure of cells to EGF activated p38 for up to 5 h. Furthermore genetic or pharmacological blockade of p38 signaling inhibited the ability of EGF to accelerate wound closure. Interestingly p38 inhibition was associated with increased EGF-stimulated ERK1/ERK2 phosphorylation and cell proliferation, suggesting that p38 regulates the balance of proliferation/migration signaling in response to EGF receptor activity. Activation of p38 in intestinal epithelial cells through EGF receptor was abolished by blockade of Src family tyrosine kinase signaling but not inhibition of phosphatidylinositol 3-kinase or protein kinase C. Taken together, these data suggest that Src family kinase-dependent p38 activation is a key component of a signaling switch routing EGF-stimulated responses to epithelial cell migration/restitution rather than proliferation during wound closure.     

Insulin-like growth factor binding protein-3 stimulates phosphatidylinositol 3-kinase in MCF-7 breast carcinoma cells

Insulin-like growth factor binding protein-3 (IGFBP-3) is the most abundant IGFBP in serum and other biological fluids. Apart from its capacity for specific and high-affinity binding to IGFs, it also has so-called "IGF-independent" activities that modulate cell proliferation and survival/apoptosis. However, the molecular elements of the IGFBP-3 signalling pathway remain obscure. In this study, we investigated the possible implication of phosphatidylinositol 3-kinase (PI 3-kinase) activity in MCF-7 breast carcinoma cells. In cells incubated with IGFBP-3, both total and insulin receptor substrate-1 (IRS-1)-associated PI 3-kinase activities were rapidly stimulated, with maximal effects after 3 and 10min of incubation, respectively. IGFBP-3-induced PI 3-kinase activity was unaffected by the state of IRS-1 tyrosine phosphorylation. Since IGFBP-3 failed to stimulate PI 3-kinase activity in MDA-MB 231 breast carcinoma cells, its effects in MCF-7 cells could be considered as cell-type-specific. Pertussis toxin abolished IGFBP-3-stimulation of PI 3-kinase activity, suggesting that this IGFBP-3 signalling pathway depends upon a pertussis toxin-sensitive G protein. Our results provide further evidence that IGFBP-3 directly triggers a specific intracellular signal in MCF-7 cells.     

ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells

Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.     

ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor.

Conflicting results concerning the ability of the epidermal growth factor (EGF) receptor to associate with and/or activate phosphatidylinositol (PtdIns) 3-kinase have been published. Despite the ability of EGF to stimulate the production of PtdIns 3-kinase products and to cause the appearance of PtdIns 3-kinase activity in antiphosphotyrosine immunoprecipitates in several cell lines, we did not detect EGF-stimulated PtdIns 3-kinase activity in anti-EGF receptor immunoprecipitates. This result is consistent with the lack of a phosphorylated Tyr-X-X-Met motif, the p85 Src homology 2 (SH2) domain recognition sequence, in this receptor sequence. The EGF receptor homolog, ErbB2 protein, also lacks this motif. However, the ErbB3 protein has seven repeats of the Tyr-X-X-Met motif in the carboxy-terminal unique domain. Here we show that in A431 cells, which express both the EGF receptor and ErbB3, PtdIns 3-kinase coprecipitates with the ErbB3 protein (p180erbB3) in response to EGF. p180erbB3 is also shown to be tyrosine phosphorylated in response to EGF. In contrast, a different mechanism for the activation of PtdIns 3-kinase in response to EGF occurs in certain cells (PC12 and A549 cells). Thus, we show for the first time that ErbB3 can mediate EGF responses in cells expressing both ErbB3 and the EGF receptor.     

E-cadherin homophilic ligation directly signals through Rac and phosphatidylinositol 3-kinase to regulate adhesive contacts.

Classical cadherins mediate cell recognition and cohesion in many tissues of the body. It is increasingly apparent that dynamic cadherin contacts play key roles during morphogenesis and that a range of cell signals are activated as cells form contacts with one another. It has been difficult, however, to determine whether these signals represent direct downstream consequences of cadherin ligation or are juxtacrine signals that are activated when cadherin adhesion brings cell surfaces together but are not direct downstream targets of cadherin signaling. In this study, we used a functional cadherin ligand (hE/Fc) to directly test whether E-cadherin ligation regulates phosphatidylinositol 3-kinase (PI 3-kinase) and Rac signaling. We report that homophilic cadherin ligation recruits Rac to nascent adhesive contacts and specifically stimulates Rac signaling. Adhesion to hE/Fc also recruits PI 3-kinase to the cadherin complex, leading to the production of phosphatidylinositol 3,4,5-trisphosphate in nascent cadherin contacts. Rac activation involved an early phase, which was PI 3-kinase-independent, and a later amplification phase, which was inhibited by wortmannin. PI 3-kinase and Rac activity were necessary for productive adhesive contacts to form following initial homophilic ligation. We conclude that E-cadherin is a cellular receptor that is activated upon homophilic ligation to signal through PI 3-kinase and Rac. We propose that a key function of these cadherin-activated signals is to control adhesive contacts, probably via regulation of the actin cytoskeleton, which ultimately serves to mediate adhesive cell-cell recognition.     

Insulin-like growth factor I-stimulated melanoma cell migration requires phosphoinositide 3-kinase but not extracellular-regulated kinase activation

Dysregulated signaling contributes to altered cellular growth, motility, and survival during cancer progression. We have evaluated the ability of several factors to stimulate migration in WM1341D, a cell line derived from an invasive human vertical growth phase melanoma. Basic fibroblast growth factor, hepatocyte growth factor, interleukin-8, and CCL27 each slightly increased migration. Insulin-like growth factor I (IGF-I), however, stimulated a 15-fold increase in migration. This response required the IGF-I receptor, which activates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. Both pathways have been implicated in migration in a variety of cell types, but the signaling required for IGF-I-induced melanoma cell migration is not well defined. IGF-I-stimulated activation of MAPK/ERK signaling in WM1341D cells was inhibited by U0126, but a 33-fold higher dose of U0126 was needed to inhibit IGF-I-stimulated cellular migration. In contrast, similar concentrations of either wortmannin or LY294002 were required to inhibit both IGF-I-induced PI3K activation and migration. These results indicate that IGF-I-stimulated migration of WM1341D cells requires PI3K activation but is independent of MAPK/ERK signaling. Determining the contributions of IGF-I signaling pathways to migration will help us to understand melanoma progression and may lead to new therapeutic targets of this highly metastatic cancer.     

Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.     

Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration.

Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a "scattering" response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.