Ability Of Epidermal Growth Factor Research Articles

A Rat Experimental Model for Investigation of the Effect of Diabetes on Submandibular Salivary Glands Treated with Epidermal Growth Factor

Background: Despite the plethora of research around the negative effects of diabetes on different body organs, this topic still attracts a lot of attention in order to find potential remedies that could counteract or reverse the damaging effect of diabetes.
 Aim: In this study, we developed a reliable experimental rat model that can be used for investigation of the ability of epidermal growth factor (EFG) in restoring the normal architecture of oral tissues after being damaged by diabetes.
 Methods: Eighty adult male albino rats (average weight ±220 gm) were used in the current study. Twenty rats served as control and received no treatment. Diabetes was induced in forty rats using a single injection of 65mg/kg of Streptozotocin (STZ). Out of the forty diabetic rats, twenty rats received a single daily intraperitoneal injection of EGF (10 µg/Kg) for 8 weeks. Furthermore, twenty healthy rats received the same dose of EGF and served as positive controls. The submandibular salivary glands of all rats were examined for Immunohistochemical detection of myosin in the glandular structure.
 Results: The EGF treated group showed comparable myosin expression to the control group. The diabetic group revealed deterioration of all components of the submandibular salivary glands. Finally, the diabetic + EGF group has demonstrated restoration of the myosin expression levels in the submandibular salivary glands to a level that is not significantly different from healthy (non-diabetic) rats in the control group (p>0.05) and significantly higher than the diabetic group (p<0.0001).
 Conclusion: The findings of the present study confirm previous studies and validates the use of our animal model as predictable experimental tool to investigate the effects of diabetes and EGF on different oral tissues. It also highlights the importance of further research investigating EGF as a promising treatment modality for restoration of the condition and functions of tissues damaged by diabetes not only in the oral cavity but also around the whole body.

Epidermal Growth Factor based Therapy Promotes Intracellular Trafficking and Accumulation of its Receptor in the Nucleus of Fibroblasts from Diabetic Foot Ulcers

Objective: To gain a better understanding of the Epidermal Growth Factor (EGF) Receptor (EGFR) activation, trafficking and biological response in diabetic foot ulcers (DFU), exposed to recombinant human EGF via intra-ulcer infiltration as a healing alternative. Methods: We studied by immunoelectron microscopy the intracellular localization of the EGFR and Proliferating Cell Nuclear Antigen (PCNA) in fibroblast-like cells (FLC) from granulation tissue of DFU patients, collected before and at different time points after EGF treatment. Results: EGF therapy appears to increase EGFR immunolabeling. At early time-points, EGFR labeling is observed predominantly in the nucleus, suggesting a fast EGFR internalization and nuclear translocation. Interestingly EGFR is also detected in the mitochondrial outer membrane. PCNA expression and trafficking were also detected in a time-dependent manner after EGF infiltration. Conclusion: Differential subcellular distribution of EGFR and PCNA and accumulation in the nucleus, in a timepoint specific manner, supports the induction of an EGF-mediated activation program that is sustained for at least 24 hours after the EGF administration. These findings substantiate the therapeutic ability of EGF to restore the healing process in DFU.

Additive effects of EGF and IL-1β regulate tumor cell migration and invasion in gastric adenocarcinoma via activation of ERK1/2

Growth and inflammatory factors are associated with poor prognosis in gastric adenocarcinoma (GA); however, the additive effects of growth and inflammatory factors in GA remain unclear. In this study, we investigated the ability of epidermal growth factor (EGF) and interleukin (IL-1β) to activate extracellular signal-regulated kinase (ERK)1/2 in GA cells, and correlated the relationships between their roles with the metastatic potential both in GA cells and GA tissues. The effects of EGF, IL-1β and EGF plus IL-1β in AGS and MKN-45 GA cells were examined using western blotting, Transwell migration and invasion assays, immunocytochemical staining and an activator protein (AP)-1 luciferase reporter gene assay, and was further characterized in GA tissues by immunohistochemistry. The results exhibited that EGF and IL-1β additively activated ERK1/2, increased migration and invasion than either EGF or IL-1β alone in AGS and MKN-45 cells. The mechanisms were involved in upregulating MMP-9 expression through increasing AP-1 transcriptional activity via ERK1/2 pathway; these effects were dose-dependently inhibited by silencing ERK1/2 or using U0126. In vivo data also confirmed that the overexpression of p-ERK1/2 in GA tissues correlated well with the EGF, IL-1β, EGF plus IL-1β, and was associated with metastasis, which was well correlation with the expression of MMP-9 and c-fos (AP-1). The results demonstrate that growth and inflammatory factors play an important role in metastasis of GA by additively activating ERK-1/2 and AP-1, and upregulating MMP-9. As both cytokines contribute to the migration and invasion of GA cells, EGF/IL-1β/ERK1/2 pathways may be key pathways closely associated with GA progression.

Abstract P6-01-05: Enhancement of18F-FDG uptake and glycolysis by epidermal growth factor via PI3K activation in T47D breast cancer cells

Abstract It is well known that the binding of epidermal growth factor (EGF) to EGF receptors (EGFR) stimulates proliferation of breast cancer cells via activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathways. There was a report that glucose consumption and lactate production were increased in MDA-MB-468 breast cancer cells following EGF exposure. However, the effect of EGF in enhancing glucose uptake and signaling pathways that are involved have not been clearly revealed. We thus investigated the role of EGF in stimulation of18F-FDG uptake in T47D breast cancer cells, and further elucidated the molecular mechanisms that are involved. Exposure of T47D cells to 100 ng/mL EGF for 24 h caused a substantial augmentation of18F-FDG uptake to even greater extents to 310.8% ± 30.1% of control cells (P < 0.001), and this effect of EGF showed dose- and time-dependency. Increased glucose uptake by EGF occurred through enhanced membrane GLUT-1 expression as well as hexokinase activity. Inhibition experiments with cycloheximide showed that the metabolic effect by EGF required new protein biosynthesis. Stimulation of glucose uptake by EGF was dependent on sufficient EGFR expression because18F-FDG uptake in MCF-7 cells weakly expressing EGFR was not affected by EGF treatment. Although EGF was also a stimulator of T47D cell proliferation (137.3 ± 7.3% of basal levels at 24 h, P < 0.001), the metabolic effect occurred in a manner that clearly preceded and surpassed the proliferative effect. EGF-stimulated proliferation was significantly inhibited by the EGFR inhibitor BIBX1382, the PI3K inhibitor LY294002 and the specific MAPK inhibitor PD98059. Unlike proliferation, the ability of EGF to augment18F-FDG uptake was found to be dependent on PI3K but not on MAPK activity. These findings yield the insight into our understanding of the role of EGF and the molecular basis that are involved in glucose metabolism of breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-05.

Epidermal growth factor’s activation of Ras is inhibited by four cardiac hormones

Four cardiac hormones synthesized by the same gene, i.e. atrial natriuretic peptide, vessel dilator, kaliuretic peptide and long-acting natriuretic peptide, have anticancer effects in vitro and in vivo. Epidermal growth factor's mechanism of cancer formation involves the activation of Ras. These four cardiac hormones were evaluated for their ability to inhibit mitogen (epidermal growth factor) activation of Ras. Epidermal growth factor increased the activation of Ras by 68%, 85% and 90% at its 1, 2 and 5 ng mL(-1) concentrations. Vessel dilator, long-acting natriuretic peptide, atrial natriuretic peptide and kaliuretic peptide inhibited 5 ng mL(-1) epidermal growth factor's stimulation of Ras by 73%, 79%, 33% and 45%, respectively, at their 1 microM concentrations. Their effects on epidermal growth factor's activation of Ras were specific with addition of the cardiac hormones' respective antibodies (5 microM) blocking 95%, 93%, 100% and 100% (P < 0.001 for each) of their ability to inhibit epidermal growth factor's stimulation of Ras. Four cardiac hormones specifically inhibit epidermal growth factor's activation of Ras. This investigation would suggest that these cardiac hormones' anticancer effects involve the inhibition of mitogens such as epidermal growth factor's ability to activate Ras as well as inhibiting unstimulated basal activity of Ras.

Metabolic Targets of Cardiac HormonesTherapeutic Anti-Cancer Effects

The heart is a sophisticated endocrine gland synthesizing a family of peptide hormones by three different genes. These cardiac hormones are stored as 3 prohormones, i.e. atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) prohormones. Within the ANP prohormones are 4 peptide hormones, i.e. atrial natriuretic peptide, vessel dilator, kaliuretic peptide and long-acting natriuretic peptide (LANP) which decrease up to 97% of human pancreatic, breast, colon, prostate, kidney and ovarian carcinomas as well as small-cell and squamous cell lung cancer cells within 24 hours in cell culture. In vivo these 4 cardiac hormones eliminate up to 80% of human pancreatic adenocarcinomas, 2/3rds of human breast cancers, and up to 86% of human small-cell lung cancers in athymic mice. Their anticancer mechanism(s) target the Ras-MEK 1/2-ERK 1/2 kinase cascade in cancer cells. These 4 cardiac hormones inhibit up to 95% of the basal activity of Ras, 98% of the phosphorylation of MEK 1/2 and 97% of the activation of basal activity of ERK 1/2. They also completely block the activity of mitogens such as epidermal growth factor's ability to stimulate ERK. They do not inhibit the activity of ERK in healthy cells such as human fibroblasts. The final step in their anticancer mechanism of action is that they enter the nucleus as demonstrated by immunfluorescence to inhibit DNA synthesis within cancer cells.

IQGAP1 Integrates Ca2+/Calmodulin and B-Raf Signaling

Ca(2+) and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca(2+) and calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates Ca(2+) and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca(2+) promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca(2+)-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca(2+) ionophore A23187. In contrast, chelation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, Ca(2+) regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca(2+)](i) with Ca(2+) ionophores significantly reduces co-immunoprecipitation of B-Raf and IQGAP1, whereas chelation of Ca(2+) enhances the interaction. Consistent with these findings, increasing and decreasing [Ca(2+)](i) increase and decrease, respectively, co-immunoprecipitation of calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples Ca(2+) and calmodulin signaling to B-Raf function.

Epidermal Growth Factor Inhibits Campylobacter jejuni -Induced Claudin-4 Disruption, Loss of Epithelial Barrier Function, and Escherichia coli Translocation

Campylobacter jejuni is a leading cause of acute bacterial enteritis in humans. Poultry serves as a major reservoir of C. jejuni and is thought to act as a principal vehicle of transmission to humans. Epidermal growth factor (EGF) is a small amino acid peptide that exerts a broad range of activities on the intestinal epithelium. The aims of this study were to determine the effect of EGF on C. jejuni intestinal colonization in newly hatched chicks and to characterize its effects on C. jejuni-induced intestinal epithelial barrier disruption. White Leghorn chicks were treated with EGF daily, starting 1 day prior to C. jejuni infection, and were compared to control and C. jejuni-infected, EGF-treated chicks. Infected chicks shed C. jejuni in their feces throughout the study period. C. jejuni colonized the small intestine and cecum, disseminated to extraintestinal organs, and caused jejunal villus atrophy. EGF reduced jejunal colonization and dissemination of C. jejuni to the liver and spleen. In EGF-treated C. jejuni-infected chicks, villus height was not significantly different from that in untreated C. jejuni-infected chicks or controls. In vitro, C. jejuni attached to and invaded intestinal epithelial cells, disrupted tight junctional claudin-4, and increased transepithelial permeability. C. jejuni also promoted the translocation of noninvasive Escherichia coli C25. These C. jejuni-induced epithelial abnormalities were abolished by pretreatment with EGF, and the effect was dependent upon activation of the EGF receptor. These findings highlight EGF's ability to alter colonization of C. jejuni in the intestinal tract and to protect against pathogen-induced barrier defects.

Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

ERK Negatively Regulates the Epidermal Growth Factor-mediated Interaction of Gab1 and the Phosphatidylinositol 3-Kinase

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 μg/mL LH and 0.5 μg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, ocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.

ERK2 Mitogen-activated Protein Kinase Binding, Phosphorylation, and Regulation of the PDE4D cAMP-specific Phosphodiesterases: THE INVOLVEMENT OF COOH-TERMINAL DOCKING SITES AND NH2-TERMINAL UCR REGIONS

The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42(MAPK)). These straddle the target residue, Ser(579), for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH(2)-terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOH-terminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.

Epidermal growth factor-induced heterologous desensitization of the luteinizing hormone/choriogonadotropin receptor in a cell-free membrane preparation is associated with the tyrosine phosphorylation of the epidermal growth factor receptor.

Epidermal growth factor (EGF) attenuated hCG-stimulated adenylyl cyclase activity in rat luteal and follicular membranes. H7, an equipotent serine/threonine protein kinase inhibitor of cAMP-dependent protein kinases, cGMP-dependent protein kinases, and lipid-dependent protein kinase C, did not effect the ability of EGF to decrease hCG-responsive adenylyl cyclase activity, suggesting that a serine/threonine phosphorylation event catalyzed by these kinases was not critically involved in EGF-induced desensitization. Likewise, pertussis toxin-catalyzed ADP-ribosylation of a 40-kDa luteal membrane protein, which exhibited immunoreactivity with an antibody against Gi alpha, did not hinder the ability of EGF to attenuate hCG-stimulated adenylyl cyclase activity, indicating that Gi did not mediate EGF-induced desensitization. Rather, EGF-induced heterologous desensitization of LH/CG receptor in ovarian membranes was closely associated with the specific and prominent tyrosine phosphorylation of the 170-kDa EGF receptor. Both EGF-stimulated autophosphorylation of EGF receptor and EGF-induced LH/CG receptor desensitization were attenuated by genistein, a tyrosine kinase inhibitor. These results suggest that tyrosine phosphorylation of the 170-kDa EGF receptor is a necessary component of the signaling pathway in EGF-induced heterologous desensitization of the LH/CG receptor.

Hormonal regulation of epidermal growth factor receptor content and signaling in bovine mammary tissue.

Mammary tissue from midpregnant heifers was cultured with epidermal growth factor (EGF) or transforming growth factor alpha for 1-3 days. After 1 day, 10 nM EGF or transforming growth factor alpha doubled DNA synthesis, whereas lower concentrations (0.1 or 1 nM) increased DNA synthesis 2- to 3-fold after 2-3 days in culture. In other studies, bovine mammary tissue was transplanted to ovariectomized athymic mice and treated for 10 days with saline, estradiol (1 microg/day), progesterone (1 mg/day), or estradiol + progesterone. Subsequent explant culture of the bovine tissue indicated that estradiol + progesterone augmented the ability of EGF to stimulate DNA synthesis. The increased response to EGF was associated with increased EGF binding and with increased EGF-induced tyrosine kinase that paralleled the increased EGF binding. In other studies, athymic mice bearing xenografted bovine mammary tissue were primed for 10 days with estradiol and progesterone, followed by 2-day treatment with saline (control), hydrocortisone (200 microg/day), PRL (1 mg/day), or hydrocortisone + PRL. Hydrocortisone and PRL alone decreased, and PRL + hydrocortisone eliminated, EGF-induced DNA synthesis. EGF receptor content was unaffected by hydrocortisone but was reduced by PRL or hydrocortisone + PRL. Furthermore, the ability of EGF to induce tyrosine kinase activity was decreased by PRL and by hydrocortisone + PRL. The decreased kinase activity was greater than the decrease in receptor binding, suggesting a specific modulation of EGF receptor kinase activity in response to lactogenic hormones.

Effects of epidermal growth factor (EGF) and prolactin on EGF receptor cytoskeletal association in mammary epithelial cells.

Prolactin treatment of NMuMG mammary epithelial cells inhibits the ability of epidermal growth factor (EGF) to transduce a variety of signals, possibly by interfering with receptor tyrosine phosphorylation. However, the mechanism by which prolactin inhibits EGF receptor signaling is unclear. The objective of this study was to evaluate the effects of prolactin on the dynamics of EGF receptor degradation and resynthesis, and on the association of the receptor with the cytoskeleton. EGF decreased the EGF receptor content of NMuMG cells, and this decrease was unaffected by prolactin treatment. Subsequent to the decrease in EGF receptors, cells reaccumulated EGF receptors, and this re-accumulation was also unaffected by prolactin. In other studies, EGF induced a rapid association of EGF receptor with Triton X-100-insoluble (cytoskeletal) elements. The cytoskeletally associated receptors were more heavily tyrosine phosphorylated than soluble receptors in the absence of prolactin. In the presence of prolactin, similar amounts of EGF receptor associated with the cytoskeleton, but both cytoskeletal and soluble receptors exhibited decreased tyrosine phosphorylation. These studies indicate that the effects of prolactin on EGF receptor signaling are not likely to be due to altered receptor dynamics or cytoskeletal association but are more likely due to an alteration in receptor kinase activity.

Differential Effects of EGF and Amphiregulin on Adhesion Molecule Expression and Migration of Colon Carcinoma Cells

Epidermal growth factor (EGF) is a potent morphogen affecting cell shape and motility through regulation of adhesive interactions. We have characterized the morphological effects of EGF on GP2d and GP5d colon carcinoma cell lines and have compared the ability of the heparin-binding EGF receptor ligand amphiregulin (AR) to elicit the same effects. EGF induced a marked epithelial–mesenchymal transition in both cell lines. This effect was evident at 7 pMEGF and was associated with a reduction in cellular adherens junctions and diminished cell–cell contact; it was also associated with an increase in expression of α2-integrin as well as enhanced adhesion to the substratum and cell spreading. These changes in adhesion molecule expression were accompanied by enhanced migration on collagen. Blockade of cell growth with mitomycin C did not prevent the EGF-induced morphological change, showing that the mitogenic and morphogenic responses of the GP cells were separable. The phosphatidyl inositol (PI) 3-kinase inhibitor wortmannin inhibited basal proliferation but had no effect on the EGF-induced morphological change, further suggesting that the PI 3-kinase pathway was not involved in the morphogenic response of these cells. Amphiregulin stimulated proliferation of both cell lines, but could only elicit a modest morphological change if used at considerably higher doses or if growth was blocked with mitomycin C. In cells treated with 55 nMAR, α2-integrin expression was slightly increased; however, unlike the EGF case, adherens junctions remained intact. These differences in the ability of EGF and amphiregulin to affect cellular adhesion and migration may be significant factors influencing normal and tumor cell behavior.

Secondary Dimerization between Members of the Epidermal Growth Factor Receptor Family

Growth factor receptors of the epidermal growth factor (EGF) receptor family play pivotal roles in the regulation of cell proliferation and differentiation and are involved in the development of human cancers. It has been well documented that these receptors undergo growth factor-stimulated homo- and heterodimerization as a first step in the initiation of signaling cascades. Here we provide evidence for a new mechanism for growth factor-stimulated receptor dimer formation, designated secondary dimerization. The growth factor-induced dimerization and ensuing receptor trans-autophosphorylation results in the dissociation of the original (primary) receptor dimer. Each phosphorylated receptor monomer then interacts with a new (nonphosphorylated) receptor to form a secondary dimer. Treatment of cells with EGF yields Neu-ErbB3 secondary dimers, and heregulin treatment induces the formation of Neu-EGF receptor (secondary) dimers. The ability of EGF and heregulin to stimulate a cascade of dimerization events points to a novel mechanism by which multiple signaling activities and diverse biological responses are initiated by members of the EGF receptor family.

Decreased binding and autophosphorylation of the epidermal growth factor receptor in ethanol-fed rats

We have shown previously that binding and processing of epidermal growth factor are impaired in livers of ethanol-fed rats. In the current study, we examined these ethanol-induced alterations in greater detail by studying both high and low affinity epidermal growth factor binding as well as the ability of added ligand to stimulate receptor autophosphorylation. We also measured the binding of anti-receptor antibody to intact and permeabilized cells in order to determine more accurately the levels of receptor protein. Hepatocytes were isolated from ethanol-fed and pair-fed control rats. Ligand binding, antibody binding, and ligand-induced receptor autophosphorylation were measured in the respective cell populations. In ethanol-fed animals, binding to both high and low affinity states of the hepatic epidermal growth factor receptor was decreased by 40–50% ( P < 0.01). This ethanol-induced decrease in ligand binding was accompanied by a reduced ability of epidermal growth factor to stimulate receptor autophosphorylation (32% decrease, P < 0.01). In contrast, binding of anti-receptor antibody was not altered in ethanol-fed animals. In conclusion, chronic ethanol feeding decreased epidermal growth factor binding with a concomitant decrease in the ability of the receptor tyrosine kinase to phosphorylate tyrosine residues. These changes were not accompanied by an actual decrease in receptor protein content. These findings could be relevant to modified responses to this growth factor in the livers of chronic ethanol-fed animals.

Tyrosine Phosphorylation of Gsα and Inhibition of Bradykinin-induced Activation of the Cyclic AMP Pathway in A431 Cells by Epidermal Growth Factor Receptor

An increasing amount of experimental data suggest that cross-talk exists between pathways involving tyrosine kinases and heterotrimeric G proteins. In a previous study, we demonstrated that bradykinin (BK) increases the intracellular accumulation of cAMP in the human epidermoid carcinoma cell line A431 by stimulating adenylate cyclase activity via a stimulatory G protein (Gsalpha) (Liebmann, C., Graness, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.-D., Nürnberg, B., and Wetzker, R. (1996) Biochem. J. 313, 109-118). Here, we present several lines of evidence indicating the ability of epidermal growth factor (EGF) to suppress BK-induced activation of the cAMP pathway in A431 cells via tyrosine phosphorylation of Gsalpha. Gsalpha was specifically immunoprecipitated from A431 cells using the anti-alphas antiserum AS 348. Tyrosine phosphorylation of Gsalpha was detectable in EGF-pretreated cells with monoclonal anti-phosphotyrosine antibodies. Additionally, A431 cells were labeled with [32P]orthophosphate in vivo and treated with EGF, and the resolved immunoprecipitates were subjected to amino acid analysis. The results clearly indicate that EGF induces tyrosine phosphorylation of Gsalpha in A431 cells. Treatment of A431 cells with EGF decreased BK-induced cAMP accumulation in intact cells as well as the stimulation of adenylate cyclase by BK, NaF, and guanyl nucleotides, but not by forskolin. Also, EGF treatment abolished both the BK- and isoprenaline-induced stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate) binding to Gsalpha. In contrast, the BK-evoked, Gq-mediated stimulation of inositol phosphate formation in A431 cells was not affected by EGF pretreatment. Thus, EGF-induced tyrosine phosphorylation of Gsalpha is accompanied by a loss of its susceptibility to G protein-coupled receptors and its ability to stimulate adenylate cyclase via guanyl nucleotide exchange. We propose that Gsalpha may represent a key regulatory protein in the cross-talk between the signal transduction pathways of BK and EGF in A431 cells.

Role of epidermal growth factor receptor in osteoblastic differentiation of rat bone marrow stromal cells

In an attempt to understand the role of epidermal growth factor (EGF) and its receptor (EGF-R) in osteoblastic cell differentiation, the changes in [125I]-EGF binding capacity, synthesis of EGF-R protein, and expression of EGF-R mRNA were investigated during osteoblastic differentiation of cultured bone marrow stromal cells which were collected from the femora of young adult rats. In addition, the ability of EGF to suppress osteoblastic differentiation was also studied. Dexamethasone at a concentration of 0.1 mM increased the expression of osteoblastic markers by bone marrow stromal cells cultured in alpha-modified minimum essential medium (α-MEM) con taining 1% fetal bovine serum (FBS), 50 mg/ml ascorbic acid, and 10 mM β-glycerophosphate, as revealed by elevated alkaline phosphatase activity, an increase in osteopontin mRNA expression, and bone nodule formation. This osteoblastic differentiation was accompanied by a decreased expression of EGF-R mRNA, decreased synthesis of EGF-R protein, and a decreased number of EGF-binding sites without any change in affinity. When these cells were incubated with dexamethasone and EGF in combination throughout the culture, they exhibited significantly lower levels of all osteoblastic markers than did dexamethasonetreated cells, indicating suppression of osteoblastic differentiation by EGF. In contrast, EGF treatment of the cells induced expression of EGF-R mRNA. Thus, a decrease in EGF binding associated with osteoblastic differentiation could lead to decreased responsiveness of bone marrow cells to EGF, whereas the EGF-induced increase in expression of EGF-R could facilitate the inhibition of cell differentiation by EGF. These findings suggested that upregulation of EGF-R on bone marrow stromal cells antagonizes their differentiation, and thus possibly functions as a negative regulator of osteoblastic differentiation.