Epidermal Growth Factor Binding Research Articles

INHIBITION OF PRESSURE INDUCED BLADDER SMOOTH MUSCLE CELL HYPERPLASIA USING CRM197

In vivo the effects of sustained hydrostatic pressure on the bladder wall and its components are evident under physiological and pathological conditions. We previously reported that exposure of bladder smooth muscle cells to 20 and 40 cm. H2O hydrostatic pressure for as little as 1 hour resulted in the up-regulation of heparin binding epidermal growth factor messenger RNA in a time dependent fashion as well as in activation of the heparin binding epidermal growth factor growth factor gene. In our current study we investigated the use of CRM197 as an agent for blocking undesirable cellular level events, such as smooth muscle cell hyperplasia, eliminating the irreversible alterations in bladder and kidney function that result from chronic and/or severe bladder outlet obstruction. Control and experimental neonatal ovine smooth muscle cells were exposed to 0.3 pressure and 8.5 cm. H2O, respectively, for 7 days. We evaluated the mitogenic activity of the supernatant medium from bladder smooth muscle cells exposed to 8.5 cm. H2O for 5 days (conditioned medium) before and after the addition of 0.1 mg./ml. CRM197. Bladder smooth muscle cell apoptosis was also assessed after CRM197 exposure. Statistical analysis was performed using the Student t test with p <0.05 considered significant. Exposing bladder smooth muscle cells to sustained 8.5 cm. H2O hydrostatic pressure for 7 days resulted in increased cell proliferation. Conditioned medium contained mitogenic activity, which was ablated after CRM197 was added. No direct toxic effect of CRM197 on bladder smooth muscle cell growth was appreciated (no apoptosis). We demonstrated a proliferative response of neonatal bladder smooth muscle cells after exposure to sustained hydrostatic pressure. This response was partially due to the release of heparin binding epidermal growth factor and was blocked by adding CRM197. These data support the potential use of CRM197 in drug targeted therapy for diseases involving bladder outlet obstruction.

The Asn-420-linked Sugar Chain in Human Epidermal Growth Factor Receptor Suppresses Ligand-independent Spontaneous Oligomerization: POSSIBLE ROLE OF A SPECIFIC SUGAR CHAIN IN CONTROLLABLE RECEPTOR ACTIVATION

To elucidate a role(s) of Asn-linked sugar chain(s) in the function of epidermal growth factor receptor (EGFR), a series of the EGFR mutants were prepared in which potential glycosylation sites in the domain III were eliminated by site-directed mutagenesis. Although the wild-type and mutants of Asn-328, Asn-337, and Asn-389 underwent autophosphorylation in response to epidermal growth factor (EGF), the Asn-420 --> Gln mutant was found to be constitutively tyrosine-phosphorylated. This abnormal ligand-independent phosphorylation of the mutant appears to be due to a ligand-independent spontaneous oligomer formation, as shown by a cross-linking experiment using the purified soluble extracellular domain (sEGFR). As revealed by the dissociation of the Asn-420 --> Gln sEGFR oligomer by simple dilution, it seems likely that the equilibrium is shifted toward oligomer formation to an unusual degree. Furthermore, it was also found that the mutation caused a loss of the ability to bind EGF. These findings suggest that the sugar chain linked to Asn-420 plays a crucial role in EGF binding and prevents spontaneous oligomerization of the EGFR, which may otherwise lead to uncontrollable receptor activation, and support the view of a specific role of an Asn-linked sugar chain in the function of a glycoprotein.

ONCOSTATIN M SUPPRESSES EGF-MEDIATED PROTEIN TYROSINE PHOSPHORYLATION IN BREAST CANCER CELLS

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.

Meltrin gamma(ADAM-9) mediates cellular adhesion through alpha(6)beta(1 )integrin, leading to a marked induction of fibroblast cell motility.

The ADAMs (A Disintegrin and Metalloprotease Domains) are a family of membrane-anchored proteins that play a role in fertilisation, myoblast fusion and ectodomain shedding of cell surface proteins. Meltrin gamma (ADAM-9) is a widely expressed member of this family and is involved in the shedding of heparin binding epidermal growth factor. Here we report that meltrin gamma can function as a cell adhesion molecule via its disintegrin domain. Using solid-phase binding assays and antibody inhibition experiments, we demonstrate that a murine meltrin gamma-Fc (Mel gamma -Fc) fusion protein binds to the integrin alpha(6)beta(1) on the surface of fibroblast cell lines, HT1080 and Wehi 164 in a specific manner. Since alpha(6)beta(1) is important for the motility of several cell types on laminin, cell migration studies using time-lapse video microscopy were performed. Cells adhering to Mel gamma-Fc displayed a rounded morphology and a marked increase (eight- to tenfold) in their motility compared to that on laminin. Furthermore, the p160 ROCK kinase inhibitor Y-27632 specifically reduced the migration of cells on meltrin gamma but had no effect on migration of cells on laminin, whilst the general tyrosine phoshorylation inhibitor, genistein, inhibited cell migration on both substrates. These results together suggest that meltrin gamma may play a role in regulating the motility of cells by binding to alpha(6)beta(1) integrin and this may be important during a variety of biological and pathological processes.

Immunoelectron microscope analysis of epidermal growth factor receptor (EGFR) in isolated Mytilus galloprovincialis (Lam.) digestive gland cells: evidence for ligand-induced changes in EGFR intracellular distribution.

In mammalian cells, the binding of epidermal growth factor (EGF) to its receptor (EGFR), a glycoprotein with intrinsic tyrosine kinase activity, leads to the pleiotropic responses to EGF. Among these, a negative feedback response by stimulation of receptor internalization and lysosomal degradation, this attenuating signal transduction. In this work, data are reported on the identification of specific EGFRs in isolated digestive gland cells from the marine mussel (Mytilus galloprovincialis Lam.) By immunoelectron microscopy. In control digestive cells, EGFR immunoreactivity was mainly associated with cytoplasmic membrane structures and, to a lesser extent, the cell membrane. The presence of EGFR-like receptors was confirmed by Western blotting of digestive gland cell extracts with two different monoclonal antibodies that recognize either intracellular or extracellular epitopes. The addition of mammalian EGF resulted in significant time and temperature-dependent changes in EGFR subcellular distribution in mussel cells. In cells exposed to EGF for 0-15 min at 4 degrees C, the distribution of EGFR was not significantly different from that of the control cells. On the other hand, at 18 degrees C, an increased labelling along the cell membrane was observed after 5-10 min after EGF addition, with a concomitant decrease in the cytoplasmic signal. Moreover, after 20 min of exposure to EGF, ligand binding apparently resulted in EGFR compartmentation within the lysosomes. These observations were confirmed by quantitative analysis of EGFR labelling at different times of EGF exposure. Similar results were obtained utilizing the two different monoclonal antibodies. The results indicate that, in mussel digestive cells, the binding of heterologous EGF to specific receptors induces a negative feedback response by stimulating the lysosomal degradation of EGFR, thus suggesting the presence of mechanisms responsible for receptor downregulation similar to those observed in mammalian cells.

EGF Receptors of Hepatocytes from Rats Treated with Phenobarbital Are Sensitized to Down-Regulation by Phenobarbital in Culture

Hepatocytes from Fisher 344 rats treated with the liver tumor promoter phenobarbital (PhB; 0.1% in the drinking water, 2–3 months) exhibit reduced epidermal growth factor (EGF) binding and EGF-induced mitogenesis in culture. Similar responses are induced by >1 mM PhB added to the culture medium of hepatocytes from untreated rats. In this study, we demonstrated that hepatocyte EGFr protein, as determined by immunoblotting, was unchanged by treatment of rats with PhB. However, hepatocytes from PhB-treated rats are more sensitive to PhB in culture in that decreased EGF binding occurred with 0.05 mM PhB, a concentration also attained in plasma of rats exposed to PhB. Sensitization was reversible, as is tumor promotion, since hepatocytes from rats withdrawn from PhB for 1 month were unresponsive to <3 mM PhB. EGFr down-regulation by a series of barbiturates correlated well with their known activities as tumor promoters and CYP2B1/2 inducers, with pentobarbital and PhB yielding high activities, while barbital was intermediate and barbituric acid, 5-phenylbarbituric acid, and 5-ethylbarbituric acid were ineffective. Differentiated hepatocyte function was required for PhB-induced EGFr down-regulation since HepG2 and rat liver epithelial cells were unresponsive, but involvement of CYP2B1/2 activity was discounted by the failure of metyrapone to inhibit the response in PhB-induced hepatocytes. These studies support a role for impaired EGFr function in PhB liver tumor promotion due to effects on existing EGFr protein and suggest that EGFr down-regulation by PhB in culture is independent of CYP2B1/2 activity but shares mechanistic components involved in its transcriptional activation by PhB.

Transcellular transport of leptin by the short leptin receptor isoform ObRa in Madin-Darby Canine Kidney cells.

Leptin is an adipocyte-derived hormone that acts in specific regions of the brain to regulate body weight and neuroendocrine function. The mechanism by which leptin enters the brain is unknown. We previously reported that rat brain microvessels, which constitute the blood-brain barrier, contain large amounts of messenger RNA encoding a short form of the leptin receptor (ObRa), suggesting that this site may be important for receptor-mediated transport of leptin into the brain. The purpose of this study was to determine whether ObRa is capable of transcellular transport of intact leptin. A transwell system in which Madin-Darby Canine Kidney (MDCK) cells stably expressing ObRa are grown in a monolayer was used to determine receptor distribution on apical or basolateral cell surfaces and the capacity for directional transport of 125I-leptin. Binding of 125I-leptin was greater on the apical vs. the basolateral cell surface and transport of 125I-leptin occurred only in the apical to basolateral direction. 11% of transported radioactivity appearing in the basolateral chamber represented intact leptin as assessed by TCA precipitation analysis and by SDS-PAGE. Parental MDCK cells did not express leptin receptors and did not bind or transport 125I-leptin. Epidermal growth factor (EGF) binding and transport via endogenous EGF receptors in MDCK cells also was assessed. In contrast to leptin, specific binding of 125I-EGF occurred primarily on the basolateral cell surface and transport of 125I-EGF occurred predominantly in the basolateral to apical direction. These data show that ObRa is preferentially targeted to the apical cell membrane in MDCK cells and that leptin transport occurs, albeit at a low rate, in a unidirectional manner in the apical to basolateral direction. These findings may be relevant to the putative role of ObRa in receptor-mediated transport of leptin from the circulation into the brain.

Polyubiquitination of the Epidermal Growth Factor Receptor Occurs at the Plasma Membrane upon Ligand-induced Activation

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

Ancillary methods for the detection of recurrent urothelial neoplasia

Ancillary methods for the detection of recurrent urothelial neoplasia

Epidermal growth factor receptor is modulated by redox through multiple mechanisms

The cellular redox state has been shown to play an essential role in cellular signaling systems. Here we investigate the effects of reductants and H2O2 on the signaling of epidermal growth factor (EGF) in cells. H2O2 induced the phosphorylation of the EGF receptor and the formation of a receptor complex comprising Shc, Grb2, Sos, and the EGF receptor. Dimerization or oligomerization of the EGF receptor was not induced by H2O2. Protein tyrosine phosphatase (PTP) assay showed that H2O2 suppressed dephosphorylation of the EGF receptor in cell lysates, suggesting that inactivation of PTP was involved in H2O2-induced activation of the EGF receptor. In contrast, the reductants N-acetyl-L-cysteine [Cys(Ac)] and dithiothreitol markedly suppressed EGF-induced dimerization and activation of the EGF receptor in cells. In accordance with suppression of the EGF receptor, Cys(Ac) suppressed EGF-induced activation of Ras, phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Dithiothreitol completely inhibited EGF binding and kinase activation of the EGF receptor both in vitro and in vivo. In contrast, Cys(Ac) suppressed high-affinity EGF-binding sites on the cells, but had no effect on low-affinity binding sites. Furthermore, Cys(Ac) did not suppress EGF-induced kinase activation or dimerization of the EGF receptor in vitro, indicating that it suppressed the EGF receptor through a redox-sensitive cellular process or processes. Thus, the EGF receptor is regulated by redox through multiple steps including dephosphorylation by PTP, ligand binding, and a Cys(Ac)-sensitive cellular process or processes.

Tumor Necrosis Factor-α and Interleukin-1β Inhibit Apolipoprotein B Secretion in CaCo-2 Cells via the Epidermal Growth Factor Receptor Signaling Pathway

In inflammatory conditions of the gut, cytokines are released into the mucosa and submucosa propagating and sustaining the inflammatory response. In CaCo-2 cells, we have shown that various inflammatory cytokines interfere with the secretion of lipids, an effect that is likely caused by the release of a ligand to the epidermal growth factor (EGF) receptor. In the present study, the role of the EGF receptor signaling pathway and the effects of the cytokines tumor necrosis factor-alpha (TNF-alpha) and and interleukin 1beta (IL-1beta) on triacylglycerol-rich lipoprotein secretion were investigated. CaCo-2 cells were incubated with oleic acid to enhance triacylglycerol-rich lipoprotein secretion. TNF-alpha and IL-1beta significantly decreased the basolateral secretion of apolipoprotein B (apoB) mass, with IL-1beta being more potent. Tyrphostin, an inhibitor of the EGF receptor intrinsic tryosine kinase, prevented or markedly attenuated the decrease in apoB secretion by TNF-alpha or IL-1beta. Both cytokines increased the phosphorylation of the EGF receptor by 30 min. Moreover, phosphotyrosine immunoblots of the EGF receptor demonstrated an increase in tyrosine residues phosphorylated by 0.5 and 6.5 h. At both these time points, TNF-alpha and IL-1beta also decreased the binding of EGF to its cell surface receptor. At 6.5 h, activation of the EGF receptor was sustained. In contrast, the early activation of the receptor was only transient as receptor phosphorylation and binding of EGF to its receptor returned to basal levels by 2 h. Preventing ligand binding to the EGF receptor by a receptor-blocking antibody attenuated receptor activation observed after 6.5 h. This did not occur at 0.5 h, suggesting that early activation of the EGF receptor was non-ligand-mediated. Similarly, apoB secretion was inhibited by an early non-ligand-mediated process; whereas at the later time, inhibition of apoB secretion was ligand-mediated. Thus, the inflammatory cytokines TNF-alpha and IL-1beta interfere with the secretion of triacylglycerol-rich lipoproteins by both early and delayed signaling events mediated by the EGF receptor signaling pathway.

Sequence and characterization of the glyceraldehyde-3-phosphate dehydrogenase of Mycobacterium avium: correlation with an epidermal growth factor binding protein

Mycobacterium avium is a common pathogen in AIDS patients. The extracellular environment within the granuloma shown to support mycobacterial growth is in the caseous fluid. Previous work demonstrated that the presence of human epidermal growth factor (EGF), which is found in the tissue of chronic granulomous lesions, increases the growth rate of M. avium and Mycobacterium tuberculosis. Previously, a protein capable of binding recombinant human EGF (rEGF) in a western blot was identified with homology to glyceraldehyde-3-phosphate dehydrogenase (GAP) in both M. avium and M. tuberculosis but not Mycobacterium smegmatis. Surface GAPs have been identified in group A Streptococcus, enteropathogenic Escherichia coli, Candida albicans and Schistosoma mansoni. We have cloned thegap gene of M. avium. M. avium GAP has high homology with M. tuberculosis GAP. The protein was also expressed in M. smegmatis, conveying the ability to bind rEGF, but no growth increase was observed in 7H9 broth in the presence of rEGF up to 500 ng/ml. Only one copy of the GAP gene was identified in M. avium These results contribute to the understanding of M. avium pathogenesis by characterizing its interaction with a host protein present in the site of infection.

Conformationally constrained analogues of diacylglycerol (DAG). 16. How much structural complexity is necessary for recognition and high binding affinity to protein kinase C?

The design of potent protein kinase C (PK-C) ligands with low nanomolar binding affinities was accomplished by the combined use of pharmacophore- and receptor-guided approaches based on the structure of the physiological enzyme activator, diacylglycerol (DAG). Earlier use of the former approach, which was based on the structural equivalence of DAG and phorbol ester pharmacophores, identified a fixed template for the construction of a semirigid "recognition domain" that contained the three principal pharmacophores of DAG constrained into a lactone ring (DAG-lactones). In the present work, the pharmacophore-guided approach was refined to a higher level based on the X-ray structure of the C1b domain of PK-Cdelta complexed with phorbol-13-O-acetate. A systematic search that involved modifying the DAG-lactone template with a combination of linear or branched acyl and alpha-alkylidene chains, which functioned as variable hydrophobic "affinity domains", helped identify compounds that optimized hydrophobic contacts with a group of conserved hydrophobic amino acids located on the top half of the C1 domain where the phorbol binds. The hydrophilic/hydrophobic balance of the molecules was estimated by the octanol/water partition coefficients (log P) calculated according to a fragment-based approach. The presence of branched alpha-alkylidene or acyl chains was of critical importance to reach low nanomolar binding affinities for PK-C. These branched chains appear to facilitate important van der Waals contacts with hydrophobic segments of the protein and help promote the activation of PK-C through critical membrane interactions. Molecular modeling of these DAG-lactones into an empty C1b domain using the program AutoDock 2.4 suggests the existence of competing binding modes (sn-1 and sn-2) depending on which carbonyl is directly involved in binding to the protein. Inhibition of epidermal growth factor (EGF) binding, an indirect PK-C mediated response, was realized with some DAG-lactones at a dose 10-fold higher than with the standard phorbol-12, 13-dibutyrate (PDBU). Through the National Cancer Institute (NCI) 60-cell line in vitro screen, DAG-lactone 31 was identified as a very selective and potent antitumor agent. The NCI's computerized, pattern-recognition program COMPARE, which analyzes the degree of similarity of mean-graph profiles produced by the screen, corroborated our principles of drug design by matching the profile of compound 31 with that of the non-tumor-promoting antitumor phorbol ester, prostratin. The structural simplicity and the degree of potency achieved with some of the DAG-lactones described here should dispel the myth that chemical complexity and pharmacological activity go hand in hand. Even as a racemate, DAG-lactone 31 showed low namomolar binding affinity for PK-C and displayed selective antitumor activity at equivalent nanomolar levels. Our present approach should facilitate the generation of multiple libraries of structurally similar DAG-lactones to help exploit molecular diversity for PK-C and other high-affinity receptors for DAG and the phorbol esters. The success of this work suggests that substantially simpler, high-affinity structures could be identified to function as surrogates of other complex natural products.

Single-molecule imaging of EGFR signalling on the surface of living cells.

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Epiregulin, a Novel Member of the Epidermal Growth Factor Family, Is an Autocrine Growth Factor in Normal Human Keratinocytes

Epiregulin is a new member of the epidermal growth factor (EGF) family purified from conditioned medium of NIH-3T3 clone T7. Some EGF family growth factors play essential roles in human keratinocytes in an autocrine manner. We show here that epiregulin is another autocrine growth factor for human keratinocytes. Epiregulin stimulated human keratinocyte proliferation under both subconfluent and confluent culture conditions in the absence of exogenous EGF family growth factors. Immunoprecipitation of [(35)S]methionine-labeled conditioned medium revealed a 5-kDa band corresponding to epiregulin. Northern blot analysis detected a 4. 8-kilobase transcript of epiregulin, and the addition of epiregulin up-regulated epiregulin mRNA synthesis. Furthermore, an anti-epiregulin blocking antibody reduced DNA synthesis by 25%. Epiregulin up-regulated the mRNA levels of heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, and TGF-alpha. In turn, the addition of EGF, HB-EGF, amphiregulin, and TGF-alpha increased epiregulin mRNA levels. These results demonstrate that epiregulin acts as an autocrine growth factor in human epidermal keratinocytes and is part of auto- and cross-induction mechanisms involving HB-EGF, amphiregulin, and TGF-alpha. The mRNA expression profile resulting from induction of differentiation with high calcium and fetal calf serum revealed the differential expression of epiregulin, HB-EGF, amphiregulin, and TGF-alpha in keratinocytes. This indicates that these four growth factors have distinct, non-redundant biological functions.

A NONTOXIC DIPHTHERIA TOXIN ANALOGUE INHIBITS NEONATAL BLADDER SMOOTH MUSCLE CELL PROLIFERATION

The response of the neonatal bladder to infravesical obstruction, such as posterior urethral valves or detrusor-sphincter dyssynergia, may result in structural and functional changes that persist well after obstruction is treated. A pharmacological means of inhibiting smooth muscle cell proliferation would likely serve to halt or reverse this deleterious process. Heparin binding (HB) epidermal growth factor (EGF) is a known smooth muscle cell mitogen, while its membrane bound precursor serves as the diphtheria toxin receptor. We report the effects of the nontoxic diphtheria toxin analogue cross reacting material (CRM) 197 on neonatal sheep bladder smooth muscle cell proliferation. Neonatal sheep smooth muscle cell cultures were obtained from whole bladder explants. Immunohistochemical staining was performed for desmin and alpha-smooth muscle actin. HB-EGF messenger RNA was detected by reverse transcriptase polymerase chain reaction using primers to the human sequence, while pro-HB-EGF peptide was confirmed using a diphtheria toxin sensitivity assay with incorporation of tritiated leucine. Cells grown in 96 well plates were exposed to 1, 10 and 100 microg./ml. CRM 197 for 5 days, after which relative cell number was determined using an 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; thiazolyl blue based assay read at a wavelength of 550 nm. Statistical analysis was performed using Student's t test. Primary cell cultures stained positive for desmin and alpha-smooth muscle actin, confirming a smooth muscle origin. Reverse transcriptase polymerase chain reaction yielded a 453 bp product with 88% homology to human HB-EGF. Total protein synthesis significantly decreased when cells were incubated with diphtheria toxin, confirming the presence of membrane bound pro-HB-EGF. CRM 197 inhibited bladder smooth muscle cell growth in a dose dependent fashion at a concentration of 10 microg./ml., resulting in a 40% decrease in proliferation (p <0.0001). CRM 197 inhibits bladder smooth muscle cell proliferation in a dose dependent, nontoxic fashion through its interaction with HB-EGF. These data suggest that molecular strategies designed to inhibit HB-EGF mediated cell growth may prove beneficial for the prevention and/or treatment of detrusor hypertrophy secondary to anatomical or functional bladder outlet obstruction.

Removing Receptors by Ubiquitination

STKE The intracellular adaptor protein c-Cbl has been implicated in the ubiquitination and degradation of growth factor receptors. Levkowitz et al. and Lill et al. have determined that interaction between the src homology 2 (SH2) domain of c-Cbl and the cytoplasmic tail of the epidermal growth factor (EGF) receptor is essential for this effect. Binding of EGF to the receptor is known to activate the cytoplasmic tyrosine kinase domain; Levkowitz et al. showed that a phosphorylated tyrosine residue in the EGF receptor tail serves as the c-Cbl docking site and also that c-Cbl itself becomes phosphorylated on a tyrosine residue. Phosphorylation of c-Cbl was required for ubiquitination of activated EGF receptor, suggesting that the adaptor protein may act as a ubiquitin ligase. Furthermore, ubiquitination may contribute to diverting internalized growth factor receptors to the lysosomal degradation pathway because activated EGF receptors lacking the c-Cbl docking residue were neither ubiquitinated nor degraded. Instead, they were internalized and then recycled to the cell surface. Thus, ubiquitination of growth factor receptors may ensure receptor downregulation by recruiting both proteosomal and lysosomal degradation pathways.— LDC Mol. Cell 4 , 1029 (1999); J. Biol. Chem. 275 , 367 (2000).

Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.

Impaired nuclear accumulation and shortened phosphorylation of ERK after growth factor stimulation in cultured hepatocytes from rats exposed to 2-acetylaminofluorene.

The hepatic carcinogen 2-acetylaminofluorene (AAF) exerts its effect as a tumor promoter by mitoinhibition of normal hepatocytes. Initiated cells proliferate selectively and develop into preneoplastic foci and subsequently into carcinomas. To study whether some of the mitoinhibitory effects of AAF could be attributed to an influence on intracellular signal transduction, growth factor signaling was studied in cultured hepatocytes from rats fed AAF for 7 d. Activation through the epidermal growth factor receptor (EGFR) was used to probe possible changes in downstream mitogenic signaling mechanisms. The proliferative response to epidermal growth factor (EGF), measured as proliferating cell nuclear antigen expression and thymidine incorporation, was almost completely inhibited in hepatocytes exposed to AAF. Neither EGFR protein levels nor EGF binding was notably altered in AAF-exposed hepatocytes as opposed to normal hepatocytes. The initial tyrosine phosphorylation of EGFR and downstream activation of Sos, Raf-1, and extracellular signal-regulated protein kinase (ERK) were similar in AAF-treated and control hepatocytes. Even though ERK phosphorylation was unaffected, a remarkable (80%) reduction of ERK nuclear accumulation was observed in AAF-exposed hepatocytes immediately after mitogen stimulation. EGFR tyrosine phosphorylation and downstream signaling lasted 6 h in control cells versus 2 h in AAF-exposed hepatocytes. We previously demonstrated that AAF inhibits the growth factor-dependent induction of cyclin D1 and arrests hepatocyte cell-cycle progression before the p21/CIP1-controlled DNA-damage check point. The present data indicate that the DNA-damaging carcinogen AAF induces growth inhibition by a distinct inhibition of ERK nuclear accumulation after mitogen stimulation. Inhibition of intracellular signal transduction may represent a novel mechanism of growth arrest. Mol. Carcinog. 28:84-96, 2000.

MRNA EXPRESSION OF TRANSFORMING GROWTH FACTOR-α AND THE EGF RECEPTOR FOLLOWING NEPHROTOXIC RENAL INJURY

We studied gene expression for transforming growth factor (TGF)-α, epidermal growth factor (EGF), heparin binding (HB) EGF, and the EGF receptor following acute renal failure induced by mercuric chloride administration to gain insight into potential mechanisms of renal repair. Twenty four hours after HgCl2, 2 mg/kg, creatinine increased from 0.3 ± 0.01 mg/dl in controls to 2.2 ± 0.26 mg/dl in injured rats (n = 5, p < 0.01). Similar changes were observed after 3 days. Messenger RNA expression for EGF was decreased at 24 hours in HgCl2 treated rats and remained depressed for at least 3 days. On the other hand steady state mRNA for TGF-α increased nearly 2 fold at day 3 in HgCl2 treated rats 4 mg/kg. Heparin binding EGF was increased early, by day one in injured kidneys and gene expression for the EGF receptor was increased as well. Immunohistochemistry documented an increase in expression of TGF-α in injured kidneys at distal nephron sites. These studies suggest that TGF-α along with HB EGF may be important ligands for the EGF receptor during repair from renal injury.