C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.     

Activation of proteinase-activated receptor 1 promotes human colon cancer cell proliferation through epidermal growth factor receptor transactivation

Serine proteases are now considered as crucial contributors to the development of human colon cancer. We have shown recently that thrombin is a potent growth factor for colon cancer cells through activation of the aberrantly expressed protease-activated receptor 1 (PAR1). Here, we analyzed the signaling pathways downstream of PAR1 activation, which lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events on activation of PAR1 by thrombin or specific activating peptide: (a) a matrix metalloproteinase-dependent release of transforming growth factor-alpha (TGF-alpha) as shown with TGF-alpha blocking antibodies and measurement of TGF-alpha in culture medium; (b) TGF-alpha-mediated activation of epidermal growth factor receptor (EGFR) and subsequent EGFR phosphorylation; and (c) activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and subsequent cell proliferation. The links between these events are shown by the fact that stimulation of cell proliferation and ERK1/2 on activation of PAR1 is reversed by the MMP inhibitor batimastat, TGF-alpha neutralizing antibodies, EGFR ligand binding domain blocking antibodies, and the EGFR tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGFR seems to be a major mechanism whereby activation of PAR1 results in colon cancer cell growth. Finally, PAR1 activation induces Src phosphorylation, which is reversed by using the Src tyrosine kinase inhibitor PP2, suggesting that Src activation plays a permissive role for PAR1-mediated ERK1/2 activation and cell proliferation probably acting downstream of the EGFR. These data explain how thrombin exerts robust trophic action on colon cancer cells and underline the critical role of EGFR transactivation.     

Binding of epidermal growth factor to its receptor is affected by membrane phospholipid environment

Cells of epithelial origin generally require ethanolamine to grow in culture; when these cells are grown without ethanolamine, the phosphatidylethanolamine content of their membrane phospholipid becomes 1/2 to 1/3 of the normal amount, and growth stops. We have hypothesized that growth ceases because the phospholipid environment becomes unsuitable for membrane-associated function. Using ethanolamine-requiring rat mammary cells, we have investigated the possible effect of phosphatidylethanolamine deficiency on the binding characteristics of epidermal growth factor. Apparent dissociation constant for the high-affinity sites in cells having normal membrane phospholipid was 1.7 X 10(-10) M, whereas that of phosphatidylethanolamine-deficient cells was 2.7 X 10(-10) M: the difference was small, but significant. Pretreatment with phorbol ester caused the loss of high-affinity sites in cells having normal membrane, whereas binding characteristics of epidermal growth factor became refractory to the pretreatment in phosphatidylethanolamine-deficient cells. In addition, the rate of internalization of bound epidermal growth factor in phosphatidylethanolamine-deficient cells was about 1/4 of normal cells. Further, whether cells had normal or phosphatidylethanolamine-deficient membranes seemed to affect the phosphorylation patterns of membrane proteins in response to epidermal growth factor or phorbol ester. These results suggest that membrane phospholipid environment affects the activity of the epidermal growth factor receptor.     

Cyclooxygenase-2-derived prostaglandin E2 promotes human cholangiocarcinoma cell growth and invasion through EP1 receptor-mediated activation of the epidermal growth factor receptor and Akt

Cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis has recently been implicated in human cholangiocarcinogenesis. This study was designed to examine the mechanisms by which COX-2-derived prostaglandin E2 (PGE2) regulates cholangiocarcinoma cell growth and invasion. Immunohistochemical analysis revealed elevated expression of COX-2 and the epidermal growth factor (EGF) receptor (EGFR) in human cholangiocarcinoma tissues. Overexpression of COX-2 in a human cholangiocarcinoma cell line (CCLP1) increased tumor cell growth and invasion in vitro and in severe combined immunodeficient mice. Overexpression of COX-2 or treatment with PGE2 or the EP1 receptor agonist ONO-DI-004 induced phosphorylation of EGFR and enhanced tumor cell proliferation and invasion, which were inhibited by the EP1 receptor small interfering RNA or antagonist ONO-8711. Treatment of CCLP1 cells with PGE2 or ONO-DI-004 enhanced binding of EGFR to the EP1 receptor and c-Src. Furthermore, PGE2 or ONO-DI-004 treatment also increased Akt phosphorylation, which was blocked by the EGFR tyrosine kinase inhibitors AG 1478 and PD 153035. These findings reveal that the EP1 receptor transactivated EGFR, thus activating Akt. On the other hand, activation of EGFR by its cognate ligand (EGF) increased COX-2 expression and PGE2 production, whereas blocking PGE2 synthesis or the EP1 receptor inhibited EGF-induced EGFR phosphorylation. This study reveals a novel cross-talk between the EP1 receptor and EGFR signaling that synergistically promotes cancer cell growth and invasion.     

Polyubiquitination of the epidermal growth factor receptor occurs at the plasma membrane upon ligand-induced activation

We have previously shown that, although overexpression of mutant dynamin inhibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhibit ligand-induced translocation of the EGFR into clathrin-coated pits. In the present study, we demonstrate that, upon ligand binding and incubation at 37 degrees C, the EGFR was polyubiquitinated regardless of overexpression of mutant dynamin. In cells not overexpressing mutant dynamin, the EGFR was rapidly internalized and deubiquitinated. In cells being endocytosis-deficient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then eventually moved out of the coated pits and back onto the smooth plasma membrane. Polyubiquitination occurred equally efficiently in cells with or without intact clathrin-dependent endocytosis, while the kinetics of ubiquitination and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presence of endocytosis with the same kinetics as polyubiquitination of the EGFR, but that the EGF-induced monoubiquitination of Eps15 was somewhat reduced upon overexpression of mutant dynamin. Our data show that EGF-induced polyubiquitination of the EGFR occurs at the plasma membrane.     

YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR

Epidermal growth factor (EGF) is known to play key roles in skin regeneration and wound-healing. Here, we demonstrate that Pep2-YAC, a tripeptide covering residues 29-31 in the B loop of EGF, promotes the proliferation of HaCaT keratinocytes with activity comparable to EGF. The treatment of HaCaT cells with Pep2-YAC induced phosphorylation, internalization, and degradation of EGFR and organization of signaling complexes, which consist of Grb2, Gab1, SHP2, and PI3K. In addition, it sti mulated the phosphorylation of ERK1/2 at Thr 202/Tyr 204 and of Akt1 at Ser 473 and the nuclear translocation of EGFR, STAT3, c-Jun, and c-Fos. These results suggest that Pep2-YAC may be useful as a therapeutic agent for skin regeneration and wound-healing as an EGFR agonist. [BMB Reports 2014; 47(10): 581-586]     

Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor

We recently demonstrated that depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001) J. Biol. Chem. 276, 34617-34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MbetaCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MbetaCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MbetaCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MbetaCD-induced tyrosine phosphorylation of EGFR, indicating that MbetaCD activation of EGFR is ligand-independent. Moreover, MbetaCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MbetaCD-induced phosphorylation of EGFR, SHC, phospholipase C-gamma, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MbetaCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MbetaCD, in addition to its ability to disrupt membrane rafts.     

Akt-PDK1 complex mediates epidermal growth factor-induced membrane protrusion through Ral activation

We studied the spatiotemporal regulation of Akt (also called protein kinase B), phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] by using probes based on the principle of fluorescence resonance energy transfer. On epidermal growth factor (EGF) stimulation, the amount of PtdIns(3,4,5)P3 was increased diffusely in the plasma membrane, whereas that of PtdIns(3,4)P2 was increased more in the nascent lamellipodia than in the plasma membrane of the central region. The distribution and time course of Akt activation were similar to that of increased PtdIns(3,4)P2 levels, which were most prominent in the nascent lamellipodia. Moreover, we found that upon EGF stimulation 3-phosphoinositide-dependent protein kinase-1 (PDK1) was also recruited to nascent lamellipodia in an Akt-dependent manner. Because PDK1 is known to activate Ral GTPase and because Ral is required for EGF-induced lamellipodial protrusion, we speculated that the PDK1-Akt complex may be indispensable for the induction of lamellipodia. In agreement with this idea, EGF-induced lamellipodia formation was promoted by the overexpression of Akt and inhibited by an Akt inhibitor or a Ral-binding domain of Sec5. These results identified the Akt-PDK1 complex as an upstream positive regulator of Ral GTPase in the induction of lamellipodial protrusion.     

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21^WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α_vβ₃ were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.     

Integrin alpha5/beta1 mediates fibronectin-dependent epithelial cell proliferation through epidermal growth factor receptor activation

Human integrin alpha5 was transfected into the integrin alpha5/beta1-negative intestinal epithelial cell line Caco-2 to study EGF receptor (EGFR) and integrin alpha5/beta1 signaling interactions involved in epithelial cell proliferation. On uncoated or fibronectin-coated plastic, the integrin alpha5 and control (vector only) transfectants grew at similar rates. In the presence of the EGFR antagonistic mAb 225, the integrin alpha5 transfectants and controls were significantly growth inhibited on plastic. However, when cultured on fibronectin, the integrin alpha5 transfectants were not growth inhibited by mAb 225. The reversal of mAb 225-mediated growth inhibition on fibronectin for the integrin alpha5 transfectants correlated with activation of the EGFR, activation of MAPK, and expression of proliferating cell nuclear antigen. EGFR kinase activity was necessary for both MAPK activation and integrin alpha5/beta1-mediated cell proliferation. Although EGFR activation occurred when either the integrin alpha5-transfected or control cells were cultured on fibronectin, coprecipitation of the EGFR with SHC could be demonstrated only in the integrin alpha5-transfected cells. These results suggest that integrin alpha5/beta1 mediates fibronectin-induced epithelial cell proliferation through activation of the EGFR.     

Mechanisms for oncogenic activation of the epidermal growth factor receptor

The Epidermal growth factor receptor (EGFR) is a membrane spanning glycoprotein, which frequently has been implicated in various cancer types. The mechanisms by which EGFR becomes oncogenic are numerous and are often specific for each cancer type. In some tumors, EGFR is activated by autocrine/paracrine growth factor loops, whereas in others activating mutations promote EGFR signaling. Overexpression and/or amplification of the EGFR gene are prevalent in many cancer types leading to aberrant EGFR signaling. In addition, failure to attenuate receptor signaling by receptor downregulation can also lead to cellular transformation. Heterodimerization of EGFR with ErbB2 inhibits downregulation of EGFR and thereby prolongs growth factor signaling. This also indicates that cross-talk between EGFR and heterologous receptor systems serves as another mechanism for oncogenic activation of EGFR. Because of its role in tumor promotion, the EGFR has been intensely studied as a therapeutic target. There are currently two major mechanisms by which the EGFR is targeted: antibodies binding to the extracellular domain of EGFR and small-molecule tyrosine-kinase inhibitors. However, tumorigenesis is a multi-step process involving several mutations, which might explain why EGFR therapeutics has only been partially successful. This highlights the importance of pinpointing the mechanisms by which EGFR becomes oncogenic in a particular cancer. In this review, each of the above mentioned mechanisms will be discussed, as a detailed molecular and genetic understanding of how EGFR contributes to the malignant phenotype might offer new promise for the design, development and clinical evaluation of future tumor-specific anticancer approaches.     

Solubilization of membrane receptor for epidermal growth factor.

The membrane receptor for epidermal growth factor (EGF) has been solubilized from A-431 tumor cells using Triton X-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1.5 hrs and the requirement for polyethylene glycol precipitation to detect ¹²⁵I-EGF: receptor complexes on membrane filters. Properties of the solubilized EGF are characterized and compared to the properties of the particulate receptor. The specific binding capacity of the solubilized EGF receptor was 8.0 picomoles ¹²⁵I-EGF bound per mg protein--approximately 60% of the binding capacity of particulate receptor preparations. Also, solubilization of the EGF receptor resulted in a 10-fold decrease in the affinity of the receptor for ¹²⁵I-EGF.     

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.     

Chronic myelogenous leukaemia exosomes modulate bone marrow microenvironment through activation of epidermal growth factor receptor

Chronic myelogenous leukaemia (CML) is a clonal myeloproliferative disorder. Recent evidence indicates that altered crosstalk between CML and mesenchymal stromal cells may affect leukaemia survival; moreover, vesicles released by both tumour and non‐tumour cells into the microenvironment provide a suitable niche for cancer cell growth and survival. We previously demonstrated that leukaemic and stromal cells establish an exosome‐mediated bidirectional crosstalk leading to the production of IL8 in stromal cells, thus sustaining the survival of CML cells. Human cell lines used are LAMA84 (CML cells), HS5 (stromal cells) and bone marrow primary stromal cells; gene expression and protein analysis were performed by real‐time PCR and Western blot. IL8 and MMP9 secretions were evaluated by ELISA. Exosomes were isolated from CML cells and blood samples of CML patients. Here, we show that LAMA84 and CML patients’ exosomes contain amphiregulin (AREG), thus activating epidermal growth factor receptor (EGFR) signalling in stromal cells. EGFR signalling increases the expression of SNAIL and its targets, MMP9 and IL8. We also demonstrated that pre‐treatment of HS5 with LAMA84 exosomes increases the expression of annexin A2 that promotes the adhesion of leukaemic cells to the stromal monolayer, finally supporting the growth and invasiveness of leukaemic cells. Leukaemic and stromal cells establish a bidirectional crosstalk: exosomes promote proliferation and survival of leukaemic cells, both in vitro and in vivo, by inducing IL8 secretion from stromal cells. We propose that this mechanism is activated by a ligand–receptor interaction between AREG, found in CML exosomes, and EGFR in bone marrow stromal cells.