Expression and function of the bile acid receptor TGR5 in Kupffer cells

Kupffer cells are resident macrophages in the liver and play a central role in the hepatic response to injury. Bile acids can impair macrophage function leading to decreased cytokine release. TGR5 is a novel, membrane-bound bile acid receptor, and it has been suggested that the immunosuppressive effect of bile acids can be mediated by TGR5. However, the function of TGR5 in Kupffer cells has not been studied and a direct link between TGR5 and cytokine production in macrophages has not been established. The present study demonstrates that TGR5 is localized in the plasma membrane of isolated Kupffer cells and is responsive to bile acids. Furthermore, bile acids inhibited LPS-induced cytokine expression in Kupffer cells via TGR5-cAMP dependent pathways. TGR5-immunoreactivity in Kupffer cells was increased in rat livers following bile-duct ligation, suggesting that TGR5 may play a protective role in obstructive cholestasis preventing excessive cytokine production thereby reducing liver injury.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an increase in protein kinases growth hepatic associated with epidermal factor receptor in the plasma membrane

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), administered to male rats at a single intraperitoneal (IP) injection dose of 25 μg/kg causes down-regulation of epidermal growth factor (EGF) receptor in the plasma membrane of rat liver which starts after two days and continues throughout the experimental period (20 days). Using monoclonal antibody to EGF receptor, it was determined that TCDD-caused EFG receptor down-regulation in the rat liver was accompanied by increased protein kinase activity. Such an increase in the protein kinase activity involves, at least in part, an activation of protein tyrosine kinase. Examination of serum samples from control and treated rats revealed no detectable difference in the level of EGF itself or EGF receptor-reacting substances (eg, hormones and other growth factors). In vivo TCDD caused early eye opening and tooth eruption and poor body weight gain and hair growth in mouse neonates similar to those observed with exogenously administered EGE The results indicate that such EGF receptor–mediated effect of TCDD has some toxocilogical significance in vivo. Although TCDD causes significant reduction in [¹²⁵I]-EGF binding in the hepatic plasma membrane in susceptible strains of mice, it has only modest effects in tolerant strains. The results are consistent with the idea that the action of TCDD on the EGF receptor is mediated through the cytosoliclnuclear TCDD receptor, which is known to be regulated by the Ah locus.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Binding properties of biotinylated epidermal growth factor to its receptor on cultured cells and tissue sections

A biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N-biotinyl-epsilon-aminocaproyl-N-hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl-epsilon-caproyl-EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using the BioEGF probe.     

Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells

Class B scavenger receptor type I (SR-BI) is a multiligand membrane protein expressed in a variety of cell types. This receptor is responsible for the incorporation of lipids from high density lipoprotein (HDL) by steroidogenic cells, as well as for the phosphatidylserine (PS)-mediated phagocytosis of apoptotic cells by some phagocytic cell types, such as testicular Sertoli cells. Although SR-BI directly binds to PS present on the surface of apoptotic cells, as to whether SR-BI transmits signals to induce engulfment has not been clear. In the present study, we examined this issue using a monoclonal antibody that neutralizes SR-BI activity and a chemical known to be an inhibitor of the SR-BI-mediated incorporation of HDL lipids. The chemical compound inhibited the incorporation of HDL lipids and PS-containing liposomes by an SR-BI-expressing culture cell line, with no effect on the binding of these targets. Similarly, the phagocytosis of PS-exposing apoptotic cells by primary cultured rat Sertoli cells was inhibited in the presence of either reagent, not at the recognition but at the engulfment step. The addition of apoptotic cells or PS-containing liposomes caused a temporal increment of the phosphorylation of all three mitogen-activated protein kinases, p38, extracellular-signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK), in Sertoli cells. The increase of phosphorylated p38 and ERK, but not of phosphorylated JNK, was cancelled in the presence of the monoclonal antibody. Furthermore, the level of Sertoli cell phagocytosis of PS-exposing apoptotic cells, as well as that of PS-containing liposomes, was reduced only when the actions of p38 and ERK were simultaneously repressed. In conclusion, these results indicate that SR-BI, when it binds to PS, transmits signals to activate the mitogen-activated protein kinase pathway, which leads to the induction of the engulfment of PS-exposing apoptotic cells by phagocytic cells.     

Substrate specificities of insulin and epidermal growth factor receptor kinases

The abilities of insulin and EGF stimulated protein kinases to phosphorylate a series of exogenous substrates were compared using wheat germ lectin purified preparations of solubilized rat liver membranes. Three different kinds of substrates were found: substrates phosphorylated primarily by insulin stimulated kinase, substrates phosphorylated primarily by EGF stimulated kinase and substrates phosphorylated by both kinases to a similar extent. These results indicate that the insulin and the EGF receptor kinase have different, but overlapping, substrate specificities. In vivo, phosphorylation of cellular proteins by various hormone receptor kinases may be part of the signal transmission process for actions of the hormones. Different substrate specificities of kinases of different hormone receptors may therefore represent an important mechanism to preserve the specificity of the hormonal signal at the post receptor level.     

RME-8 regulates trafficking of the epidermal growth factor receptor

We recently identified receptor-mediated endocytosis 8 (RME-8), a DnaJ domain protein localized to endosomes. We now demonstrate that RME-8 depletion leads to decreased levels of epidermal growth factor receptor (EGFR) without influencing receptors that primarily recycle to the plasma membrane. Decreases in EGFR are detected at both surface and intracellular pools and result from increased rates of EGFR degradation. Interestingly, RME-8 depletion also decreases EGFR levels in breast cancer cell lines in which overexpression of the EGFR family member ErbB2 has been shown to protect EGFR from degradation. These data implicate RME-8 in sorting decisions influencing EGFR at the level of endosomes and point to RME-8 as a potential regulatory target in ErbB2-positive breast cancers.     

Effect of cycloheximide on epidermal growth factor receptor trafficking and signaling

Cycloheximide is the most common protein synthesis inhibitor, and is believed to specifically inhibit the cytoplasmic protein synthesis. Here we demonstrate that cycloheximide induces internalization and redistribution of EGF receptor to early endosomes in HeLa cells independent of receptor tyrosine phosphorylation, but dependent on p38 MAPK activity. Degradation of EGF receptor or its downstream effectors was not observed. EGF-induced activation of ERK1/2 was inhibited upon pre-treatment with cycloheximide, but did not activate JNK. The observed effects of treatment with cycloheximide alone are significant and therefore results involving the use of cycloheximide for inhibition of protein synthesis must be interpreted with caution.

Structured summary of protein interactions

EEA1 and EGFRcolocalize by fluorescence microscopy (View interaction).     

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.     

Involvement of hepatocyte epidermal growth factor receptor mediated activation of mitogen-activated protein kinase signaling pathways in response to growth inhibition by a novel K vitamin

Compound 5 (Cpd 5), a synthetic K vitamin analogue, or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is a potent inhibitor of epidermal growth factor (EGF)-induced rat hepatocyte DNA synthesis and induces EGF receptor (EGFR) tyrosine phosphorylation. To understand the cellular responses to Cpd 5, its effects on the EGF signal transduction pathway were examined and compared to those of the stimulant, EGF. Cpd 5 induced a cellular response program that included the induction of EGFR tyrosine phosphorylation and the activation of the mitogen-activated protein kinase (MAPK) cascade. EGFR tyrosine phosphorylation was induced by Cpd 5 in a time- and dose-dependent manner. Coimmunoprecipitation studies demonstrated that both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR was associated with increased amounts of adapter proteins Shc and Grb2, and the Ras GTP-GDP exchange protein Sos, indicating the formation of functional EGFR complexes. Although EGFR phosphorylation was induced both by the stimulant EGF and the inhibitor Cpd 5, the timing and intensity of activation by EGF and Cpd 5 were different. EGF activated EGFR transiently, whereas Cpd 5 induced an intense and sustained activation. Cpd 5-altered cells had a decreased ability to dephosphorylate tyrosine phosphorylated EGFR, providing evidence for an inhibition of tyrosine phosphatase activity. Both EGF and Cpd 5 caused an induction of phospho-extracellular response kinase (ERK), which was also more sustained with Cpd 5. Moreover, whereas Cpd 5 induced a striking translocation of phosphorylated ERK from cytosol to the nucleus, no significant nuclear translocation occurred after stimulation with EGF. The data suggest that this novel compound causes growth inhibition through antagonism of EGFR phosphatases and consequent induction of EGFR and ERK phosphorylation. This is supported by experiments with PD 153035 and PD 098059, antagonists of phosphorylation of EGFR and MAP kinase kinase (MEK), respectively, which both antagonized Cpd 5-induced phosphorylation and the inhibition of DNA synthesis. These results imply a mechanism of cell growth inhibition associated with intense and prolonged protein tyrosine phosphorylation. Protein tyrosine phosphatases may thus be a novel target for drugs designed to inhibit cell growth.     

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y₂ or P2Y₄ receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y₂ or P2Y₄ receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR.     

Insulin-like growth factor-binding protein-3 potentiates epidermal growth factor action in MCF-10A mammary epithelial cells. Involvement of p44/42 and p38 mitogen-activated protein kinases

Insulin-like growth factor-binding protein-3 (IGFBP-3) is inhibitory to the growth of many breast cancer cells in vitro; however, a high level of expression of IGFBP-3 in breast tumors correlates with poor prognosis, suggesting that IGFBP-3 may be associated with growth stimulation in some breast cancers. We have shown previously in MCF-10A breast epithelial cells that chronic activation of Ras-p44/42 mitogen-activated protein (MAP) kinase confers resistance to the growth-inhibitory effects of IGFBP-3 (Martin, J. L., and Baxter, R. C. (1999) J. Biol. Chem. 274, 16407-16411). Here we show that, in the same cell line, IGFBP-3 potentiates DNA synthesis and cell proliferation stimulated by epidermal growth factor (EGF), a potent activator of Ras. A mutant of IGFBP-3, which fails to translocate to the nucleus and has reduced ability to cell-associate, similarly enhanced EGF action in these cells. By contrast, the structurally related IGFBP-5, which shares many functional features with IGFBP-3, was slightly inhibitory to DNA synthesis in the presence of EGF. IGFBP-3 primes MCF-10A cells to respond to EGF because pre-incubation caused a similar degree of EGF potentiation as co-incubation. In IGFBP-3-primed cells, EGF-stimulated EGF receptor phosphorylation at Tyr-1068 was increased relative to unprimed cells, as was phosphorylation and activity of p44/42 and p38 MAP kinases, but not Akt/PKB. Partial blockade of the p44/42 and p38 MAP kinase pathways abolished the potentiation by IGFBP-3 of EGF-stimulated DNA synthesis. Collectively, these findings indicate that IGFBP-3 enhances EGF signaling and proliferative effects in breast epithelial cells via increased EGF receptor phosphorylation and activation of p44/42 and p38 MAP kinase signaling pathways.     

2,3,7,8-tetrachlorodibenzo-p- dioxin reduces high-affinity binding of epidermal growth factor to cell surface receptors in C3H 10T1/2 cells

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10^?12M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.     

Identification of a second egfr gene in Xiphophorus uncovers an expansion of the epidermal growth factor receptor family in fish

The epidermal growth factor receptor (EGFR) gives name to a family of receptors formed by four members in mammals (EGFR, ErbB2, ErbB3, and ErbB4). Members of this family can be activated to become potent oncogenes, and many human and animal tumors express qualitatively or quantitatively altered receptors from this group. We have isolated and characterized a second egfr gene in the melanoma model fish Xiphophorus. Both Xiphophorus egfra and egfrb duplicates are co-orthologs of the mammalian egfr gene. Database analysis showed that not only egfr but also erbB3 and erbB4 are present as duplicates in some fish species. They originated from ancient duplication events that might be consistent with the hypothesis of a fish-specific genome duplication. In Xiphophorus, the egfrb gene underwent a second duplication that generated the melanoma-inducing oncogene Xmrk. The study and comparison of some of the functional characteristics of both Xiphophorus EGF receptors, including expression profile, ligand-binding abilities, and intracellular signal transduction revealed that Xiphophorus Egfra not only shares common features with Egfrb and the human EGFR but also shows significant differences in its functional characteristics. The mechanism of maintenance of these duplicates remains to be clarified.