Antenatal steroid and tracheal occlusion restore vascular endothelial growth factor receptors in congenital diaphragmatic hernia rat model

OBJECTIVE: Investigate the effects of antenatal steroids and tracheal occlusion on pulmonary expression of vascular endothelial growth factor receptors in rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN: Fetuses were exposed to nitrofen at embryonic day 9.5. Subgroups received dexamethasone or were operated on for tracheal occlusion, or received combined treatment. Morphologic variables were recorded. To analyze vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, we performed Western blotting and immunohistochemistry. Morphologic variables were analyzed by analysis of variance and immunohistochemistry by Kruskal-Wallis test. RESULTS: Congenital diaphragmatic hernia decreased body weight, total lung weight, and lung-to-body weight ratio. Tracheal occlusion increased total lung weight and lung-to-body weight ratio (P < .05). Fetuses with congenital diaphragmatic hernia had reduced vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, whereas steroids and tracheal occlusion increased their expression. Combined treatment increased expression of receptors, but had no additive effect. CONCLUSION: Vascular endothelial growth factor signaling disruption may be associated with pulmonary hypertension in congenital diaphragmatic hernia. Tracheal occlusion and steroids provide a pathway for restoring expression of vascular endothelial growth factor receptors.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of `B` and `C` splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the `B` and `C` spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Basic fibroblast growth factor and fibroblast growth factor receptors in adult olfactory epithelium

Basic fibroblast growth factor (FGF2) stimulates proliferation of the globose basal cells, the neuron:ll precursor in the olfactory epithelium. The present study investigates the expression of basic fibroblast growth factor and fibroblast growth factor receptors in the adult olfactory epithelium. FGF2 immunoreactivity was expressed widely in the olfactory epithelium, with the highest density of immunoreactivity in the supporting cells. In contrast, most cells in the epithelium expressed FGF2 mRNA. Fibroblast growth factor receptor-1 (FGFr1) immunoreactivity was densest in the basal cell and neuronal layers of the olfactory epithelium and on the apical surface of supporting cells. In the lamina propria FGF2 immunoreactivity and mRNA were densest in cells close to the olfactory nerve bundles. FGFr1 immunoreactivity was heaviest on the olfactory ensheathing cells. Using reverse transcriptase-polymerase chain reaction analysis, the olfactory epithelium was shown to express only three receptor splice variants, including one (FGFr1c) with which basic fibroblast growth factor has high affinity. Other receptor splice variants were present in the lamina propria. Taken together, these observations indicate endogenous sources of FGF? within the olfactory epithelium and lamina propria and suggest autocrine and paracrine pathways via which FGF2 might regulate olfactory neurogenesis. The observation of only three receptor splice variants in the olfactory epithelium limits the members of the fibroblast growth factor family which could act in the olfactory epithelium. The widespread distribution of receptors suggests that fibroblast growth factors may have roles other than proliferation of globose basal cells. (C) 2001 Published by Elsevier Science B.V.

Epidermal growth factor receptors, testosterone levels and parotid gland changes in rats infected with Trypanosoma cruzi

It has been demonstrated that parotid glands of rats infected with Trypanosoma cruzi present severe histological alterations; changes include reduction in density and volume of the acini and duct systems and an increase in connective tissue. We evaluated the association between morphological changes in parotid glands, circulating testosterone levels and epidermal growth factor receptor (EGF-R) expression in experimental Chagas disease in rats. Animals at 18 days of infection (acute phase) showed a significant decrease in body weight, serum testosterone levels and EGF-R expression in the parotid gland compared with a control group. Since decreases in body weight could lead to a reduction in circulating testosterone concentration, we believe that the reduction in EGF-R expression in parotid glands of infected rats is due to alterations in testosterone levels and atrophy of parotid glands is caused by changes in EGF-R expression. Additionally, at 50 days (chronic phase) of infection parotid glands showed a normal histological aspect likely due to the normalization of the body weight. These findings suggest that the testosterone-EGF-R axis is involved in the histological changes.

Early investigational drugs that target epidermal growth factor receptors for the treatment of head and neck cancer.

INTRODUCTION: Squamous-cell carcinoma of the head and neck (SCCHN) remains a challenging clinical problem, due to the persistent high rate of local and distant failures and the occurrence of secondary primaries. For locally advanced SCCHN, a combination of chemotherapy (CT), radiation or surgery is often used, but there are limitations, which may reduce compliance. Molecular targeted therapies, namely anti-EGFR treatments, are in development with the aim of improving clinical outcomes and mitigating treatment-related toxicities. AREAS COVERED: This review provides an overview of early investigational drugs that target EGFR for the treatment of SCCHN and discusses the ongoing trials in this domain. EXPERT OPINION: Targeted therapies are increasingly used in oncology, especially in SCCHN. Cetuximab has demonstrated a significant improvement in the treatment outcome, both as a curative treatment in combination with radiation therapy and as a palliative treatment in combination with CT; however, it failed to show any benefit in combination with concomitant chemoradiotherapy. Presently, there are many new agents, including monoclonal antibodies and small-molecule tyrosine kinase inhibitors, which are either currently under investigation for or which warrant further investigation for treating SCCHN. The discovery of predictive factors that help to identify patients most likely to respond to EGFR inhibitors as well as patient-customized therapies would help to improve patient outcomes in the future.

Hydrogen peroxide enhances the expression and function of Giα proteins in aortic vascular smooth muscle cells from Sprague-Dawley rats : role of growth factor receptors transactivation

Nous avons récemment démontré que les espèces réactives oxygénées induisent une augmentation de l’expression des protéines Giα dans les cellules du muscle lisse vasculaire (CMLV) provenant d’aortes de rats spontanément hypertendus (SHR, de l’anglais spontaneously hypertensive rats). La présente étude a pour but d’étudier les effets du peroxyde d’hydrogène (H2O2), un oxydant qui induit le stress oxydatif, sur l’expression de Giα et sur l’activité de l’adénylate cyclase, et d’explorer les voies de signalisation sous-jacentes responsables de cette réponse. Nos résultats montrent que H2O2 induit une augmentation de l’expression des protéines Giα-2 et Giα-3 de manière dose- et temps-dépendante avec une augmentation maximale de 40-50% à 100 µM après 1 heure, sans affecter l’expression de Gsα. L’expression des protéines Giα a été maintenue au niveau normal en presence de AG 1478, AG1295, PD98059 et la wortmannine, des inhibiteurs d’EGF-R (de l’anglais epidermal growth factor receptor), PDGFR-β (de l’anglais platelet-derived growth factor receptor β), de la voie de signalisation ras-ERK1/2 (de l’anglais extracellular regulated kinase1/2), et de la voie de la PI3Kinase-AKT (de l’anglais phosphatidyl inositol-3 kinase), respectivement. En outre, le traitement des CMLV avec H2O2 a induit une augmentation du degré de phosphorylation d’EGF-R, PDGF-R, ERK1/2 et AKT; et cette expression a été maintenue au niveau témoin par leurs inhibiteurs respectifs. Les inhibiteurs d’EGF-R et PDGF-R ont aussi induit une diminution du degré de phosphorylation de ERK1/2, et AKT/PKB. En outre, la transfection des cellules avec le siRNA (de l’anglais, small interfering ribonucleic acid) de EGF-R et PDGFR-β a atténué la surexpression des protéines Giα-2 et Giα-3 induite par le traitement au H2O2. La surexpression des protéines Giα induite par H2O2 a été corrélée avec une augmentation de la fonction de la protéine Giα. L’inhibition de l’activité de l’adénylate cyclase par de faibles concentrations de GTPγS après stimulation par la forskoline a augmenté de 20% dans les cellules traitées au H2O2. En outre, le traitement des CMLV au H2O2 a aussi accru l’inhibition de l’activité de l’adénylate cyclase par les hormones inhibitrices telles que l’angiotensine II, oxotrémorine et C-ANP4-23. D’autre part, la stimulation de l’adénylate cyclase induite par GTPγS, glucagon, isoprotérénol, forskoline, et le fluorure de sodium (NaF) a été atténuée de façon significative dans les cellules traitées au H2O2. Ces résultats suggèrent que H2O2 induit la surexpression des protéines Giα-2 and Giα-3 via la transactivation des récepteurs des facteurs de croissance EGF-R, PDGFR-β et l’activation des voies de signalisation ras-ERK1/2 et PI3K-AKT Mot-cles: Protéines Giα, peroxyde d’hydrogène, stress oxydant, récepteurs des facteurs de croissance, MAP kinases, adénylate cyclase, hypertension

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

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.

Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Distinct Endocytic Responses of Heteromeric and Homomeric Transforming Growth Factor β Receptors

Transforming growth factor β (TGFβ) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGFβ receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGFβ receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor α or β receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGFβ receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGFβ receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGFβ receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGFβ receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGFβ receptors and that TGFβ receptor heteromers and homomers show distinct trafficking behavior.

Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2α leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2α:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.