Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase Cgamma1. Consistent with these findings, the VEGFR-1-specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2-activated phospholipase Cgamma1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (−) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER− and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER− and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER− cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER− human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF–EGFR interaction is mediated via activation of nuclear factor κB (NF-κB). The basal level of active NF-κB in ER− cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-κB activation factor. NF-κB transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER− cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294–002, blocked this event, suggesting a role of the former in the activation of NF-κB by EGF. Go6976, a well-characterized NF-κB inhibitor, blocked EGF-induced NF-κB activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER− cells. Thus Go6976 and similar NF-κB inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER− breast cancer patients.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear H-1-N-15 NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta -strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. N-15 longitudinal and transverse relaxation rates, and {H-1}-N-15 heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85 +/- 0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides. (C) 2001 Academic Press.

Premature calvarial synostosis and epidermal hyperplasia (Beare-Stevenson syndrome-like anomalies) resulting from a P250R missense mutation in the gene encoding fibroblast growth factor receptor 3

We report on a patient with a severe premature calvarial synostosis and epidermal hyperplasia. The phenotype was consistent with that of a mild presentation of Beare-Stevenson syndrome but molecular analysis of the IgIII-transmembrane linker region and the transmembrane domain of the gene encoding the FGFR2 receptor, revealed wild-type sequence only. Subsequently, molecular analysis of the FGFR3 receptor gene identified a heterozygous P250R missense mutation in both the proposita and her mildly affected father. This communication extends the clinical spectrum of the FGFR3 P250R mutation to encompass epidermal hyperplasia and documents the phenomenon of activated FGFR receptors stimulating common downstream developmental pathways, resulting in overlapping clinical outcomes. (C) 2001 Wiley-Liss, Inc.

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.

A single in vivo administration of bombesin antagonist RC-3095 reduces the levels and mRNA expression of epidermal growth factor receptors in MXT mouse mammary cancers

Epidermal growth factor (EGF) and its receptors (EGFR) play important roles in tumorigenesis. In various experimental cancers, treatment with antagonists of bombesin/gastrin-releasing peptide (BN/GRP) produces a reduction in EGFRs, concomitant to inhibition of tumor growth. To investigate the mechanisms involved, we monitored concentrations of BN/GRP antagonist RC-3095 in serum of mice, rats, and hamsters given a single subcutaneous or intravenous injection of this analog. In parallel studies, we measured levels and mRNA expression of EGFRs in estrogen-dependent and independent MXT mouse mammary cancers, following a single subcutaneous administration of RC-3095 to tumor-bearing mice. Peak values of RC-3095 in serum were detected 2 min after intravenous or 15 min after subcutaneous injection. The levels of RC-3095 declined rapidly and became undetectable after 3–5 hr. In the estrogen-dependent MXT tumors, the concentration of EGF receptors was reduced by about 60% 6 hr following injection and returned to original level after 24 hr. Levels of mRNA for EGFR fell parallel with the receptor number and were nearly normal after 24 hr. In the hormone-independent MXT cancers, the number of EGFRs decreased progressively, becoming undetectable 6 hr after injection of RC-3095, and returned to normal values at 24 hr, but EGFR mRNA levels remained lower for 48 hr. Thus, in spite of rapid elimination from serum, BN/GRP antagonist RC-3095 can induce a prolonged decrease in levels and mRNA expression of EGFRs. These findings may explain how single daily injections of BN/GRP antagonists can maintain tumor growth inhibition.

Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

Stimulation of β1-Integrin Function by Epidermal Growth Factor and Heregulin-β Has Distinct Requirements for erbB2 but a Similar Dependence on Phosphoinositide 3-OH Kinase

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the β1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest β1-dependent increases in cell adhesion and motility, heregulin-β (HRGβ) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGβ-mediated adhesion and potently blocked HRGβ- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate β1-integrin function.

Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor–ErbB2 Heterodimers in Response to EGF Stimulation

Epidermal growth factor (EGF) stimulates the homodimerization of EGF receptor (EGFR) and the heterodimerization of EGFR and ErbB2. The EGFR homodimers are quickly endocytosed after EGF stimulation as a means of down-regulation. However, the results from experiments on the ability of ErbB2 to undergo ligand-induced endocytosis are very controversial. It is unclear how the EGFR–ErbB2 heterodimers might behave. In this research, we showed by subcellular fractionation, immunoprecipitation, Western blotting, indirect immunofluorescence, and microinjection that, in the four breast cancer cell lines MDA453, SKBR3, BT474, and BT20, the EGFR–ErbB2 heterodimerization levels were positively correlated with the ratio of ErbB2/EGFR expression levels. ErbB2 was not endocytosed in response to EGF stimulation. Moreover, in MDA453, SKBR3, and BT474 cells, which have very high levels of EGFR–ErbB2 heterodimerization, EGF-induced EGFR endocytosis was greatly inhibited compared with that in BT20 cells, which have a very low level of EGFR–ErbB2 heterodimerization. Microinjection of an ErbB2 expression plasmid into BT20 cells significantly inhibited EGF-stimulated EGFR endocytosis. Coexpression of ErbB2 with EGFR in 293T cells also significantly inhibited EGF-stimulated EGFR endocytosis. EGF did not stimulate the endocytosis of ectopically expressed ErbB2 in BT20 and 293T cells. These results indicate that ErbB2 and the EGFR–ErbB2 heterodimers are impaired in EGF-induced endocytosis. Moreover, when expressed in BT20 cells by microinjection, a chimeric receptor composed of the ErbB2 extracellular domain and the EGFR intracellular domain underwent normal endocytosis in response to EGF, and this chimera did not block EGF-induced EGFR endocytosis. Thus, the endocytosis deficiency of ErbB2 is due to the sequence of its intracellular domain.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins.

Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wild-type counterpart. Immunocytochemistry confirmed ligand-induced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.

Epidermal growth factor in liposomes may enhance osteoclast recruitment during tooth movement in rats

Objective: To evaluate the effects of local administration of epidermal growth factor (EGF) located within liposomes on recruitment of osteoclasts during mechanical force in rats. Materials and Methods: An orthodontic elastic band was inserted between the left upper first and second molars, to move mesially the first molar. Rats were randomly divided into 4 groups (n = 8): EGF (2 ng/mu L) located within liposomes (group 1), liposomes only (group 2), soluble EGF (2 ng/mu L; group 3), or vehicle alone (group 4). The solutions were injected into the region of the root furcation of the left first molar after elastic band insertion. Tooth movement was measured using a plaster model of the maxilla, and the number of osteoclasts recruited at the pressure side of the first molar was histologically evaluated. Results: Intergroup analysis showed that there was no significant difference between group 2 and group 4 (P >.05) and between group 1 and group 3 (P >.05). However, group 1 and group 3 exhibited greater differences in tooth movement than group 2 and group 4 (P <.05). On the other hand, group 1 showed greater tooth movement than groups 2 and 4 with statistical significance (P <.01). The increase in the number of osteoclasts in group 1 was significantly higher than in the other groups (P <.05). Conclusion: Exogenous EGF-liposome administration has an additive effect when compared with soluble EGF on the rate of osteoclast recruitment, producing faster bone resorption and tooth movement.

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

Differential expression of notch signaling-related transcripts accompanies Pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.