Src tyrosine kinase inhibitor PP2 suppresses ERK1/2 activation and epidermal growth factor receptor transactivation by X-irradiation

Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.     

Matrix metalloproteinases 2 and 9 mediate epidermal growth factor receptor transactivation by gonadotropin-releasing hormone

Rapid engagement of the extracellular signal-regulated kinase (ERK) cascade via the Gq/11-coupled GnRH receptor (GnRHR) is mediated by transactivation of the epidermal growth factor receptor (EGFR). Here we show that the cross-talk between GnRHR and EGFR in gonadotropic cells is accomplished via gelatinases A and B (matrix metalloproteinases (MMPs) 2 and 9), identifying gelatinases as the first distinct members of the MMP family mediating EGFR transactivation by G protein-coupled receptors. Using a specific MMP2 and MMP9 inhibitor, Ro28-2653, GnRH-dependent EGFR transactivation was abrogated. Proving the specificity of the effect, transient transfection of alphaT3-1 cells with ribozymes directed against MMP2 or MMP9 specifically blocked EGFR tyrosine phosphorylation in response to GnRH stimulation. GnRH challenge of alphaT3-1 cells furthered the release of active MMP2 and MMP9 and increased their gelatinolytic activities within 5 min. Rapid release of activated MMP2 or MMP9 was inhibited by ribozyme-targeted down-regulation of MT1-MMP or MMP2, respectively. We found that GnRH-induced Src, Ras, and ERK activation were also gelatinase-dependent. Thus, gelatinase-induced EGFR transactivation was required to engage the extracellular-signal regulated kinase cascade. Activation of c-Jun N-terminal kinase and p38 MAPK by GnRH was unaffected by EGFR or gelatinase inhibition that, however, suppressed GnRH induction of c-Jun and c-Fos. Our findings suggest a novel role for gelatinases in the endocrine regulation of pituitary gonadotropes.     

mu-Opioid receptor-mediated ERK activation involves calmodulin-dependent epidermal growth factor receptor transactivation.

Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors involves multiple signaling pathways. One of these pathways entails growth factor receptor transactivation followed by ERK activation. This study demonstrates that a similar signaling pathway is used by the mu-opioid receptor (MOR) expressed in HEK293 cells and involves calmodulin (CaM). Stimulation of MOR resulted in both epidermal growth factor receptor (EGFR) and ERK phosphorylation. Data obtained with inhibitors of EGFR Tyr kinase and membrane metalloproteases support an intermediate role of EGFR activation, involving release of endogenous membrane-bound epidermal growth factor. Previous studies had demonstrated a role for CaM in opioid signaling based on direct CaM binding to MOR. To test whether CaM contributes to EGFR transactivation and ERK phosphorylation by MOR, we compared wild-type MOR with mutant K273A MOR, which binds CaM poorly, but couples normally to G proteins. Stimulation of K273A MOR with [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin (10-100 nm) resulted in significantly reduced ERK phosphorylation. Furthermore, wild-type MOR stimulated EGFR Tyr phosphorylation 3-fold more than K273A MOR, indicating that direct CaM-MOR interaction plays a key role in the transactivation process. Inhibitors of CaM and protein kinase C also attenuated [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin-induced EGFR transactivation in wild-type (but not mutant) MOR-expressing cells. This novel pathway of EGFR transactivation may be shared by other G protein-coupled receptors shown to interact with CaM.     

Maximal beta3-adrenergic regulation of lipolysis involves Src and epidermal growth factor receptor-dependent ERK1/2 activation

Catecholamine-stimulated lipolysis is primarily a beta-adrenergic and cAMP-dependent event. In previous studies we established that the beta(3)-adrenergic receptor (beta(3)AR) in adipocytes utilizes a unique mechanism to stimulate extracellular signal-regulated kinases 1 and 2 (ERK) by direct recruitment and activation of Src kinase. Therefore, we investigated the role of the ERK pathway in adipocyte metabolism and found that the beta(3)AR agonist CL316,243 regulates lipolysis through both cAMP-dependent protein kinase (PKA) and ERK. Inhibition of PKA activity completely eliminated lipolysis at low (subnanomolar) CL316,243 concentrations and by 75-80% at higher nanomolar concentrations. The remaining 20-25% of PKA-independent lipolysis, as well as ERK activation, was abolished by inhibiting the activity of either Src (PP2 or small interfering RNA), epidermal growth factor receptor (EGFR with AG1478 or small interfering RNA), or mitogen-activated protein kinase kinase 1 or 2 (MKK1/2 with PD098059). PD098059 inhibited lipolysis by 53% in mice as well. Finally, the effect of estradiol, a reported acute activator of ERK and lipolysis, was also totally prevented by PP2, AG1478, and PD098059. These results suggest that ERK activation by beta(3)AR depends upon Src and epidermal growth factor receptor kinase activities and is responsible for the PKA-independent portion of the lipolytic response. Together these results illustrate the distinct and complementary roles for PKA and ERK in catecholamine-stimulated lipolysis.     

Roles of Gab1 and SHP2 in paxillin tyrosine dephosphorylation and Src activation in response to epidermal growth factor

Epidermal growth factor (EGF) induces paxillin tyrosine dephosphorylation and Src activation, but the signaling pathways that mediate these responses were largely undefined. We found that Gab1, a docking protein for the SHP2 protein-tyrosine phosphatase in EGF-stimulated cells, was associated with paxillin. SHP2 dephosphorylated paxillin and caused dissociation of Csk, a negative regulator of Src, from paxillin but had no effect on paxillin-Src association. A lower level of Src Tyr-530 phosphorylation was detected in paxillin-associated Src in EGF-stimulated cells. Expression of an SHP2 binding defective mutant of Gab1 (Gab1FF) or a catalytically inactive mutant of SHP2 (SHP2DN) prevented paxillin tyrosine dephosphorylation and Src activation induced by EGF. Importantly, Gab1FF blocked paxillin-SHP2 complex formation, Src Tyr-530 dephosphorylation, Erk activation, and cell migration induced by EGF. Inhibition of Src tyrosine kinase activity abrogated EGF-stimulated Erk activation and cell migration. Together, these results reveal that Gab1 recruits SHP2 to dephosphorylate paxillin, leading to dissociation of Csk from the paxillin-Src complex and Src activation and that Src is an SHP2 effector involved in EGF-stimulated Erk activation and cell migration.     

Dependence of gonadotropin-releasing hormone-induced neuronal MAPK signaling on epidermal growth factor receptor transactivation

The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1-7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr(402). This was reduced by Ca(2+) chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKC alpha and -epsilon to the cell membrane and enhanced the association of Src with PKC alpha and PKC epsilon, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.     

Retinoids suppress epidermal growth factor-associated cell proliferation by inhibiting epidermal growth factor receptor-dependent ERK1/2 activation.

Human papillomavirus (HPV) is an important etiological agent in the genesis of cervical cancer. HPV-positive cervical tumors and human papillomavirus-positive cell lines display increased epidermal growth factor receptor (EGFR) expression, which is associated with increased cell proliferation. ECE16-1 cells are an HPV-immortalized human ectocervical epithelial cell line that is a model of HPV-associated cervical neoplasia and displays elevated EGFR levels. In the present study, we evaluated the effects of receptor-selective retinoid ligands on EGFR-associated signal transduction. We show that retinoic acid receptor (RAR)-selective ligands reduce EGFR level and the magnitude and duration of EGFR activation in EGF-stimulated cells. These effects are reversed by cotreatment with an RAR antagonist. To identify the mechanism, we examined the effects of retinoid treatments on EGF-dependent signaling. Stimulation with EGF causes a biphasic activation of the ERK1/2 MAPK. The first peak of activation is present at 20 min, and the second is present at 36 h. This activation subsequently leads to an increase in the cyclin D1 level and increased cell proliferation. Simultaneous treatment with EGF and a RAR-selective retinoid inhibits both phases of ERK1/2 activation, completely eliminates the cyclin D1 induction, and suppresses EGF-dependent cell proliferation. This effect is specific as retinoid treatment does not alter the level or activity of other EGFR-regulated kinases, including AKT and the MAPKs p38 and JNK. Retinoid X receptor-selective ligands, in contrast, did not regulate these responses. These results suggest that RAR ligand-associated down-regulation of EGFR activity reduces cell proliferation by reducing the magnitude and duration of EGF-dependent ERK1/2 activation.     

c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.     

Interferon gamma-dependent transactivation of epidermal growth factor receptor

The present report provides evidence that, in A431 cells, interferon gamma (IFNgamma) induces the rapid (within 5 min), and reversible, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). IFNgamma-induced EGFR transactivation requires EGFR kinase activity, as well as activity of the Src-family tyrosine kinases and JAK2. Here, we show that IFNgamma-induced STAT1 activation in A431 and HeLa cells partially depends on the kinase activity of both EGFR and Src. Furthermore, in these cells, EGFR kinase activity is essential for IFNgamma-induced ERK1,2 activation. This study is the first to demonstrate that EGFR is implicated in IFNgamma-dependent signaling pathways.     

Three-dimensional matrix induces sustained activation of ERK1/2 via Src/Ras/Raf signaling pathway

Research in cell signaling often depends on tissue culture, but the artificial substrates used to grow cells in vitro are likely to distort the conclusions, particularly when adhesion-mediated signaling events are investigated. Studies of signal transduction pathways operating in cells grown in three-dimensional (3D) matrices provide a better system, giving a closer insight of the cell signaling in vivo. We compared the steady-state levels of ERK1/2 activity in primary human fibroblasts, induced by cell-derived 3D fibronectin matrix or fibronectin, coated on flat surfaces. 3D environment caused ERK1/2 stimulation concomitant with a 2.5-fold increase in Ras GTP loading and Src activation. Under these conditions FAK autophosphorylation was suppressed. Treatment with Src inhibitor PP2 abolished these effects indicating that 3D fibronectin matrix activated ERK1/2 through Src/Ras/Raf pathway, bypassing FAK. These observations suggest that within in vivo-like conditions Src may have a leading role in the induction of sustained ERK1/2 activation.     

Epidermal growth factor receptor tyrosine kinase mediates Ras activation by gonadotropin-releasing hormone

Gonadotropin releasing hormone (GnRH) contributes to the maintenance of gonadotrope function by increasing extracellular signal-regulated kinase (ERK) activity subsequent to binding to its cognate G-protein-coupled receptor. As the GnRH receptor exclusively interacts with G(q/11) proteins and as receptor expression is regulated in a beta-arrestin-independent fashion, it represents a good model to systematically dissect underlying signaling pathways. In alphaT3-1 gonadotropes endogenously expressing the GnRH receptor, GnRH challenge resulted in a rapid increase in ERK activity which was attenuated by the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor AG1478. In COS-7 cells transiently expressing the human GnRH receptor, agonist-induced ERK activation was independent of free Gbetagamma subunits but could be mimicked by short-term phorbol ester treatment. Most notably, G(q/11)-induced ERK activation was sensitive to N17-Ras and to expression of the C-terminal Src kinase but also to other dominant negative mutants of signaling components localized upstream of Ras, like Shc and the EGFR. GnRH as well as phorbol esters led to Ras activation in COS-7 and alphaT3-1 cells, which was dependent on Src and EGFR tyrosine kinases, indicating that both tyrosine kinases act downstream of protein kinase C (PKC) and upstream of Ras. However, Src did not contribute to Shc tyrosine phosphorylation. GnRH or phorbol ester challenge resulted in PKC-dependent EGFR autophosphorylation. Furthermore, a 5-min phorbol ester treatment was sufficient to trigger tyrosine phosphorylation of the platelet-derived growth factor-beta receptor in L cells. Thus, in several cell systems PKC is able to stimulate Ras via activation of receptor tyrosine kinases.     

Redox-sensitive transactivation of epidermal growth factor receptor by tumor necrosis factor confers the NF-kappa B activation

Cross-communication between different signaling systems allows the integration of the great diversity of stimuli that a cell receives under varying physiological situations. In this paper we have explored the possibility that tumor necrosis factor (TNF) receptor signal cross-talks with epidermal growth factor (EGF) receptor signal on the nuclear factor-kappa B (NF-kappa B) activation pathway. We have demonstrated that overexpression of the EGF receptor (EGFR) in NIH3T3 cells significantly enhances TNF-induced NF-kappa B-dependent luciferase activity even without EGF, that EGF treatment has a synergistic effect on the induction of the reporter activity, and that this enhancement is suppressed by AG1478, EGFR-specific tyrosine kinase inhibitor. We also have shown that TNF induces tyrosine phosphorylation and internalization of the overexpressed EGFR in NIH3T3 cells and the endogenously expressed EGFR in A431 cells and that the transactivation by TNF is suppressed by N-acetyl-l-cysteine or overexpression of an endogenous reducing molecule, thioredoxin, but not by phosphatidylinositol 3-kinase inhibitors and protein kinase C inhibitor. Taken together, this evidence strongly suggests that EGFR transactivation by TNF, which is regulated in a redox-dependent manner, is playing a pivotal role in TNF-induced NF-kappa B activation.     

Macrophage-inflammatory protein-3alpha mediates epidermal growth factor receptor transactivation and ERK1/2 MAPK signaling in Caco-2 colonic epithelial cells via metalloproteinase-dependent release of amphiregulin

Previously, we reported that normal colonocytes produce the memory CD4(+) T cell-directed chemokine MIP-3alpha, and that epithelial MIP-3alpha levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3alpha, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3alpha may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3alpha can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3alpha can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3alpha-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3alpha-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3alpha can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.     

Sustained and transient downregulation of Src kinases in response to nerve growth factor and epidermal growth factor, respectively

Within a search for novel points of divergence in the signalling of differentiative nerve growth factor (NGF) and mitogenic epidermal growth factor (EGF) in PC12 cells, in the present study, I comparatively analysed the impact of these growth factors on the in situ activation status of Src kinases. By probing total cell extracts and anti-P-Tyr immunoprecipitates with a phosphorylation site-specific antibody targeting a conserved site of positive regulation of the overall family of Src kinases, a sustained and a transient downregulation of the immunoprecipitable subpopulation of nominally active Src kinases is detected in response to NGF and EGF, respectively. As the recovery of immunoreactive Src kinases is greater from cells being lysed while in suspension than from adherent cells, the possibility that NGF reduces the stability of Src kinases by upregulating cell adhesiveness is preliminarily explored and discussed.     

Growth hormone-releasing hormone induced transactivation of epidermal growth factor receptor in human triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subset of breast cancers which is negative for expression of estrogen and progesterone receptors and human epidermal growth factor receptor-2 (HER2). Chemotherapy is currently the only form of treatment for women with TNBC. Growth hormone-releasing hormone (GHRH) and epidermal growth factor (EGF) are autocrine/paracrine growth factors in breast cancer and a substantial proportion of TNBC expresses receptors for GHRH and EGF. The aim of this study was to evaluate the interrelationship between both these signaling pathways in MDA-MB-468 human TNBC cells. We evaluated by Western blot assays the effect of GHRH on transactivation of EGF receptor (EGFR) as well as the elements implicated. We assessed the effect of GHRH on migration capability of MDA-MB-468 cells as well as the involvement of EGFR in this process by means of wound-healing assays. Our findings demonstrate that in MDA-MB-468 cells the stimulatory activity of GHRH on tyrosine phosphorylation of EGFR is exerted by two different molecular mechanisms: i) through GHRH receptors, GHRH stimulates a ligand-independent activation of EGFR involving at least cAMP/PKA and Src family signaling pathways; ii) GHRH also stimulates a ligand-dependent activation of EGFR implicating an extracellular pathway with an important role for metalloproteinases. The cross-talk between EGFR and GHRHR may be impeded by combining drugs acting upon GHRH receptors and EGFR family members. This combination of GHRH receptors antagonists with inhibitors of EGFR signalling could enhance the efficacy of both types of agents as well as reduce their doses increasing therapeutic benefits in management of human breast cancer.