Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor activation in primary hepatocytes. Role of acidic sphingomyelinase-mediated ceramide generation in FAS receptor activation

We have shown previously that bile acids can activate the JNK pathway and down-regulate cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis. In this study, the mechanism(s) by which deoxycholic acid (DCA) activates the JNK pathway were examined. FAS receptor (FAS-R) and acidic sphingomyelinase (ASM)-deficient hepatocytes were resistant to DCA-induced activation of the JNK pathway. Activation of the JNK pathway (2-3-fold) in response to tumor necrosis factor-alpha was similar in both wild-type and FAS-R(-/-) hepatocytes. In wild-type and FAS-R(-/-) hepatocytes, ceramide elevation was detected as early as 2 min and peaked at 10 min after DCA treatment. In contrast, ASM(-/-) hepatocytes were defective in DCA-induced ceramide generation. Treatment with DCA resulted in movement of FAS-R to the cell surface, which was blocked upon treatment with brefeldin A. However, brefeldin A failed to block DCA-mediated JNK activation in wild-type hepatocytes. DCA-induced JNK activation was independent of either the epidermal growth factor receptor activation or free radical generation. Addition of ASM to rat hepatocytes activated JNK and down-regulated CYP7A1 mRNA levels. In conclusion, these results show that DCA activates JNK and represses CYP7A1 mRNA levels in primary hepatocytes via an ASM/FAS-R-dependent mechanism that is independent of either the epidermal growth factor receptor or free radical generation.     

Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.     

UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation

Whereas poly-ubiquitination targets protein substrates for proteasomal degradation, mono-ubiquitination is known to regulate protein trafficking in the endosomal system and to target cargo proteins for lysosomal degradation. The role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal trafficking of cargo proteins such as the epidermal growth factor receptor (EGFR) has only very recently been the subject of study and is already a matter of debate. Although one report (Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and Komada, M. (2005) Mol. Biol. Cell 16, 5163-5174) concludes that UBPY negatively regulates EGFR degradation by de-ubiquitinating the EGFR on endosomes, another report (Row, P. E., Prior, I. A., McCullough, J., Clague, M. J., and Urbe, S. (2006) J. Biol. Chem. 281, 12618-12624) concludes that UBPY-mediated EGFR de-ubiquitination is essential for EGFR degradation. Here, we demonstrate that Usp8/UBPY, the mammalian ortholog of budding yeast Ubp4/Doa4, constitutively co-precipitates in a bivalent manner with the EGFR. Moreover, UBPY is a substrate for Src-family tyrosine kinases that are activated after ligand-induced EGFR activation. Using overexpression of three different recombinant dominant negative UBPY mutants (UBPY C748A mutant, UBPY 1-505, and UBPY 640-1080) in NIH3T3 and HEK293 cells, we demonstrate that UBPY affects both constitutive and ligand-induced (i) EGFR ubiquitination, (ii) EGFR expression levels, and (iii) the appearance of intermediate EGFR degradation products as well as (iv) downstream mitogen-activated protein kinase signal transduction. Our findings provide further evidence in favor of the model that UBPY-mediated EGFR de-ubiquitination promotes EGFR degradation.     

CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Pharmacogenomics of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors

The EGFR is a validated anticancer target whose successful exploitation has added novel agents to our current treatment protocols. Subsets of patients have shown to benefit the most from these therapies, and though these differential responses have yet to be completely defined, they are mostly of genetic nature. Egfr amplifications have shown to increase sensitivity to both small molecule inhibitors and specific monoclonal antibodies targeting the EGFR. A somatic/germline egfr intron 1 CA repeat sequence polymorphism has shown to have an important role in the control of EGFR protein expression, and has been linked to an increased risk of familial breast cancer, a worse outcome in patients with colorectal cancer, and anti-EGFR treatment efficacy in preclinical models. Egfr activating mutations have been recently described in lung cancer linking a cluster of genotypes with sensitivity to EGFR tyrosine kinase pharmacological inhibition. Despite the initial excitement that this discovery elicited, follow-up reports have not unequivocally confirmed this finding, and these drugs have been solidly efficacious both in individual patients and in diseases generally lacking egfr mutations such as pancreas cancer. We are witnessing exciting developments in the field of the pharmacogenomics of cancer, and this has particularly evolved in the area pertaining EGFR tyrosine kinase inhibitors. This review will discuss the background and currently available preclinical and clinical data.     

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.     

An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family

We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.     

Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination

Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.     

Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection

This paper presents an electrochemical immune sensor for label free detection of epidermal growth factor receptor (EGFR) by immobilizing anti-EGFR antibody (Anti-EGFRab) on dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) on gold (Au) electrode. Electrochemical studies show that increased surface concentration of redox moieties onto Anti-EGFRab/DTSP immuno-electrode leads to high electron transport and improved sensing performance. The antigen-antibody complex demonstrates a high association constant (5×10(12)L/mol) that results in high affinity of Anti-EGFRab to EGFR, confirming that the DTSP-SAM provides a conducive environment for anti-EGFR immobilization. The electrochemical response of EA/Anti-EGFRab/DTSP/Au electrode as a function of EGFR concentrations exhibits a linear range from 1pg/mL to 100ng/mL, a detection limit of 1pg/mL at a sensitivity of 2.02μAM(-1)at a regression coefficient of 0.99.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

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.     

Pasteurella multocida toxin stimulates mitogen-activated protein kinase via G(q/11)-dependent transactivation of the epidermal growth factor receptor

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.     

Four-membered heterocycles-containing 4-anilino-quinazoline derivatives as epidermal growth factor receptor (EGFR) kinase inhibitors

We report herein the design and synthesis of novel azaspirocycle or azetidine substituted 4-anilinoquinazoline derivatives. The EGFR inhibitory activities and in vitro antitumor potency of these newly synthesized compounds against two lung cancer cell lines HCC827 and A549 were evaluated. Most of the target compounds possess good inhibitory potency. In particular, compounds 21g with 2-oxa-6-azaspiro[3.4]octane substituent was found to possess higher EGFR inhibitory activities and similar antitumor potency comparing to the lead compound gefitinib with improved water solubility.     

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.