Agonist-specific transactivation of phosphoinositide 3-kinase signaling pathway mediated by the dopamine D2 receptor

Bromocriptine, acting through the dopamine D2 receptor, provides robust protection against apoptosis induced by oxidative stress in PC12-D2R and immortalized nigral dopamine cells. We now report the characterization of the D2 receptor signaling pathways mediating the cytoprotection. Bromocriptine caused protein kinase B (Akt) activation in PC12-D2R cells and the inhibition of either phosphoinositide (PI) 3-kinase, epidermal growth factor receptor (EGFR), or c-Src eliminated the Akt activation and the cytoprotective effects of bromocriptine against oxidative stress. Co-immunoprecipitation studies showed that the D2 receptor forms a complex with the EGFR and c-Src that was augmented by bromocriptine, suggesting a cross-talk between these proteins in mediating the activation of Akt. EGFR repression by inhibitor or by RNA interference eliminated the activation of Akt by bromocriptine. D2 receptor stimulation by bromocriptine induced c-Src tyrosine 418 phosphorylation and EGFR phosphorylation specifically at tyrosine 845, a known substrate of Src kinase. Furthermore, Src tyrosine kinase inhibitor or dominant negative Src interfered with Akt translocation and phosphorylation. Thus, the predominant signaling cascade mediating cytoprotection by the D2 receptor involves c-Src/EGFR transactivation by D2 receptor, activating PI 3-kinase and Akt. We also found that the agonist pramipexole failed to stimulate activation of Akt in PC12-D2R cells, providing an explanation for our previous observations that, despite efficiently activating G-protein signaling, this agonist had little cytoprotective activity in this experimental system. These results support the hypothesis that specific dopamine agonists stabilize distinct conformations of the D2 receptor that differ in their coupling to G-proteins and to a cytoprotective c-Src/EGFR-mediated PI-3 kinase/Akt pathway.     

Control of FLIPL expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP_L) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP_L levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP_L upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP_L expression. FLIP_L upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP_L upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP_L levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.     

EGF mediates calcium-activated chloride channel activation in the human bronchial epithelial cell line 16HBE14o-: involvement of tyrosine kinase p60c-src

Particulate atmospheric pollutants interact with the human airway epithelium, which releases cytokines, chemokines, and EGF receptor (EGFR) ligands leading to proinflammatory responses. There is little information concerning the short-term effects of EGFR activation by extracellular ligands on ionic regulation of airway surface lining fluids. We identified in the membrane of human epithelial bronchial cells (16HBE14o(-) line) an endogenous calcium- and voltage-dependent, outwardly rectifying small-conductance chloride channel (CACC), and we examined the effects of EGF on CACC activity. Ion channel currents were recorded with the patch-clamp technique. In cell-attached membrane patches, CACC were activated by exposure of the external surface of the cells to physiological concentrations of EGF without any change in cytosolic Ca(2+) concentration ([Ca(2+)](i)) and inhibited by tyrphostin AG-1478 (an inhibitor of EGFR that also blocks EGF-dependent Src family kinase activation). EGF activation of c-Src protein in 16HBE14o(-) cells was observed, and the signaling pathway elicited by EGFR was blocked by tyrphostin AG-1478. In excised inside-out membrane patches CACC were activated by exposure of the cytoplasmic face of the channels to the human recombinant Src(p60(c-src)) kinase with endogenous or exogenous ATP and inhibited by lambda-protein phosphatase. Secretion of EGFR ligands by epithelial airway cells exposed to pollutants would then elicit a rapid and direct ionic response of CACC mediated by EGFR activation via a Src kinase family-dependent signaling pathway.     

Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.     

Inhibition of the EGF receptor and ERK1/2 signaling pathways rescues the human epidermoid carcinoma A431 cells from IFNγ-induced apoptosis

Interferon gamma (IFNγ) has been demonstrated to inhibit tumor growth in vivo as well as proliferation of multiple types of cultured transformed cells. In this study, we showed that IFNγ promoted progressive death in A431 cells, overexpressing EGF receptor (EGFR). Based on the data provided by evaluating cell morphology, MTT assay, FACS analysis, and cleaved caspase-3 staining we concluded that the major cause of IFNγ-induced A431 cell growth inhibition was not cell cycle arrest, but apoptosis. We investigated a role for the EGFR and ERK1/2 MAPK signaling pathways in IFNγ-induced apoptosis of A431 cells. IFNγ-induced cell death was accompanied by both an increase of the ERK1/2 MAPK activation and a simultaneous reduction of the EGFR activation. Activation of ERK1/2 was crucial for IFNγ-induced cell death because MEK1/2 inhibitors, PD0325901 and U0126 efficiently protected cells from apoptosis by suppressing caspase-3 activation. Even though EGFR tyrosine kinase inhibitor AG1478 also rescued A431 cells from IFNγ-induced apoptosis, unlike MEK1/2 inhibitors, it initiated G₁ arrest. Together, these results suggest that sustained inhibition of both EGFR and ERK1/2 leads to significant protection of the cells from IFNγ-induced apoptosis, indicating important roles for the EGFR tyrosine kinase and ERK1/2 MAP-kinases in regulating A431 cell death.     

STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.     

Regulation of Mammalian Target of Rapamycin Complex 1 by Bcl-2 and Bcl-XL Proteins

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism. Its activity is controlled by various types of signals, including growth factors, nutrients, and stresses. In this study, we show that changes in expression levels of two antiapoptotic proteins, Bcl-2 and Bcl-X_L, also affect mTORC1 signaling activity. In cells overexpressing Bcl-X_L, mTORC1 activity is increased and becomes less sensitive to growth factor or nutrient conditions. In contrast, reduction in expression levels of the two antiapoptotic proteins inhibits mTORC1 signaling activity. Our results suggest that the effect of Bcl-2 and Bcl-X_L on mTORC1 is mediated by FKBP38, an inhibitor of mTORC1. The two proteins compete with mTORC1 for FKBP38 binding and hence alter mTORC1 activity. This study reveals a novel cross-talk between Bcl-2/X_L and mTORC1 signaling, which is likely to contribute to cancer development.     

c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling

c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.     

Lysophosphatidylcholine stimulates EGF receptor activation and mesangial cell proliferation: regulatory role of Src and PKC

Lysophosphatidylcholine (LPC), a major component of oxidized-low density lipoproteins (ox-LDL), modulates various pathobiological processes involved in vascular and glomerular diseases. Although several studies have shown increased plasma concentrations of ox-LDL as well as LPC in patients with renal disease, the role of LPC in mesangial cell proliferation and associated signaling mechanisms are not clearly understood. In this study, we have shown that LPC induced the phosphorylation of epidermal growth factor receptor (EGFR), as well as the p42/44 MAP kinases. LPC activated Src-kinase and protein kinase C (PKC), and both Src kinase inhibitor PP-2 and PKC inhibitor inhibited the activation of EGFR by LPC. LPC (5-25 microM) stimulated human mesangial cell proliferation by 4-5 fold. Preincubation of mesangial cells with the Src inhibitor (PP-2), or PKC inhibitor (bisindolylmaleimide GF109203-X), or EGF receptor kinase inhibitor (AG1478), or MEK inhibitor (PD98059) significantly inhibited LPC-mediated mesangial cell proliferation. The data suggest that LPC, by activating Src and PKC signaling pathways, stimulates EGF receptor transactivation and down-stream MAP kinase signaling resulting in mesangial hypercellularity, which is a characteristic feature of diverse renal diseases.     

Signal characteristics of G protein-transactivated EGF receptor.

The epidermal growth factor receptor (EGFR) tyrosine kinase recently was identified as providing a link to mitogen-activated protein kinase (MAPK) in response to G protein-coupled receptor (GPCR) agonists in Rat-1 fibroblasts. This cross-talk pathway is also established in other cell types such as HaCaT keratinocytes, primary mouse astrocytes and COS-7 cells. Transient expression of either Gq- or Gi-coupled receptors in COS-7 cells allowed GPCR agonist-induced EGFR transactivation, and lysophosphatidic acid (LPA)-generated signals involved the docking protein Gab1. The increase in SHC tyrosine phosphorylation and MAPK stimulation through both Gq- and Gi-coupled receptors was reduced strongly upon selective inhibition of EGFR function. Inhibition of phosphoinositide 3-kinase did not affect GPCR-induced stimulation of EGFR tyrosine phosphorylation, but inhibited MAPK stimulation, upon treatment with both GPCR agonists and low doses of EGF. Furthermore, the Src tyrosine kinase inhibitor PP1 strongly interfered with LPA- and EGF-induced tyrosine phosphorylation and MAPK activation downstream of EGFR. Our results demonstrate an essential role for EGFR function in signaling through both Gq- and Gi-coupled receptors and provide novel insights into signal transmission downstream of EGFR for efficient activation of the Ras/MAPK pathway.     

Inhibition of E-Cadherin Dependent Cell-Cell Contact Promotes MUC5AC Mucin Production through the Activation of Epidermal Growth Factor Receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

Human epidermal growth factor receptor-1 expression renders Chinese hamster ovary cells sensitive to alternative aldosterone signaling

The epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport using ERK1/2 as a downstream effector. Furthermore, the EGF receptor (EGFR) is involved in signaling by G-protein-coupled receptors, growth hormone, and cytokines via transactivation. It has been suggested that steroids interact with peptide hormones. Previously, we have shown that aldosterone modulates EGF responses in Madin-Darby canine kidney cells (Gekle, M., Freudinger, R., Mildenberger, S., and Silbernagl, S. (2002) Am. J. Physiol. 282, F669-F679). Here, we tested the hypothesis that human EGFR-1 can confer alternative aldosterone responsiveness with respect to ERK1/2 phosphorylation to Chinese hamster ovary cells, which do not express EGFR. Wild-type Chinese hamster ovary cells did not respond to EGF or aldosterone. After transfection of human EGFR-1, the cells responded to EGF, but not to aldosterone. However, when submaximal concentrations of EGF were used, nanomolar concentrations of aldosterone potentiated the action of EGF within minutes, resulting in a leftward shift of the EGF dose-response curve. This was not the case in mock-transfected cells. The EGFR kinase inhibitor tyrphostin AG1478 or the MEK1/2 inhibitor U0126 completely prevented the effect. Furthermore, aldosterone enhanced Tyr phosphorylation of c-Src and EGFR, and an inhibitor of cytosolic tyrosine kinases (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriociaine) prevented the action of aldosterone. Our data show that aldosterone uses the EGF-EGFR-MEK1/2-ERK1/2 signaling cascade to elicit its alternative effects. In the presence of EGF, aldosterone leads to EGFR transactivation via cytosolic tyrosine kinases of the Src family.     

Cytoskeletal and Morphological Changes Associated with the Specific Suppression of the Epidermal Growth Factor Receptor Tyrosine Kinase Activity in A431 Human Epidermoid Carcinoma

The epidermal growth factor (EGF) receptor is well known as a mediator of mitogenic signaling and its tyrosine kinase activity has been suggested as a viable target in cancer chemotherapy. To explore the consequences of abolishing the kinase activity of this receptor, we have utilized a potent and specific inhibitor of the enzyme, PD 153035, to sustain a long-term suppression of its activity. This compound inhibits EGF receptor autophosphorylation in cells with an IC₅₀in the low nanomolar range and does not block PDGF or FGF receptor kinase until concentrations are greater than 10 μM.[1] Human epidermoid carcinoma A431 cells were grown in the presence of PD 153035 and were passed weekly until cells grew in the presence of 1 μMinhibitor. These cells, referred to as A431R, showed a remarkable change in morphology, becoming flattened and spread out. A comparison of the sensitivity of EGF receptor autophosphorylation to PD 153035 between A431 and A431R showed a similar dose response, indicating that the cells had not developed any defect in the kinase which might make it resistant to the inhibitor. Likewise, EGF receptor autophosphorylation in response to exogenously added EGF, as well as receptor internalization, was similar between the two cell lines. Furthermore, analysis of A431R cells by flow cytometry showed no significant change in DNA content or percentage of cells in any one phase of the cell cycle compared to the parent line.¹²⁵I-labeled EGF/receptor binding studies showed that receptor number in the A431R cells was equivalent to that of the parent line; however, the Scatchard plot was linear, in contrast to the typical biphasic plot obtained with the parent cells, implying a loss of high-affinity receptors. Cytoskeletal preparations from both cell lines indicated that the A431R had fourfold less EGF receptor associated with the cytoskeleton than A431. This was accompanied by a remarkable increase in polymerized actin stress fibers throughout the A431R cells, which most likely accounts for their flattened morphology. The A431R cells also exhibited a twofold increase in the expression of focal adhesion kinase, which is consistent with a greater contact area for their cell surface and increase in focal adhesions. Finally, although the A431R cells have a doubling time of 24 h, similar to that of the parent line, these cells stop growing as the monolayer approaches confluence, reminiscent of the contact inhibition seen in nontransformed cells. These data indicate that long-term suppression of the EGF receptor tyrosine kinase activity in A431 human epidermoid carcinoma results in certain cellular properties which are more consistent with a differentiated and nontransformed phenotype.     

Inhibition of E-cadherin dependent cell-cell contact promotes MUC5AC mucin production through the activation of epidermal growth factor receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation

Background

Substance P (SP) is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC) aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R) signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator) and of metalloproteinases (ligand-dependent mediators) in HER2/EGFR activation.

Results and Discussion

Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4’-phenoxyanilino)-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1–10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.

Conclusion

Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.     

Role of epidermal growth factor and ErbB2 receptors in 3T3-L1 adipogenesis

Objective: Epidermal growth factor (EGF) stimulates proliferation in 3T3‐L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance. Research Methods and Procedures: We compared the effects of low (0.1 nM) vs. high (10 nM) EGF on the activation of EGF receptors, proximal signaling molecules Src and Shc, and the downstream mitogen‐activated protein kinase (MAPK) pathways extracellular regulated kinase (ERK) and p38 in proliferating and differentiated 3T3‐L1 cells. Results: Both low and high EGF activated ERK and p38 in preadipocytes. Src inhibitors PP1 and PP2 blocked ERK and p38 activation by low but not high EGF, and only high EGF increased Shc phosphorylation. Selective inhibition of the EGF receptor (EGFR) with AG1478 blocked ERK and p38 activation at both concentrations; however, selective inhibition of the ErbB2 receptor (EB2R) with AG825 or small interfering RNA (siRNA) blocked low but not high EGF activation of ERK and p38. Coimmunoprecipitation of EGFR with EB2R and Src was observed with low EGF in preadipocytes but at both concentrations in adipocytes. EB2R inhibition during differentiation decreased p38 activity and peroxisome proliferator‐activated receptor γ (PPARγ) abundance. Discussion: Our results show that EGFR homodimers mediate action of EGF at high abundance, but at low abundance, EGF promotes differentiation through EGFR/EB2R heterodimer activation of Src and p38. These results may partially explain the observations that high EGF concentrations inhibit, whereas low concentrations support, preadipocyte differentiation.     

Src kinase mediates the regulation of phospholipase C-gamma activity by glycosphingolipids

Glucosylceramide-based glycosphingolipids have been previously demonstrated to regulate negatively the formation of inositol 1,4,5-trisphosphate by phospholipase C-gamma1. In the present study, the depletion of endogenous glucosylceramide by D-t-EtDO-P4 in cultured ECV304 cells induced autophosphorylation of Src kinase at tyrosine residue 418 within the catalytic loop and dephosphorylation of Src kinase at tyrosine residues 529 within the carboxyl-terminal regulatory region. Phosphotransferase activities of Src kinase were also induced in the glucosylceramide-depleted cells. c-Src kinase activity and phosphorylations at Src Tyr-418 and epidermal growth factor (EGF) receptor Tyr-1068 were significantly enhanced by bradykinin in response to 100 nm D-t-EtDO-P4 compared with control cells. The phosphorylation and dephosphorylation on Tyr-418 and Tyr-529 residues of c-Src were reversed by treatment of 4-amino-5-(4-chlorophenyl)-7-t-butyl(pyrazolo)[3,4-d]pyrimidine (PP2), an inhibitor of Src kinase, in control cells. Glucosylceramide-depleted cells resisted treatment with PP2, and both phosphorylation of Tyr-418 and dephosphorylation of Tyr-529 induced by depletion of glucosylceramide were maintained. Compared with untreated cells, tyrosine phosphorylation of phospholipase C-gamma1 was enhanced by EGF stimulation in glucosylceramide-depleted cells, associated with enhanced tyrosine phosphorylation of the EGF receptor at Tyr-1068 and Tyr-1086 stimulated by EGF. The Src inhibitor, PP2, significantly blocked EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 in control cells, whereas in glucosylceramide-depleted cells, suppression of Src kinase activity by PP2 toward EGF-induced tyrosine phosphorylation of phospholipase C-gamma1 was less significant. Thus the activation of Src kinase by depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells is a critical up-stream event in the activation of phospholipase C-gamma1.     

Potentiation of Epidermal Growth Factor-Mediated Oncogenic Transformation by Sialidase NEU3 Leading to Src Activation

We previously demonstrated that sialidase NEU3, a key glycosidase for ganglioside degradation, is up-regulated in various human cancers, leading to increased cell invasion, motility and survival of cancer cells possibly through activation of EGF signaling. Its up-regulation is also important for promotion of the stage of colorectal carcinogenesis in vivo in human NEU3 transgenic mice treated with azoxymethane for the induction of aberrant crypt foci in the colon mucosa, accompanied by enhanced phosphorylation of EGF receptor (EGFR). To address whether the activation of EGF signaling by the sialidase is associated with oncogenic transformation, we here analyzed the effects of overexpression of NEU3 and EGFR in NIH-3T3 cells. When NEU3 was stably transfected with or without EGFR, it was associated with significant increases in clonogenic growth, clonogenicity on soft agar and in vivo tumor growth in nude mice either with or without the receptor overexpression in the presence of EGF, compared with the levels in their vector controls. Despite the fact that the endogenous level of EGFR is known to be extremely low in these cells, NEU3 significantly enhanced the phosphorylation of Akt and ERK, as well as that of the receptor. The NEU3-mediated activation was largely abrogated by the EGFR inhibitor AG1478 or PD153035, but significant clonogenic growth still remained. NEU3 was then found to activate Src kinase, and the clonogenicity was completely suppressed by an Src inhibitor, PP2. The activity-null mutants failed to activate Src and EGFR, indicating that ganglioside modulation by NEU3 may be necessary for the activation. NEU3 and Src were co-immunoprecipitated with EGFR in NEU3- and EGFR- transfected cells. These findings identify NEU3 as an essential participant in tumorigenesis through the EGFR/Src signaling pathway and a potential target for inhibiting EGFR-mediated tumor progression.     

TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src

TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. Here, we demonstrate that TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases.     

Antagonistic effects of protein kinase C alpha and delta on both transformation and phospholipase D activity mediated by the epidermal growth factor receptor.

Downregulation of protein kinase C delta (PKC delta) by treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418-3428, 1997). We extended these studies to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor (EGFR cells); like c-Src, the EGF receptor is overexpressed in several human tumors. In contrast with expectations, downregulation of PKC isoforms with TPA did not transform the EGFR cells; however, treatment with EGF did transform these cells. Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC delta- and PKC alpha-specific inhibitors and the expression of dominant negative mutants for both PKC delta and alpha. Consistent with a tumor-suppressing function for PKC delta, the PKC delta-specific inhibitor rottlerin and a dominant negative PKC delta mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC alpha-specific inhibitor Go6976 and expression of a dominant negative PKC alpha mutant blocked the transformed phenotype induced by both EGF and PKC delta inhibition. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity, which is commonly elevated in response to mitogenic stimuli. The elevation of PLD activity in response to inhibiting PKC delta, like transformation, was dependent upon PKC alpha and restricted to the EGFR cells. These data demonstrate that PKC isoforms alpha and delta have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC delta that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.