Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(q/11 )mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.     

Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.     

G protein-coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes

G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 alpha-subunit (Galphaq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Galphaq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Galphaq/11 signaling, including tyrosine phosphorylation of Galphaq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Galphaq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.     

The osmotic shock-induced glucose transport pathway in 3T3-L1 adipocytes is mediated by gab-1 and requires Gab-1-associated phosphatidylinositol 3-kinase activity for full activation

Osmotic shock treatment of 3T3-L1 adipocytes causes an increase in glucose transport activity and translocation of GLUT4 protein similar to that elicited by insulin treatment. Insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, however, activation by osmotic shock was only partially blocked. Additionally, we have found that the newly identified insulin receptor substrate Gab-1 (Grb2-associated binder-1) is tyrosine-phosphorylated following sorbitol stimulation. Treatment of cells with the tyrosine kinase inhibitor genistein inhibited osmotic shock-stimulated Gab-1 phosphorylation as well as shock-induced glucose transport. Furthermore, pretreatment with the selective Src family kinase inhibitor PP2 completely inhibited the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. We have also shown that microinjection of anti-Gab-1 antibody inhibits osmotic shock-induced GLUT4 translocation. Furthermore, phosphorylated Gab-1 binds and activates phosphatidylinositol 3-kinase (PI3K) in response to osmotic shock. The PI3K activity associated with Gab-1 was 82% of that associated with anti-phosphotyrosine antibodies, indicating that Gab-1 is the major site for PI3K recruitment following osmotic shock stimulation. Although wortmannin only causes a partial block of osmotic shock-stimulated glucose uptake, wortmannin completely abolishes Gab-1 associated PI3K activity. This suggests that other tyrosine kinase-dependent pathways, in addition to the Gab-1-PI3K pathway, contribute to osmotic shock-mediated glucose transport. To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway.     

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes

We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active G(q) (CA-G(q); Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-G(q) expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G alpha(q/11) in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-G(q), and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C lambda (PKC lambda) antibody or overexpression of a kinase-deficient PKC lambda construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active G(q) expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC lambda inhibition blocks CA-cdc42, CA-G(q), and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G alpha(q/11) and upstream of PI3-kinase and PKC lambda in this stimulatory pathway.     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

PYK2 as a mediator of endothelin-1/G alpha 11 signaling to GLUT4 glucose transporters

Endothelin-1 (ET-1) signaling through G alpha(q/11) stimulates translocation of intracellular GLUT4 glucose transporters to the plasma membrane of 3T3-L1 adipocytes by an unknown mechanism that requires protein tyrosine phosphorylation and ADP-ribosylation factor 6 (ARF6) but is independent of phosphatidylinositol 3 (PI3)-kinase. In contrast, insulin action on this process requires PI3-kinase but not ARF6. Here we report the identification of two proteins selectively tyrosine-phosphorylated in response to ET-1 but not insulin: the Ca(2+)-activated tyrosine kinase PYK2 and its physiological substrate, the adhesion scaffold protein paxillin. Endogenous paxillin as well as expressed Myc-tagged PYK2 or a Myc-tagged kinase-deficient PYK2 protein were acutely directed to F-actin-rich adhesion sites from the adipocyte cytoplasm in response to ET-1 but not insulin. CADTK-related non-kinase (CRNK) is a dominant negative form of PYK2 containing the C-terminal portion of the protein, which binds paxillin but lacks the PYK2 autophosphorylation site (Tyr(402)). CRNK expression in 3T3-L1 adipocytes inhibited ET-1-mediated F-actin polymerization and translocation of Myc-tagged GLUT4-enhanced green fluorescent protein (EGFP) to the plasma membrane without disrupting insulin action on these processes. These data reveal the tyrosine kinase PYK2 as a required signaling element in the regulation of GLUT4 recycling in 3T3-L1 adipocytes by ET-1, whereas insulin signaling is directed through a different pathway.     

Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4.

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.     

APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes

APS is a Cbl-binding protein that is tyrosine phosphorylated by the insulin receptor kinase. Insulin-stimulated phosphorylation of tyrosine 618 in APS is necessary for its association with c-Cbl and the subsequent tyrosine phosphorylation of Cbl by the insulin receptor in both 3T3-L1 adipocytes and CHO-IR cells. When overexpressed in these cells, wild-type APS but not an APS/Y(618)F mutant facilitated the tyrosine phosphorylation of coexpressed Cbl and its association with Crk upon insulin stimulation. APS-facilitated phosphorylation occurred on tyrosines 371, 700, and 774 in the Cbl protein. APS also interacted directly with the c-Cbl-associated protein (CAP) and colocalized with the protein in cells. The association was dependent on the SH3 domains of CAP and was independent of insulin treatment. Overexpression of the APS/Y(618)F mutant in 3T3-L1 adipocytes blocked the insulin-stimulated tyrosine phosphorylation of endogenous Cbl and binding to Crk. Moreover, the translocation of GLUT4 from intracellular vesicles to the plasma membrane was also inhibited by overexpression of the APS/Y(618)F mutant. These data suggest that APS serves as an adapter protein linking the CAP/Cbl pathway to the insulin receptor and, further, that APS-facilitated Cbl tyrosine phosphorylation catalyzed by the insulin receptor is a crucial event in the stimulation of glucose transport by insulin.     

Endothelin-1-stimulated glucose uptake is desensitized by tumor necrosis factor-alpha in 3T3-L1 adipocytes

Tumor necrosis factor-alpha (TNF-alpha) is a potent inducer of insulin resistance, and increased TNF-alpha expression is associated with impaired glucose disposal. Although insulin is the primary regulator of glucose transport in adipose, endothelin-1, a vasoconstrictor peptide that signals through the heterotrimeric G proteins Galphaq/11, potently stimulates glucose uptake in 3T3-L1 adipocytes by a mechanism independent of phosphatidylinositol (PI) 3-kinase. Here, we report that exposure of 3T3-L1 adipocytes to TNF-alpha for 48 h dose-dependently decreased endothelin-1-stimulated glucose uptake and translocation of GLUT4 to the plasma membrane. TNF-alpha exposure had no effect on endothelin-1 receptor number at the cell surface. In contrast, TNF-alpha treatment reduced the quantity of Galphaq/11 and proline-rich tyrosine kinase 2 (PYK2) and decreased endothelin-1-stimulated PYK2-Tyr402 tyrosine phosphorylation. Taken together, these results suggest that TNF-alpha-induced desensitization of endothelin-1-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes is due, at least in part, to a decreased expression of Galphaq/11, leading to a suppression in tyrosine phosphorylation of PYK2.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Natriuretic peptides inhibit G protein activation. Mediation through cross-talk between cyclic GMP-dependent protein kinase and regulators of G protein-signaling proteins

Atrial natriuretic peptide (ANP) inhibits the proliferation of many cells, in part through interfering with signal transduction enacted by G protein-coupled growth factor receptors. Signaling interactions between ANP and the G protein-coupled growth factor receptor ligand, endothelin-3 (ET-3), regulate astrocyte proliferation at a very proximal but undefined point. Here, we find that ANP inhibits the ability of ET-3 to activate Galpha(q) and Galpha(i) in these cells. ANP stimulated the translocation of endogenous regulators of G protein-signaling (RGS) proteins 3 and 4 from the cytosol to the cell membrane, and enhanced their association with Galpha(q) and Galpha(i). ANP effects were significantly blocked by HS-142-1, an inhibitor of guanylate cyclase activation, or by ET-3. KT5823, an inhibitor of cyclic GMP-dependent protein kinase (PKG) reversed the RGS translocation induced by ANP; conversely, expression of an active catalytic subunit of PKG-I, or 8-bromo-cyclic GMP stimulated RGS translocation. ANP caused the phosphorylation of both RGS proteins in a PKG-dependent fashion, and the expressed PKG (in the absence of ANP) also stimulated RGS phosphorylation. A novel cross-talk between PKG and RGS proteins is stimulated by ANP and leads to the increased translocation and association of RGS proteins with Galpha. The rapid inactivation of G proteins provides a mechanism by which ANP inhibits downstream signaling to the cell proliferation program.     

Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells

We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.     

Proximal events in signaling by plasma membrane estrogen receptors

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.     

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

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

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

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.     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。