Transactivation of the epidermal growth factor receptor mediates parathyroid hormone and prostaglandin F2 alpha-stimulated mitogen-activated protein kinase activation in cultured transgenic murine osteoblasts

Recent data suggest that G protein-coupled receptors (GPCRs), including those for PTH and prostaglandins (PGs), contribute to the proliferation and differentiation of osteoblasts in vivo. To understand how these signals are transduced, we studied activation of the ERK1/2 MAPK cascade in cultures of differentiating TMOb murine osteoblasts. In TMOb cells, stimulation of endogenous Gs/Gq-coupled PTH receptors, Gq-coupled PGF2 alpha receptors, and Gi/Gq-coupled lysophosphatidic acid receptors, but not Gs-coupled PGE2 receptors, caused a rapid 5- to 10-fold increase in ERK1/2 phosphorylation. GPCR-stimulated ERK1/2 activation coincided with increased tyrosine phosphorylation of epidermal growth factor (EGF) receptors and was blocked by the EGF receptor inhibitor, tyrphostin AG1478, and the metalloprotease inhibitor, batimastat, suggesting that the response involved transactivation of EGF receptors through the proteolytic release of an EGF receptor ligand. To further examine the mechanism of PTH-stimulated EGF receptor transactivation, we employed COS-7 cells expressing the rat PTH receptor. Here, stimulation with PTH(1-34) caused proteolysis of hemagglutinin epitope-tagged heparin binding-EGF, increased tyrosine autophosphorylation of EGF receptors, and AG1478-sensitive ERK1/2 activation. When PTH receptor-expressing COS-7 cells were placed in a mixed culture with cells lacking the PTH receptor but expressing a green fluorescent protein-tagged ERK2, stimulation with PTH(1-34) induced phosphorylation of green fluorescent protein-ERK2 that was abolished by either batimastat or tyrphostin AG1478. These data suggest that autocrine/paracrine cross-talk between EGF receptors and Gi- or Gq/11-coupled GPCRs represents the predominant mechanism of GPCR-mediated activation of ERK1/2 in cultured TMOb osteoblasts.     

Insulin-like growth factor-I (IGF-I) induces epidermal growth factor receptor transactivation and cell proliferation through reactive oxygen species

Insulin-like growth factor-I (IGF-I) plays an important role in proliferation of vascular smooth muscle cells (VSMCs). However, the mechanism that IGF-I induces VSMCs proliferation is not completely understood. In this study, we determined (a) whether and how IGF-I induces transactivation of epidermal growth factor receptor (EGFR) in primary rat aortic VSMCs, (b) the contribution of EGFR to IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK) and cell proliferation, and (c) the role of reactive oxygen species (ROS) in the cellular function. We showed that IGF-I induced phosphorylation of EGFR and ERK1/2 in VSMCs. AG1478, an EGFR inhibitor, inhibited IGF-I-induced phoshorylation of EGFR and ERK1/2. IGF-I stimulated ROS production and Src activation. Antioxidants inhibited IGF-I-induced ROS generation and activation of EGFR, ERK, and Src. Src kinase inhibitor PP1 and Src siRNA blocked IGF-I-induced activation of EGFR and ERK1/2. Inhibition of IGF-I-stimulated EGFR activation inhibited IGF-I-induced VSMC proliferation. These results suggest that (1) IGF-I induces EGFR activation through production of ROS and ROS-mediated Src activation in VSMCs, and (2) EGFR transactivation is required for IGF-I-induced VSMC proliferation.     

Activation of extracellular signal-regulated kinase by ultraviolet is mediated through Src-dependent epidermal growth factor receptor phosphorylation. Its implication in an anti-apoptotic function.

Ultraviolet (UV) irradiation stimulates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which is a member of the mitogen-activated protein kinase (MAPK) superfamily and implicated in stress-induced apoptosis. UV also induces the activation of another MAPK member, extracellular signal-regulated kinase (ERK), which is typically involved in a growth-signaling cascade. However, the UV-induced signaling pathway leading to ERK activation, together with the physiological role, has remained unknown. Here we examined the molecular mechanism and physiological function of UV-induced ERK activation in human epidermoid carcinoma A431 cells that retain a high number of epidermal growth factor (EGF) receptors. UV-induced ERK activation was accompanied with the Tyr phosphorylation of EGF receptors, and both responses were completely abolished in the presence of a selective EGF receptor inhibitor (AG1478) or the Src inhibitor PP2 and by the expression of a kinase-dead Src mutant. On the other hand, SAPK/JNK activation by UV was partially inhibited by these inhibitors. UV stimulated Src activity in a manner similar to the ERK activation, but the Src activation was insensitive to AG1478. UV-induced cell apoptosis measured by DNA fragmentation and caspase 3 activation was enhanced by AG1478 and an ERK kinase inhibitor (U0126) but inhibited by EGF receptor stimulation by the agonist. These results indicate that UV-induced ERK activation, which provides a survival signal against stress-induced apoptosis, is mediated through Src-dependent Tyr phosphorylation of EGF receptors.     

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.     

cag+ Helicobacter pylori induce transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells

The gastric pathogen Helicobacter pylori is known to activate epithelial cell signaling pathways that regulate numerous inflammatory response genes. The aim of this study was to elucidate the pathway leading to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in H. pylori-infected AGS gastric epithelial cells. We find that H. pylori, via activation of the epidermal growth factor (EGF) receptor activates the small GTP-binding protein Ras, which in turn, mediates ERK1/2 phosphorylation. cag+ strains of H. pylori are able to induce greater EGF receptor activation than cag- strains, and studies with isogenic mutants indicate that an intact type IV bacterial secretion system is required for this effect. Blockade of EGF receptor activation using tyrphostin AG1478 prevents H. pylori-mediated Ras activation, inhibits ERK1/2 phosphorylation, and substantially decreases interleukin-8 gene expression and protein production. Investigations into the mechanism of EGF receptor activation, using heparin, a metalloproteinase inhibitor and neutralizing antibodies reveal that H. pylori transactivates the EGF receptor via activation of the endogenous ligand heparin-binding EGF-like growth factor. Transactivation of gastric epithelial cell EGF receptors may be instrumental in regulating both proliferative and inflammatory responses induced by cag+ H. pylori infection.     

Elastase-released epidermal growth factor recruits epidermal growth factor receptor and extracellular signal-regulated kinases to down-regulate tropoelastin mRNA in lung fibroblasts

Elastase/anti-elastase imbalance is a hallmark of emphysema, a chronic obstructive pulmonary disease associated with the rupture and inefficient repair of interstitial elastin. We report that neutrophil elastase (NE) at low physiologic concentrations, ranging from 35 nm to 1 microm, invokes transient, peaking at 15 min, activation of extracellular signal-regulated kinases 1 and 2 (ERK) in elastogenic lung fibroblasts. ERK activation is preceded by the release of soluble 25-26-kDa forms of epidermal growth factor (EGF) and transactivation of EGF receptor (EGFR) in NE-exposed cells. The stimulatory effect of NE on ERK is abrogated in the presence of anti-EGF-neutralizing antibodies, EGFR tyrosine kinase inhibitor (AG1478), and ERK kinase inhibitor (PD98059), as well as abolished in both EGFR-desensitized and endocytosis-arrested fibroblasts. Nuclear accumulation of activated ERK is associated with transient, peaking at 30 min, induction of c-Fos and sustained, observed at 24-48 h, decrease of tropoelastin mRNA levels in NE-challenged cells. Pretreatment of fibroblasts with AG1478 or PD98059 abrogates the NE-initiated tropoelastin mRNA suppression. We conclude that proteolytically released EGF signals directly via EGFR and ERK to down-regulate tropoelastin mRNA in NE-challenged lung fibroblasts.     

Epidermal growth factor receptor-dependent and -independent cell-signaling pathways originating from the urokinase receptor

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.     

Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Cao2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Cao2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Cao2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Cao2+. This is consistent with the known expression of TGFalpha by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Cao2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.     

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.     

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.     

Fluid flow-induced tyrosine phosphorylation and participation of growth factor signaling pathway in osteoblast-like cells

To investigate how mechanical loading stimulates bone cells, we subjected murine osteoblast-like cells, MC3T3E1, to fluid flow generated by shaking culture dishes. Since we had previously found that egr-1 mRNA is up-regulated by the flow, and that the regulation involves tyrosine kinase, we examined which proteins are tyrosine-phosphorylated by flow. Western blotting and immunoprecipitation of cell lysates showed tyrosine phosphorylation enhancement of many proteins, including ERK2 and Shc, and activation of ERK1/2. Although these responses did not occur in serum-free media, addition of EGF or bFGF recovered the responses. AG1478, an inhibitor of EGF receptor kinase activity, abolished tyrosine phosphorylation enhancement, ERK1/2 activation, and egr-1 mRNA accumulation induced by the flow of EGF-containing serum-free media. These results suggest that growth factor signaling pathways are involved in these responses. Repetition of fluid flow induced repeatedly up-regulation of egr-1 mRNA. Such events may also occur in bone under mechanical loading.     

Insulin-like growth factor-I induces alpha(1B)-adrenergic receptor phosphorylation through G beta gamma and epidermal growth factor receptor transactivation

IGF-I induces alpha(1B)-adrenoceptor (alpha(1B)-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC(50) approximately 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased alpha(1B)-adrenergic effect on intracellular calcium and production of inositol phosphates. The effect of IGF-I was markedly decreased in cells treated with pertussis toxin suggesting involvement of pertussis toxin-sensitive G proteins. Transfection of the carboxyl terminus of the beta-adrenergic receptor kinase or the Deltap85 mutant of phosphoinositide 3-kinase (PI3K) markedly decreased the alpha(1B)-AR phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline. Inhibitors of PI3K and protein kinase C blocked IGF-I-induced alpha(1B)-AR phosphorylation. In addition, it was observed that AG1478, an inhibitor of the epidermal growth factor (EGF) receptor kinase, and BB-94, a metalloproteinase inhibitor, also diminished IGF-I-induced adrenoceptor phosphorylation. The data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of a serpentine G protein-coupled receptor, suggesting the following hypothetical model: 1) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; 2) the growth factor action activates metalloproteinases, which catalyze heparin binding-EGF shedding, and transactivation of EGF receptors, and 3) dissociated Gbetagamma subunits and phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of protein kinase C, resulting in alpha(1B)-AR phosphorylation and desensitization.     

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3-kinase in rat hepatocyte primary culture.

Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1-18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15-30 s and with similar EC(50)s in the nM range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor beta-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.     

Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes

The hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy-induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum-free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty-eight hour co-treatment with 1,25(OH)(2)D(3) enhanced KGFR-mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c-Jun N-terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)(2)D(3) increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)(2)D(3) increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand-induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)(2)D(3) on KGFR-mediated ERK activation. Our results support the notion that the paracrine KGF-KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury.     

Platelet-derived growth factor requires epidermal growth factor receptor to activate p21-activated kinase family kinases

The platelet-derived growth factor (PDGF) receptor (PDGFR) transactivates the epidermal growth factor (EGF) receptor (ErbB1) to stimulate the cell migration of fibroblasts through an unknown mechanism (Li, J., Kim, Y. N. & Bertics, P. (2000) J. Biol. Chem. 275, 2951-2958). In this paper we provide evidence that the transactivation of the EGF receptor (EGFR) by PDGFR is essential for PDGF to activate p21-activated kinase (PAK) family kinases. Fetal calf serum (10%) transiently stimulates the PAK activity in NIH 3T3 fibroblasts. The activation of PAK was completely inhibited by either PDGFR-specific inhibitor (AG1295) or EGFR-specific inhibitor (AG1478), suggesting that serum requires either the PDGF- or EGF-dependent pathway or the combination of both to activate PAK. PDGF-induced activation of PAK is completely inhibited by either AG1295 or AG1478, indicating that PDGF requires both PDGFR and EGFR for PAK activation. In support of this notion, a mouse embryo fibroblast cell line derived from the EGFR -/- mouse (from Dr. Erwin Wagner) doesn't activate PAK in response to PDGF. Expression of human EGFR in this cell line restores the ability of the PDGF to induce PAK activation. Our results indicate that PDGF activates PAK through transactivation of ErbB1.     

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells

We investigated a signaling pathway leading to activation of extracellular signal-regulated protein kinase (Erk) 1 and 2 in Rat-2 cells stimulated with sphingosine 1-phosphate (S1P). S1P treatment transiently activated Erk-1/-2 in a dose-dependent manner, and its activation was blocked by pertussis toxin, expression of RasN17, or inhibition of Raf or MEK-1/-2. S1P-induced activation of Erk-1/-2 was also suppressed by the inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with the specific inhibitor AG1478, suggesting that activation of EGF receptor tyrosine kinase was involved in the signaling pathway. S1P-induced Erk-1/-2 activation was enhanced up to 2-fold by inhibiting protein kinase C (PKC) with GF109203X, and PKC inhibition in the absence of S1P treatment also activated Erk-1/-2. The stimulatory effects of Erk-1/-2 activation by PKC inhibition was blocked by treating cells with AG1478, suggesting the involvement of PKC in the regulation of EGF receptor tyrosine kinase activation that leads to Erk-1/-2 activation. Together, these results suggest that S1P activates the EGF receptor through a PKC-dependent pathway that links Ras signaling to the activation of Erk-1/-2 in Rat-2 cells.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Dopamine D2 receptor stimulation of mitogen-activated protein kinases mediated by cell type-dependent transactivation of receptor tyrosine kinases

Dopamine D2 receptor activation of extracellular signal-regulated kinases (ERKs) in non-neuronal human embryonic kidney 293 cells was dependent on transactivation of the platelet-derived growth factor (PDGF) receptor, as demonstrated by the effect of the PDGF receptor inhibitors tyrphostin A9 and AG 370 on quinpirole-induced phosphorylation of ERKs and by quinpirole-induced tyrosine phosphorylation of the PDGF receptor. In contrast, ectopically expressed D2 receptor or endogenous D2-like receptor activation of ERKs in NS20Y neuroblastoma cells, which express little or no PDGF receptor, or in rat neostriatal neurons was largely dependent on transactivation of the epidermal growth factor (EGF) receptor, as demonstrated using the EGF receptor inhibitor AG 1478 and by quinpirole-induced phosphorylation of the EGF receptor. The D2 receptor agonist quinpirole enhanced the coprecipitation of D2 and EGF receptors in NS20Y cells, suggesting that D2 receptor activation induced the formation of a macromolecular signaling complex that includes both receptors. Transactivation of the EGF receptor also involved the activity of a matrix metalloproteinase. Thus, although D2 receptor stimulation of ERKs in both cell lines was decreased by inhibitors of ERK kinase, Src-family protein tyrosine kinases, and serine/threonine protein kinases, D2-like receptors activated ERKs via transactivation of the EGF receptor in NS20Y neuroblastoma cells and rat embryonic neostriatal neurons, but via transactivation of the PDGF receptor in 293 cells.