EGF-mediated phosphorylation of extracellular signal-regulated kinases in osteoblastic cells

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal-regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time-dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF-stimulated cells is not due to an increase in ERK protein, since EGF-treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (～45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1-selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12-myristate 13-acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lesser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF-stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down-regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF-mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback. © 1995 Wiley-Liss, Inc.     

Activation of extracellular-regulated kinases by normal and mutant EGF receptors

Glioblastoma cells express a mutant EGF receptor (EGFRvIII) that has constitutive tyrosine kinase activity and enhances their tumorigenicity. Here we show that EGFRvIII promotes constitutive phosphorylation of extracellular regulated kinases (ERKs) in glioblastoma cells in the absence of EGF. EGFRvIII also promoted constitutive activation of phosphoinositide 3-kinase in these cells, as assessed by phosphorylation of protein kinase B/akt. As expected, phosphorylation of protein kinase B/akt was blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Less expectedly, we found that this treatment also blocked EGFRvIII-induced phosphorylation of ERKs. In contrast, ERK phosphorylation induced by EGF-activated normal EGF receptor in the same cells was largely unaffected by treatment with phosphoinositide 3-kinase inhibitors. This difference in behavior between the normal receptor and EGFRvIII was not due to differences in the levels of activated EGFRvIII and wild-type EGF receptor, as the two types of receptor were tyrosine phosphorylated to a similar extent under the experimental conditions used. EGFRvIII activation of ERKs was also sensitive to the phospholipase C inhibitor U73122, whereas ERK activation by normal EGF receptor was not. These results show that EGFRvIII and wild-type EGF receptor preferentially use different signaling pathways to induce ERK phosphorylation. The different mechanisms of ERK activation used by normal and mutant EGF receptors may be important in understanding the potent tumorigenic activity of EGFRvIII.     

Involvement of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase 1/2 in EGF‐induced angiogenesis

Angiogenesis is a process during which endothelial cells divide and migrate to form new capillaries from the preexisting blood vessels. The present study was designed to investigate whether MAPKs (mitogen‐activated protein kinases) play crucial roles in regulating EGF (epidermal growth factor)‐induced endothelial cell angiogenesis. Our results showed that EGF stimulated HUVEC (human umbilical vein endothelial cells) proliferation in a concentration‐dependent manner, of which the maximum effective concentration of EGF was 10 ng/ml. Western blot analysis showed that EGF at 10 ng/ml significantly induced the phosphorylation of ERK1/2 (extracellular signal‐regulated kinase 1 and 2) and p38 kinase at 5 min, while it induced the phosphorylation of JNK/SAPK (c‐Jun N‐terminal kinase/stress‐activated protein kinase) at 15 min. Further results showed that a JNK/SAPK inhibitor, SP600125, and a specific siRNA JNK/SAPK could both significantly inhibit EGF‐induced tube formation in HUVEC cells, and an ERK1/2 inhibitor PD098059 could also block the tube formation in some content, while a p38 inhibitor SB203580 failed to do so. Furthermore, only SP600125 significantly inhibited EGF‐induced HUVEC cell proliferation under no cytotoxic concentration, so did JNK/SAPK siRNA. In conclusion, JNK/SAPK and ERK1/2 signals therefore play critical roles in EGF‐mediated HUVEC cell angiogenesis.     

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.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP.

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10(-8) M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line,LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10^?8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10^-8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

beta-Adrenergic-responsive activation of extracellular signal-regulated protein kinases in salivary cells: role of epidermal growth factor receptor and cAMP

The -adrenergic receptor agonist isoproterenol exerts growth-promoting effects on salivary glands. In this study, activation of ERKs, members of the mitogen-activated protein kinase family, by isoproterenol was examined in a human salivary gland cell line (HSY). Immunoblot analysis indicated that isoproterenol (10^–5 M) induced transient activation of ERK1/2 (4.4-fold relative to basal at 10 min) similar to that caused by EGF (6.7 fold). Isoproterenol, like EGF, also induced phosphorylation of the EGF receptor. However, inhibition of EGF receptor phosphorylation by the tyrphostin AG-1478 only partially attenuated isoproterenol-induced ERK phosphorylation, whereas EGF-responsive ERK activation was completely blocked. The G_i inhibitor pertussis toxin also caused partial inhibition of isoproterenol-stimulated ERK activation. The cAMP analog 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and the cAMP-elevating agents IBMX and cholera toxin produced transient ERK1/2 activation, similar to the effect of isoproterenol, in HSY cells. The stimulatory effects of isoproterenol and cAMP on ERK phosphorylation were not reduced by the PKA inhibitor H-89, whereas the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2) and transfection of a dominant-negative Src construct diminished isoproterenol-induced ERK activation. Isoproterenol induced marked overexpression of the cell growth-related adhesion molecule CD44, and this effect of isoproterenol was abolished by the ERK pathway inhibitor PD-98059. In summary, we show a dual mechanism of isoproterenol-induced ERK phosphorylation in HSY cells—one pathway mediated by EGF receptor transactivation and the other by an EGF receptor-independent pathway possibly mediated by cAMP. Our results also suggest that isoproterenol-induced growth of salivary tissue may involve ERK-mediated CD44 expression. mitogen-activated protein kinase; CD44     

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.     

2-Methoxyestradiol, an endogenous estradiol metabolite, differentially inhibits granulosa and endothelial cell mitosis: a potential follicular antiangiogenic regulator

2-Methoxyestradiol (2-ME) is an estradiol metabolite with antiangiogenic and antitumor activity. It is formed by granulosa cell (GC) catechol-O-methyltransferase activity and is present in the normal follicle at high concentrations. In this unique microenvironment, it may regulate selected cell types via autocrine and/or paracrine action. To assess the possibility that 2-ME or estradiol might exert differential mitotic and/or apoptotic effects on endothelial cells and GCs, we compared their actions on primary cultures of hormone- and/or growth factor-stimulated porcine GCs (pGCs) as well as two types of endothelial cells, primary cultures of porcine endothelial cells (pECs), and a spontaneously transformed rabbit endothelial vascular cell (REVC) line. The 2-ME, but not estradiol, dose dependently suppressed tritiated thymidine ((3)H-T) incorporation into epidermal growth factor (EGF)-stimulated REVCs and EGF/insulin (INS)-stimulated pECs. In contrast, 2-ME did not attenuate incorporation in FSH/INS-stimulated pGCs. It reduced incorporation by approximately 50% in EGF/INS-stimulated pGCs, indicating that responsiveness to 2-ME in normal cells can be modulated by hormone and growth factor treatment. Estradiol was not antimitotic to pGCs. As indicated by 4',6-diamido-2-phenylindole hydrochloride nuclear staining, estradiol was nonapoptotic in either cell type, and 2-ME significantly increased apoptosis of REVCs, but not of pGCs. In a cell migration assay, REVC movement was attenuated by 2-ME, but not by estradiol. In summary, the results show that antimitotic as well as proapoptotic responses to 2-ME vary with cell type and, in the case of pGC antimitotic activity, with the regulatory microenvironment. Thus, they provide a rationale for autocrine and/or paracrine action of 2-ME at its site of production in vivo, and they strongly support the concept of 2-ME as a candidate ovarian angiogenesis inhibitor.     

A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

It is well established that epidermal growth factor (EGF) is a potent mitogen in cells expressing EGF receptor (EGFR). However, a body of evidence indicated that the effects of mitogenic EGF signaling exhibit a non-monotonic, or biphasic dose response curve; EGF at low concentrations elicits a mitogenic signaling pathway to stimulate cell proliferation while at high concentrations, EGF inhibits cell growth. However, the molecular mechanism underlying this paradoxical effect of EGF on cell proliferation remains largely unknown. Here, we investigated the molecular mechanisms underlying the biphasic EGF signaling in ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells, both of which express endogenous EGFR. We found that EGF at low concentrations induced the phosphorylation of the Src-Y416 residue, an event to activate Src, while at high concentrations allowed Src-Y527 phosphorylation that inactivates Src. EGF at 10 ng/ml also induced phosphorylation of the MAPK/ERK and activated cyclin D1 promoter activity through the Src/EGFR/STAT5 pathways but not at a higher concentration (500 ng/ml). Our results thus demonstrated that Src functions as a switch of EGF signaling depending on concentrations of EGF.     

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.     

Growth hormone signalling and apoptosis in neonatal rat cardiomyocytes

Growth hormone (GH) has been reported to be useful to treat heart failure. To elucidate whether GH has direct beneficial effects on the heart, we examined effects of GH on oxidative stress-induced apoptosis in cardiac myocytes. TUNEL staining and DNA ladder analysis revealed that hydrogen peroxide (H₂O₂)-induced apoptosis of cardiomyocytes was significantly suppressed by the pretreatment with GH. GH strongly activated extracellular signal-regulated kinases (ERKs) in cardiac myocytes and the cardioprotective effect of GH was abolished by inhibition of ERKs. Overexpression of dominant negative mutant Ras suppressed GH-stimulated ERK activation. Overexpression of Csk that inactivates Src family tyrosine kinases also inhibited ERK activation evoked by GH. A broad-spectrum inhibitor of protein tyrosine kinases (PTKs), genistein, strongly suppressed GH-induced ERK activation and the cardioprotective effect of GH against apoptotic cell death. GH induced tyrosine phosphorylation of EGF receptor and JAK2 in cardiac myocytes, and an EGF receptor inhibitor tyrphostin AG1478 and a JAK2 inhibitor tyrphostin B42 completely inhibited GH-induced ERK activation. Tyrphostin B42 also suppressed the phosphorylation of EGF receptor stimulated by GH. These findings suggest that GH has a direct protective effect on cardiac myocytes against apoptosis and that the effect of GH is attributed at least in part to the activation of ERKs through Ras and PTKs including JAK2, Src, and EGF receptor tyrosine kinase.     

ERK phosphorylation is dependent on cell adhesion in a subset of pediatric sarcoma cell lines

Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.     

Betagamma subunits of G(i/o) suppress EGF-induced ERK5 phosphorylation, whereas ERK1/2 phosphorylation is enhanced

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation and gene expression. ERK5 is twice the size of ERK1/2, the amino-terminal half contains the kinase domain that shares the homology with ERK1/2 and TEY activation motif, whereas the carboxy-terminal half is unique. In this study, we examined the cross-talk mechanism between G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, focusing on ERK1/2 and 5. The pretreatment of rat pheochromocytoma cells (PC12) with pertussis toxin (PTX) specifically enhanced epidermal growth factor (EGF)-induced ERK5 phosphorylation. In addition, lysophosphatidic acid (LPA) attenuated the EGF-induced ERK5 phosphorylation in LPA(1) receptor- and G(i/o)-dependent manners. On the other hand, LPA alone activated ERK1/2 via Gbetagamma subunits and Ras and potentiated EGF-induced ERK1/2 phosphorylation at late time points. These results suggest G(i/o) negatively regulates ERK5, while it positively regulates ERK1/2. LPA did not affect cAMP levels after EGF treatment, and the reagents promoting cAMP production such as forskolin and cholera toxin also attenuated the EGF-induced ERK5 phosphorylation, indicating that the inhibitory effect of LPA on ERK5 inhibition via G(i/o) is not due to inhibition of adenylyl cyclase by Galpha(i/o). However, the inhibitory effect of LPA on ERK5 was abolished in PC12 cells stably overexpressing C-terminus of GPCR kinase2 (GRK2), and overexpression of Gbeta(1) and gamma(2) subunits also suppressed ERK5 phosphorylation by EGF. In response to LPA, Gbetagamma subunits interacted with EGF receptor in a time-dependent manner. These results strongly suggest that LPA negatively regulates the EGF-induced ERK5 phosphorylation through Gbetagamma subunits.     

IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation

IEX-1 is an early response and NF-kappaB target gene implicated in the regulation of cellular viability. We show here that IEX-1 is a substrate for ERKs and that IEX-1 and ERK regulate each other's activities. IEX-1 was isolated by phosphorylation screening with active ERK2 and found subsequently phosphorylated in vivo upon ERK activation. IEX-1 interacts with phosphorylated ERKs but not with c-jun N-terminal kinase (JNK) or p38. Upon phosphorylation by ERKs, IEX-1 acquires the ability to inhibit cell death induced by various stimuli. In turn, IEX-1 potentiates ERK activation in response to various growth factors. By using various IEX-1 mutants in which the ERK phosphoacceptor and/or ERK docking sites were mutated, we show that the IEX-1 pro-survival effect is dependent on its phosphorylation state but not on its ability to potentiate ERK activation. Conversely, IEX-1-induced modulation of ERK activation requires ERK-IEX-1 association but is independent of IEX-1 phosphorylation. Thus, IEX-1 is a new type of ERK substrate that has a dual role in ERK signaling by acting both as an ERK downstream effector mediating survival and as a regulator of ERK activation.     

Autocrine extracellular signal-regulated kinase (ERK) activation in normal human keratinocytes: metalloproteinase-mediated release of amphiregulin triggers signaling from ErbB1 to ERK

ErbB signaling through extracellular signal-regulated kinase (ERK) has been implicated in regulating the expression of ErbB ligands in hyperproliferative skin disorders and wound healing. Here, we characterize the process of autocrine ERK activation in cultured normal human keratinocytes (NHKs) subjected to growth factor (GF) deprivation. Basal ERK phosphorylation was lower after 48 h than after 24 h of GF deprivation, and lowest at 30-60 min after an additional medium change. ERK phosphorylation was markedly increased by low concentrations of epidermal growth factor (EGF) (0.2-1 ng/ml) that provoked only a limited increase in ErbB1 tyrosine phosphorylation and internalization. Basal ErbB tyrosine phosphorylation and ERK phosphorylation were inhibited by two different ErbB receptor tyrosine kinase inhibitors, by the ErbB1-specific neutralizing monoclonal antibody 225 IgG, by two different metalloproteinase inhibitors, and by neutralizing antibodies against amphiregulin (AR). In contrast, these responses were unaffected by neutralizing antibodies against other ErbB1 ligands or the ErbB2 inhibitors geldanamycin and AG825. The time course of autocrine ERK phosphorylation correlated with the appearance of soluble AR, and two different metalloproteinase inhibitors blocked AR release. These results define an amphiregulin- and ErbB1-dependent mechanism by which autocrine ERK activation is maintained in NHKs, even when ErbB1 autophosphorylation and internalization are limited.     

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0·5–1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2·5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor β subunit; shc phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK. © 1998 John Wiley & Sons, Ltd.     

Tyrosine Phosphorylation of Extracellular Signal-Regulated Protein Kinase 4 in Response to Growth Factors

Abstract: Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.     

Extracellular signal-regulated protein kinases mediate H(+),K(+)-ATPase alpha-subunit gene expression.

Extracellular signal-regulated protein kinases (ERKs) are important in many cellular functions. We and others have previously reported that prolonged exposure of gastric parietal cells to epidermal growth factor (EGF) enhanced gastric acid secretion stimulated by secretagogues via ERKs. In this study, we examined whether ERKs regulated H(+),K(+)-ATPase alpha-subunit gene expression using a gastric cancer cell line, AGS. EGF induced ERK activity time- and dose-dependently with a maximal effect observed at 10 min and 10 nM, respectively. The MEK inhibitors, U0126 and PD-98059, dose-dependently inhibited the ERK activity stimulated by EGF. To test H(+),K(+)-ATPase alpha-subunit gene expression, we transfected AGS cells with a plasmid containing a canine H(+),K(+)-ATPase alpha-subunit gene promoter fused to a luciferase reporter gene. EGF induced luciferase activity in transfected cells; this effect was inhibited by the MEK inhibitors, suggesting that EGF-induced gene expression involved the ERK pathway. When AGS cells were transfected with the reporter plasmids in conjunction with an expression vector encoding constitutively active MEK1, luciferase activity was strongly enhanced; this effect was attenuated by the MEK inhibitors or by an additional cotransfection of dominant negative MEK1. Taken together, our results led us to conclude that the ERK pathway may mediate H(+),K(+)-ATPase alpha-subunit gene expression, contributing to gastric acid secretion in parietal cells.