Insulin-like effects of epidermal growth factor and insulin-like growth factor-I on [3H]2-deoxyglucose uptake, diacylglycerol generation and protein kinase C activation in BC3H-1 myocytes.
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ABSTRACT:
SUBMITTER: Farese RV
PROVIDER: S-EPMC1138918 | biostudies-other | 1989 Aug
REPOSITORIES: biostudies-other
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Project description:We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)
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Project description:Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKdelta, we disrupted its gene in mice and found that DGKdelta deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKdelta-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGKdelta knockout cells, indicating that DGKdelta regulates EGFR by modulating PKC signaling.
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Project description:Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up-regulated gene. IGFBP-3 regulates cell proliferation through both insulin-like growth factor-dependent and independent mechanisms. We found that IGFBP-3 mRNA and protein expression was increased in EGFR-overexpressing primary and immortalized human esophageal cells. IGFBP-3 was also up-regulated in EGFR-overexpressing cells in organotypic culture and in EGFR transgenic mice. Furthermore, IGFBP-3 mRNA was overexpressed in 80% of primary esophageal squamous cell carcinomas and 60% of primary esophageal adenocarcinomas. Concomitant up-regulation of EGFR and IGFBP-3 was observed in 60% of primary esophageal squamous cell carcinomas. Immunohistochemistry revealed cytoplasmic localization of IGFBP-3 in the preponderance of preneoplastic and neoplastic esophageal lesions. IGFBP-3 was also overexpressed in esophageal cancer cell lines at both mRNA (60%) and protein (40%) levels. IGFBP-3 secreted by cancer cells was capable of binding to insulin-like growth factor I. Functionally, epidermal growth factor appeared to regulate IGFBP-3 expression in esophageal cancer cell lines. Finally, suppression of IGFBP-3 by small interfering RNA augmented cell proliferation, suggesting that IGFBP-3 may inhibit tumor cell proliferation as a negative feedback mechanism. In aggregate, we have identified for the first time that IGFBP-3 is an aberrantly regulated gene through the EGFR signaling pathway and it may modulate EGFR effects during carcinogenesis.
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Project description:Insulin receptor (IR) and the epidermal growth factor receptor (EGFR) were the first receptor tyrosine kinases (RTKs) to be studied in detail. Both are important clinical targets-in diabetes and cancer, respectively. They have unique extracellular domain compositions among RTKs, but share a common module with two ligand-binding leucine-rich-repeat (LRR)-like domains connected by a flexible cysteine-rich (CR) domain (L1-CR-L2 in IR/domain, I-II-III in EGFR). This module is linked to the transmembrane region by three fibronectin type III domains in IR, and by a second CR in EGFR. Despite sharing this conserved ligand-binding module, IR and EGFR family members are considered mechanistically distinct-in part because IR is a disulfide-linked (αβ)2 dimer regardless of ligand binding, whereas EGFR is a monomer that undergoes ligand-induced dimerization. Recent cryo-electron microscopy (cryo-EM) structures suggest a way of unifying IR and EGFR activation mechanisms and origins of negative cooperativity. In EGFR, ligand engages both LRRs in the ligand-binding module, "closing" this module to break intramolecular autoinhibitory interactions and expose new dimerization sites for receptor activation. How insulin binds the activated IR was less clear until now. Insulin was known to associate with one LRR (L1), but recent cryo-EM structures suggest that it also engages the second LRR (albeit indirectly) to "close" the L1-CR-L2 module, paralleling EGFR. This transition simultaneously breaks autoinhibitory interactions and creates new receptor-receptor contacts-remodeling the IR dimer (rather than inducing dimerization per se) to activate it. Here, we develop this view in detail, drawing mechanistic links between IR and EGFR.
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Project description:Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.
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Project description:Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.
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Project description:Prostasin (PRSS8) is a serine protease that metabolizes and moderates the effect of specific substrates. Epidermal growth factor receptor (EGFR), which modulates insulin secretion and pancreatic β-cell proliferation, is regulated via proteolytic shedding by PRSS8. We first detected PRSS8 expression in β-cells of pancreatic islets of mice. To better understand the molecular processes involved in PRSS8-associated insulin secretion, pancreatic β-cell-specific PRSS8 knockout (βKO) and PRSS8-overexpressing (βTG) male mice were generated. We found that glucose intolerance and reduction in glucose-stimulated insulin secretion developed in βKO mice compared with the control subjects. A higher response to glucose was noted in islets retrieved from βTG mice. Erlotinib, a specific blocker of EGFR, blocks EGF- and glucose-stimulated secretion of insulin among MIN6 cells, and glucose improves EGF release from β-cells. After silencing PRSS8 in MIN6 cells, glucose-stimulated insulin secretion decreased, and EGFR signaling was impaired. Conversely, overexpression of PRSS8 in MIN6 cells induced higher concentrations of both basal and glucose-stimulated insulin secretion and increased phospho-EGFR concentrations. Furthermore, short-term exposure to glucose improved the concentration of endogenous PRSS8 in MIN6 cells through inhibition of intracellular degradation. These findings suggest that PRSS8 is involved in glucose-dependent physiological regulation of insulin secretion via the EGF-EGFR signaling pathway in pancreatic β-cells.
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Project description:The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr(845) and Tyr(1173), but not at Tyr(1045), suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin- and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin beta1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294-21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin beta1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin beta1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin- or hypoosmolarity-induced hepatocyte swelling triggers an integrin- and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.
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Project description:The Epidermal Growth Factor Receptor (EGFR) is frequently mutated and overexpressed in metastatic cancer. Although EGFR is a transmembrane tyrosine kinase localized to the basolateral membrane in normal epithelium, it is frequently found intracellularly localized in transformed cells. We have previously demonstrated the epithelial adaptor protein mucin 1 (MUC1) alters trafficking of EGFR, inhibiting its degradation and promoting its translocation to the nucleus, where it can directly modulate gene transcription. Here, we demonstrate that MUC1 promotes the retention of EGF-bound EGFR in Early Endosome Antigen1 (EEA1)-positive vesicles while preventing its trafficking to the lysosome. These events result in the accumulation of endosomal vesicles harboring active receptor throughout the cell and a reorganization of the actin cytoskeleton. EGF-dependent cell migration and filopodia formation is reliant upon this altered trafficking, and can be prevented by blocking retrograde trafficking. Together, these results indicate that intracellular EGFR may play an essential role in cancer metastasis and a potential mechanism for the failure of therapeutic antibodies in EGFR-driven metastatic breast cancer.
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Project description:Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake.
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