Cyclooxygenase-2 transactivates the epidermal growth factor receptor through specific E-prostanoid receptors and tumor necrosis factor-alpha converting enzyme

Cyclooxygenase-2 is often highly expressed in epithelial malignancies and likely has an active role in tumor development. But how it promotes tumorigenesis is not clearly defined. Recent evidence suggests that this may involve transactivation of the epidermal growth factor receptor through E-prostanoid receptors, but reports differ about the mechanism by which this occurs. We found that E-prostanoid receptors 2-4, but not 1, transactivated the epidermal growth factor receptor. This required metalloproteinase activity, leading to release of growth factors from the cell surface. Both transforming growth factor-alpha and amphiregulin were released in response to over-expression of cyclooxygenase-2, but betacellulin and heparin-binding EGF-like growth factor were not. The metalloproteinase tumor necrosis factor-alpha converting enzyme was required for proteolytic release of transforming growth factor-alpha. We also found that addition of epidermal growth factor receptor ligands to HEK293 cells induced cyclooxygenase-2 expression, suggesting that by activating epidermal growth factor receptor signaling, cyclooxygenase-2 potentially creates a self-perpetuating cycle of cell growth. Consistent with this, inhibition of cyclooxygenase-2 reduced growth of epidermal growth factor receptor over-expressing MCF-10A breast epithelial cells in three-dimensional culture.     

The modulation of apoptosis by cyclic AMP involves Akt and epidermal growth factor receptor

Adenosine 3',5'-cyclic monophosphate (cAMP) and transforming growth factor-beta are important regulators of many biological processes. In this study we investigated the effect and its potential mechanism of cAMP on transforming growth factor-beta1- and serum deprivation-induced apoptosis in Mv1Lu cells. Transforming growth factor-beta1 treatment or serum deprivation induces apoptotic response in Mv1Lu cells. Forskolin, a cAMP-elevating agent, or 8-Bromo-cAMP (8-B-cAMP), a cell permeable cAMP analogue, inhibited the cell proliferation and markedly enhanced apoptosis induced by transforming growth factor-beta1, but completely suppressed serum deprivation-induced apoptosis. Furthermore, forskolin decreased the Akt phosphorylation, and the inhibition of phosphatidylinositol-3 kinase by LY294002 sensitized Mv1Lu cells to transforming growth factor-beta1-induced apoptosis. In addition, forskolin treatment induced tyrosine phosphorylation of epidermal growth factor receptor. Inhibition of epidermal growth factor receptor by specific inhibitor PD153035 blocked the cAMP-mediated suppression of serum deprivation-induced apoptosis. The results indicate that cAMP exerts its opposite effects in transforming growth factor-beta1- and serum deprivation-induced apoptosis via a mechanism involving the modulation of signaling components of phosphatidylinositol-3-kinase/Akt and epidermal growth factor receptor in Mv1Lu cells.     

The effect of transforming growth factor-beta on the alkaline phosphatase activity in rabbit renal cortical tubular cell cultures

The effect of various growth regulators including epidermal growth factor and transforming growth factor-beta on the alkaline phosphatase activity of rabbit renal cortical tubular cells has been investigated in a serum-free culture. As a result, it was found that transforming growth factor-beta, known to be a growth inhibitor of renal tubular cells, increased the alkaline phosphatase activity of the tubular cells dose-dependently and that cycloheximide blocked any increase in the activity of this factor. In contrast, epidermal growth factor decreased the alkaline phosphatase activity in the tubular cells.     

Structural basis for epidermal growth factor receptor function

The receptor for epidermal growth factor (EGF) has been the subject of intense study primarily as a consequence of the pioneering studies of Cohen on growth factors and also because of its homology to the transforming protein encoded by the avian oncogene v-erbB, which is a truncated receptor, and its consequent role in cancer. Although similar structural mutation of the EGF receptor has not yet been found in human tumours, aberrant overexpression of both EGF receptors and c-erbB2, a closely related putative receptor [1], have been found to occur in squamous cell carcinomas and glial tumours, and mammary carcinomas respectively [2–4]. In addition to EGF, the related polypeptides transforming growth factor α (TGFα) and vaccinia virus growth factor [5] are also ligands for the EGF receptor. Expression of TGFα occurs during embryonal development and in specific adult tissues; it may also play a role in cellular transformation (reviewed in Ref. 6). These important properties, as well as the potential roles of both TGFα and EGF in wound repair, have emphasized the need to understand EGF receptor structure, function and regulation. This review discusses the structural properties of the EGF receptor and how these can be related to receptor function and regulation.     

Immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

This study was undertaken to determine the immunocytochemical localization of transforming growth factor α, epidermal growth factor and epidermal growth factor receptor in the endometrium of ovariectomized cats treated with oestradiol-17β and/or progesterone and in the endometrium and placenta of pregnant cats. Specific immunostaining was observed for all three antibodies. Moderate immunostaining for transforming growth factor α was observed in the epithelium of ovariectomized and oestrogen-treated cats. Dark epithelial staining was observed throughout pregnancy. The epithelial cells in progesterone-treated and peri-implantation animals contained dense deposits of reaction product, which were not reduced in intensity when immunoabsorbed antiserum was used. For epidermal growth factor, light--moderate epithelial staining was observed in ovariectomized and steroid-treated animals, and this increased in pregnant cats. Stromal staining for both the transforming and the epidermal growth factors was limited in steroid-treated animals and increased as pregnancy continued. Dark staining for epidermal growth factor receptor was observed in the epithelium and stroma in all the animals studied. The tips of surface epithelial convolutions in the non-implantation sites were always more darkly stained than in other regions of the surface epithelium. Staining in the placental trophoblast was limited to the syncytiotrophoblast for the two growth factors and the cytotrophoblast for the receptor during most of pregnancy and was absent late in pregnancy. The placental maternal giant cells contained specific immunoreactivity for all the immunogens from the middle of pregnancy to term. This study demonstrates that the two growth factors and the epidermal growth factor receptor are present in the endometrium and placenta of cats and suggests that these growth factors may play an autocrine/paracrine role during reproduction     

Growth factor receptors in helminth parasites: signalling and host-parasite relationships

Parasitic helminths remain major pathogens of both humans and animals throughout the world. The success of helminth infections depends on the capacity of the parasite to counteract host immune responses but also to exploit host-derived signal molecules for its development. Recent progress has been made in the characterization of growth factor receptors of various nematode and flatworm parasites with the demonstration that transforming growth factor beta (TGF-beta), epidermal growth factor (EGF) and insulin receptor signalling pathways are conserved in helminth parasites and potentially implicated in the host-parasite molecular dialogue and parasite development.     

An extra cysteine proximal to the transmembrane domain induces differential cross-linking of p185neu and p185neu.

The neu proto-oncogene encodes a receptor tyrosine kinase (p185) that is closely related to the epidermal growth factor receptor. It has been proposed that receptor tyrosine kinases are activated through oligomerization. Because this clustering model predicts that oligomerization of receptors is sufficient to activate them, we determined if p185 can be activated by introducing an extra cysteine proximal to the transmembrane domain. This should induce inter-receptor disulfide bonding and, according to the clustering model, activate the receptor. This amino acid substitution enhanced recovery of both normal and transforming neu proteins as dimers, with normal p185 recovered predominantly as monomers and transforming p185* as dimers. However, the cysteine substitution did not affect the transforming activity of the two proteins.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Malignant transformation of murine fibroblasts by a human c-Ha-ras-1 oncogene does not require a functional epidermal growth factor receptor.

Although mutations in ras genes are thought to be important for the development of about 20% of human tumors, almost nothing is known about the way in which these mutations lead to cellular transformation. The known biochemical properties of the 21-kilodalton ras proteins suggest that they may behave as G proteins, regulating the proliferation of cells in response to growth factor stimulation of a receptor. Although the putative receptor(s) has not been identified, several lines of evidence, in particular the fact that rodent cell lines containing ras oncogenes produce transforming growth factor alpha, have suggested that the epidermal growth factor (EGF) receptor is involved in ras transformation. Here we show that murine fibroblasts with no EGF receptors can be transformed to a completely malignant phenotype with a mutated ras gene. It appears, therefore, that the EGF receptor is not required for ras-mediated transformation of these cells.     

Epidermal growth factor: structure, location, phosphorylation and regulation of its receptor

Epidermal growth factor (EGF) is a Mr 6045 polypeptide first characterized for its ability to stimulate mitogenesis in epidermal and epithelial cells. The first step in the action of the growth factor is its binding to specific, high affinity membrane receptors. These receptors have been studied in a number of tissues and cell culture lines. The level of EGF receptors is modulated by many agents. EGF down-regulates its receptor. In addition, the number of EGF receptors is decreased by other growth factors (platelet-derived growth factor; transforming growth factor), by many tumor promoters and by viral transformation. Several hormones also can regulate EGF binding in its target tissues.     

Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.     

A high-resolution 1H-NMR study of human transforming growth factor alpha. Structure and pH-dependent conformational interconversion

The 500-MHz and 600-MHz 1H-NMR spectra of recombinant human transforming growth factor alpha have been recorded at pH values of 3.8, 6.5 and 9.4. Analysis of various two-dimensional spectra has enabled sequence-specific assignments to be made and the secondary structure to be identified. Information on the tertiary fold has also been obtained from observed nuclear Overhauser effects and titration of histidine residues. The overall fold of the protein is very similar to that of epidermal growth factor, as might be expected from the sequence similarity. However, the structure of transforming growth factor alpha at pH 3.8 is found to show interesting differences from those at the two higher pHs and from that of epidermal growth factor.     

Epidermal growth factor receptor internalization through clathrin-coated pits requires Cbl RING finger and proline-rich domains but not receptor polyubiquitylation

Cbl proteins have been implicated in the regulation of endocytic trafficking of epidermal growth factor receptor. However, the precise role of Cbl in epidermal growth factor receptor endocytosis is not defined. To directly visualize Cbl in cells and perform structure-function analysis of Cbl's role in epidermal growth factor receptor internalization, a yellow fluorescent protein-fusion of c-Cbl was constructed. Upon epidermal growth factor receptor activation, Cbl-yellow fluorescent protein moved with epidermal growth factor receptor to clathrin-coated pits and endosomes. Localization of Cbl-yellow fluorescent protein to these endocytic organelles was dependent on a proline-rich domain of c-Cbl that interacts with Grb2 as shown by fluorescence resonance energy transfer microscopy. In contrast, direct binding of Cbl to phosphotyrosine 1045 of the epidermal growth factor receptor was required for epidermal growth factor receptor polyubiquitination, but was not essential for Cbl-yellow fluorescent protein localization in epidermal growth factor receptor-containing compartments. These data suggest that the binding of Cbl to epidermal growth factor receptor through Grb2 is necessary and sufficient for Cbl function during clathrin-mediated endocytosis. Overexpression of c-Cbl mutants that are capable of Grb2 binding but defective in linker/RING finger domain function severely inhibited epidermal growth factor receptor internalization. The same dominant-negative mutants of Cbl did not block epidermal growth factor receptor recruitment into coated pits but retained receptors in coated pits, thus preventing receptor endocytosis and transport to endosomes. These data suggest that the linker and RING finger domain of Cbl may function during late steps of coated vesicle formation. We propose that the RING domain of Cbl facilitates endocytosis either by epidermal growth factor receptor monoubiquitylation or by ubiquitylation of proteins associated with the receptor.     

Heparin-binding growth factor/prostatropin attenuates inhibition of rat prostate tumor epithelial cell growth by transforming growth factor type beta

Summary Normal rat prostate epithelial cell growth requires both epidermal growth factor and heparin-binding growth factor/prostatropin. In contrast, epithelial cells derived from the transplantable Dunning R3327H rat tumor require either epidermal growth factor or heparin-binding growth factor/prostatropin. Transforming growth factor type beta inhibited normal epithelial cell growth. Transforming growth factor beta inhibited epidermal growth factor-dependent growth of tumor epithelial cells, independent of epidermal growth factor concentrations. Transforming growth factor beta increased the effective dose of heparin-binding growth factor type 1 required to support tumor epithelial cell growth by 10-fold but saturating levels of heparin-binding growth factor type 1 (290 pM) completely attenuated the inhibitory effect of transforming growth factor beta. These results suggest that prostate tumor epithelial cells may escape the inhibitory effect of transforming growth factor beta as a consequence of alteration of the concurrent requirement for both epidermal growth factor (or homologues) and heparin-binding growth factors. This work was supported by NCI Grant CA37589. Editor’s Statement The observation that heparin-binding growth factor/prostatropin can counteract the inhibitory effect of transforming growth factor beta in prostate epithelial cells may help explain how some cancers avoid the action of growth inhibitors and provides a model for studying how inhibitory peptides overcome the stimulatory signals generated by growth factors.     

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.     

Altered distribution of phospholipase C-gamma 1 in benign hyperproliferative epidermal diseases.

Phospholipase C-gamma 1 (PLC-gamma 1) is a well characterized substrate for the epidermal growth factor receptor tyrosine kinase and has been implicated in the intracellular biochemical signaling cascade which occurs following stimulation of cells with epidermal growth factor. The in vivo localization of PLC-gamma 1 was examined by immunohistochemistry in sections of normal human skin and in skin sections from a diverse series of hyperproliferative epidermal conditions (psoriasis, seborrheic keratoses, acrochordons, and margins near second-degree burns). Immunoreactive PLC-gamma 1 was detected only in the basal compartment of normal skin but was readily detectable in both the basal and outer epidermal compartment in hyperproliferative skin conditions. In addition, immunoreactive PLC-gamma 1 colocalizes with immunoreactive epidermal growth factor receptor in both normal and hyperproliferative epidermis.