OBJECTIVE

To compare the gene expression pattern of control postmortem retinas with retinas from patients with proliferative vitreoretinopathy (PVR), to determine the expression of the heparin binding epidermal growth factor-like growth factor (HB-EGF) by glial cells in fibroproliferative membranes, and to examine whether cells of the human Müller cell line, MIO-M1, respond to HB-EGF with proliferation, migration, and secretion of the vascular endothelial growth factor (VEGF).

METHODS

To identify genes that were differently expressed in PVR and control retinas, the RNA from the neural retinas of seven postmortem donors and of two patients with PVR were analyzed for differential gene expression, by hybridization of labeled cRNA probes to an Affymetrix human genome microarray set. The results were validated by real time PCR experiments investigating RNA from 6 postmortem retinas and 4 PVR retinas. Epiretinal PVR membranes were immunohistochemically stained for colocalization of HB-EGF and the glial cell marker, glial fibrillary acidic protein (GFAP). The HB-EGF evoked proliferation of cultured Müller cells was investigated by a bromodeoxyuridine immunoassay, chemotaxis was assessed with a migration assay, and the release of VEGF was evaluated by ELISA.

RESULTS

Out of the 12,600 genes and expressed sequence tags investigated, the levels of 80 showed an increased expression, and 21 were expressed at decreased levels, in the retinas of PVR patients compared to the control retinas. The upregulated signals include genes for nuclear and cell cycle related proteins, extracellular secretory proteins, cytosolic signaling proteins, and proteins of the membrane and the extracellular matrix. The genes of the hepatocyte growth factor and of HB-EGF were found to be expressed in PVR retinas but not in control retinas. In epiretinal membranes of patients with PVR, HB-EGF immunoreactivity partially colocalized with GFAP. In cultured Müller cells, HB-EGF stimulated both proliferation and chemotaxis, and the secretion of VEGF, via activation of the extracellular signal regulated kinases 1 and 2 and of the phosphatidylinositol-3 kinase.

CONCLUSIONS

The development of PVR is accompanied by complex changes of the gene expression in the neural retina, with an upregulation of genes that support cell proliferation, cell signaling, cell motility, and extracellular matrix remodeling. HB-EGF is one of the factors that are significantly upregulated in PVR retinas. HB-EGF expression in fibroproliferative tissue and its stimulatory effect on glial cell proliferation, chemotaxis, and VEGF secretion suggest that HB-EGF may be a factor mediating glial cell responses during PVR.

Intestinal ischemia/reperfusion (I/R) injury affects patients of different ages, especially premature babies and the elderly. The outcome after intestinal I/R is often dismal, which may be attributed to loss of the barrier and immune functions of the intestines, as well as development of secondary injury in remote organs. The available treatment for advanced gut ischemia mandates extensive resection, which may cause growth retardation in infants and nutritional problems in the elderly. Throughout the past decade we have been investigating the potential therapeutic role of heparin-binding epidermal growth factor-like factor (HB-EGF) in intestinal I/R. The mitogenic and chemoattractant functions of HB-EGF formed the initial rationale for our investigations. In addition, HB-EGF is a potent antiapoptotic protein that enables cells and tissues exposed to different apoptotic stimuli to survive hypoxic, oxidative, and nutritional stresses. HB-EGF is known to have a vital role in wound healing and postischemic regeneration in different organs. In the current review, we summarize the results of our findings of the beneficial effects of HB-EGF in intestinal I/R, supported by additional evidence from the literature and an explanation of different possible mechanisms of its actions. Collectively, the data strongly suggest a potential therapeutic role for the use of HB-EGF to treat intestinal ischemic diseases such as I/R and necrotizing enterocolitis.

We have previously demonstrated that enterally administered heparin-binding EGF-like growth factor (HB-EGF) produced in Escherichia coli decreases the incidence and severity of intestinal injury in a neonatal rat model of necrotizing enterocolitis (NEC). In preparation for upcoming human clinical trials, large-scale production of HB-EGF according to Good Manufacturing Practice (GMP) has been successfully accomplished using a Pichia pastoris yeast system. The current studies used a neonatal rat model of NEC to elucidate several important preclinical characteristics of HB-EGF therapy. We found that enteral administration of HB-EGF (800 microg/kg/dose) four times a day effectively reduced the incidence and severity of NEC, that Pichia-derived HB-EGF was not significantly different from E. coli-derived HB-EGF in preventing NEC, that EGF was not superior to HB-EGF in preventing NEC, and that prophylactic administration of HB-EGF added to formula starting with the first feed or 12 h later significantly reduced the incidence of NEC, with no change in the incidence of NEC noted if HB-EGF was added to the formula starting 24, 48, or 72 h after birth. Thus, large-scale production of GMP-grade HB-EGF in Pichia pastoris yeast produces a biologically active molecule suitable for human clinical trials.

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2',7'-dichlorofluorescin diacetate. N-Acetyl-L-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC. DL-Buthionine-(S, R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

To identify chemoresistance-related genes of gastric cancer, we utilized cDNA microarray technology. Thirty-five gastric cancer specimens surgically resected at our institute between 1998 and 1999 were studied for quantification of expression of 6300 genes by means of oligonucleotide microarray methods, and the results were evaluated in comparison with the chemoresistance of the specimens, which was determined by MTT (tetrazolium-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Inhibition rates (IR) were determined for cisplatin (DDP), 5-fluorouracil (5-FU), mitomycin C or doxorubicin. IR of 60% or more was regarded as sensitive to each agent, and IR of less than 40% was defined as resistant. Clustering was successfully completed for DDP, resulting in selection of 23 candidates as DDP-resistance-related genes, including vascular permeability factor, 2 membrane transporting subunits, and retinoblastoma-binding protein-1. In addition, further selection of DDP-resistance-related genes was performed according to these criteria: 1) Expression of the gene can be detected in more than 70% of resistant tumors. 2) Expression can be detected in less than 30% of sensitive tumors. 3) Expression in tumors is more than twice that of normal mucosa in more than 50% of specimens. Then, metallothionein-IG and heparin-binding epidermal growth factor-like growth factor (HB-EGF) were identified as candidate DDP-resistance-related genes. When known DDP-resistance-related genes were analyzed according to the MTT assay result, families of glutathione-S-transferase and cyclooxygenase-2 genes were also evaluated as resistance-related genes. For 5-FU resistance, dihydropyrimidine dehydrogenase and HB-EGF-like growth factor genes were also suggested to be resistance-related genes. The present study demonstrated that oligonucleotide microarrays can provide information regarding chemoresistance factors in cancer.

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA.

The levels of expression of the four receptors and eleven ligands composing the epidermal growth factor family were measured using immunohistochemical staining in one hundred cases of breast cancer. All of the family were expressed to some degree in some cases; however, individual cases showed a very wide range of expression of the family from essentially none to all the factors at high levels. The highest aggregate level of expression of a receptor was HER2 followed by HER1, then HER3, then HER4. The ligands (including two splice variants of the NRG1 and NRG2 genes) broadly fell into three groups, those with the highest aggregate expression were Epigen, Epiregulin, Neuregulin 1alpha, Neuregulin 2alpha, Neuregulin 2beta, Neuregulin 4 and TGFalpha, moderate expression was seen with EGF, Neuregulin 1beta and Neuregulin 3, and relatively low levels of expression were seen of HB-EGF, Betacellulin and Amphiregulin. Statistical analysis using Spearman's Rank Correlation showed a positive correlation of expression between each of the factors. Analysing the data using the Cox Proportional Hazards model showed that, in this dataset, the most powerful predictors of relapse free interval and overall survival were the combined measurement of only Epigen and Neuregulin 4.

Cultured rat bladder smooth muscle cells (SMC) were grown on collagen-coated silicone membranes and subjected to continuous cycles of stretch-relaxation. Semiquantitative RT-PCR analysis revealed a time-dependent increase in heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA levels after stretch, with maximal levels appearing after 4 h. Immunostaining for proHB-EGF revealed higher levels of HB-EGF protein in the stretched than in the nonstretched SMC. The ANG II receptor type 1 antagonist losartan markedly suppressed stretch-activated HB-EGF expression. ANG II levels were 3.3-fold higher in the stretch- than in the non-stretch-conditioned media. Stretch stimulation of bladder SMC that had been transiently transfected with an HB-EGF promoter-luciferase expression construct resulted in an 11-fold increase in reporter activity. Mechanical stretch induced a 4.7-fold increase in tritiated thymidine incorporation rate, and this was reduced by 25% in the presence of losartan. We conclude that mechanical stretch activates HB-EGF gene expression in bladder SMC and that this is mediated in part by autocrine ANG II secretion.

CD9 is a protein with 4 transmembrane domains, and functions as a cell surface antigen. We have previously reported that CD9 functions as an up-regulator of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) activity, which is a potent mitogen as well as a soluble HB-EGF. Anti-CD9 antibodies can neutralize the juxtacrine activity of proHB-EGF when both CD9 and proHB-EGF are coexpressed. We demonstrated here: (1) the CD9 gene was transcribed and translated in the cultured human keratinocytes; (2) anti-CD9 antibody inhibited the approximately 50% growth of human keratinocytes in culture; (3) CD9 was coprecipitated with proHB-EGF and membrane-anchored amphiregulin (proAR), and (4) the transient coexpression of CD9 with proHB-EGF or proAR in mouse L cells up-regulated their juxtacrine growth factor activities. These results suggest that CD9 would make a heterodimer and/or trimer complex with proHB-EGF and proAR, and might cooperate with proHB-EGF and proAR for human keratinocyte growth in a juxtacrine manner.

OBJECTIVE

Human corneal epithelial cells (HCECs) were functionally depleted of erbB2 to elucidate its role in epidermal growth factor (EGF) receptor (EGFR) activation-dependent cell migration.

METHODS

The retrovirus pBabe-5R, which encodes an erbB2 single-chain antibody with an endoplasmic reticulum (ER)-targeting sequence, and control pBabe-puro were used to infect THCE cells (an SV40-immortalized HCEC line). Several cell lines expressing 5R were selected along with a pBabe-puro control line. The depletion of erbB2 was verified by cell surface biotinylation of proteins, followed by streptavidin precipitation and subsequent detection of erbB2 by immunoblot analysis. Activation of erbBs was analyzed by immunoprecipitation using the phosphotyrosine antibody pY20, followed by Western blot analysis with erbB1 or erbB2 antibodies. Phosphorylation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) was analyzed by Western blot with antibodies specific to phosphorylated proteins. Effects of erbB2 depletion on heparin-binding EGF-like growth factor (HB-EGF)-induced cell migration were determined by Boyden chamber migration assay and by scratch wound assay.

RESULTS

Wounding induced erbB2 tyrosine phosphorylation. Expression of 5R encoding an erbB2 single-chain antibody with an endoplasmic reticulum-targeting sequence depleted the cell surface expression of erbB2 in HCECs. Wounding resulted in a rapid increase in the phosphorylation of erbB1 in both 5R-expressing and control cells, whereas wound-induced erbB2 phosphorylation in 5R-expressing cells was not detectable. Depletion of functional erbB2 attenuated the healing of scratch wounds in the presence of HB-EGF and impaired both chemotactic migration stimulated by HB-EGF and haptotactic migration toward a fibronectin-collagen I (3:1; FNC) coating mix. Expression of 5R affected both the intensity and the duration of wound-induced, EGFR-elicited ERK and PI3K activation. Inhibition of ERK and PI3K pathways in cultured porcine corneas impaired ex vivo epithelial wound healing.

CONCLUSIONS

ErbB2 serves as a critical component that couples erbB receptor tyrosine kinase to the migration machinery of corneal epithelial cells.

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression.

Platelet-activating factor (PAF) is a lipid mediator that has been implicated in a variety of keratinocyte functions. Keratinocytes express the specific receptor for PAF (PAF-R), a seven-transmembrane G-protein-coupled receptor. Although PAF-R-dependent stimulation of numerous signal transduction pathways has been shown in a variety of cell types, to date there has been no analysis of PAF-R signal transduction in human epidermal cells. There is also contradictory evidence that PAF acts as either a suppressor or activator of keratinocyte proliferation. Using a model system created by retroviral-mediated transduction of the PAF-R into the PAF-R-negative epidermal cell line KB, we now demonstrate that the activation of the epidermal PAF-R results in the activation of both the extracellular signal-regulated kinase (ERK) and p38, but not the jun N-terminal kinase mitogen-activated protein (MAP) kinase pathways. Additionally, we show that the activation of the PAF-R stimulates the replication of epidermal cells. The activation of the ERK signal transduction pathway, as well as the PAF-dependent increase in cell proliferation, was dependent on the transactivation of the epidermal growth factor receptor (EGF-R). PAF-R-induced transactivation of the EGF-R was blocked by pharmacologic inhibitors of matrix metalloproteinases, of heparin-binding epidermal growth factor (HB-EGF), and specific inhibitors of the EGF-R tyrosine kinase. Activation of p38 MAP kinase by the PAF-R was not dependent on EGF-R activation and represents a distinct pathway of PAF-R-mediated signal transduction. In summary, these studies provide a mechanism whereby the PAF-R can exert proliferative effects through the activation of the EGF-R.

This study was designed to investigate the role of the epidermal growth factor receptor (EGFR) and its ligands in chemically induced mouse bladder cancer. Bladder tumours were induced in C57Bl/6 and B6D2F1 mice by treatment with the carcinogen, N:-butyl-N:-(4-hydroxybutyl) nitrosamine (BBN). The levels of mRNA for EGFR and its ligands were analysed by reverse transcription-polymerase chain reaction (RT-PCR) in bladder tumours and in normal bladder urothelia. EGFR mRNA was detected in all tumours, transforming growth factor alpha (TGFalpha) mRNA levels were similar to those in normal bladder urothelia or were decreased and mRNA levels for amphiregulin, heparin-binding epidermal growth factor-like factor (HB-EGF) and betacellulin were significantly higher than those in normal urothelia. Seven cell lines were derived from chemically induced tumours. These cell lines were able to grow in serum-free conditions. All the cell lines tested expressed the genes encoding EGFR and at least one of its ligands. Proliferation of these cell lines was inhibited by AG1478, a specific EGFR tyrosine kinase inhibitor, strongly suggesting that EGFR was involved in cell growth. As expected, EGFR was found to be phosphorylated in serum-free medium, this phosphorylation being inhibited by AG1478. Conditioned medium of a bladder cancer cell line had EGFR-stimulating activity and an antibody directed against EGFR inhibited proliferation by 45%. This suggests that tumour cell growth is stimulated by an autocrine loop involving EGFR and secreted growth factors. AG1478 decreased the expression of genes for amphiregulin, HB-EGF and betacellulin, showing that EGFR activation induces up-regulation of the EGFR ligands. These results suggest that EGFR plays a critical role in bladder tumour progression.

Lysophosphatidylcholine is increased in the plasma of hypercholesterolemic patients, is a component of oxidatively modified low-density lipoprotein, and, as such, may play an important role in atherosclerosis. Here we demonstrate that in human monocytes, lysophosphatidylcholine increases the level of mRNA encoding the heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle mitogen. Lysophosphatidylcholine treatment also enhances the release of heparin-binding mitogenic activity by these cells in culture. The anti-inflammatory glucocorticoid dexamethasone inhibits the upregulation of HB-EGF mRNA induced by either lysophosphatidylcholine or bacterial lipopolysaccharide in cultured monocytes. However, the responses induced by lysophosphatidylcholine and by lipopolysaccharide differ in their kinetics. In addition, the response to lysophosphatidylcholine is resistant to the action of cycloheximide, whereas the response to lipopolysaccharide is not, suggesting that the activation mechanisms induced by these two stimuli are different. Since a nuclear run-on assay showed no effect of lysophosphatidylcholine on the transcription of the HB-EGF gene, we speculate that lysophosphatidylcholine may increase the level of HB-EGF mRNA by altering the processing or degradation of primary or mature transcripts. Lysophosphatidylcholine enhancement of monocyte production of HB-EGF may represent an important result of the interactions among oxidized low-density lipoprotein and monocyte-derived macrophages and may play a role in initiation of smooth muscle proliferation in atherogenesis.

To investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in skeletal muscle, we studied its function in skeletal myotubes in vitro using mouse C2C12 cells. Expression levels of membrane-anchored HB-EGF (proHB-EGF) protein were increased specifically during their differentiation among epidermal growth factor receptor (EGFR) ligands. Production levels of EGFR on the cell surface were constant. Tyrosine phosphorylation of EGFR, however, was constitutively increased during differentiation. Quenching of endogenous HB-EGF significantly rendered myotubes sensitive to apoptotic cell death induced by hypoxic stress, suggesting that proHB-EGF in the skeletal muscle is specifically upregulated to function as a survival factor.

The bronchial epithelium is a potential source of growth factors that could mediate airway fibrosis during the progression of diseases such as asthma and chronic bronchitis. We report that conditioned medium (CM) from normal human bronchial epithelial cells (NHBECs) contains mitogenic activity for human lung fibroblasts that is blocked by the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitor AG1478 and by neutralizing antibodies raised against heparin-binding epidermal growth factor-like growth factor (HB-EGF). Neutralizing antibodies against other EGF-R ligands (EGF and transforming growth factor-alpha) or other antibodies against growth factors (platelet-derived growth factors, insulin-like growth factor-1) had no affect on the mitogenic activity of NHBEC-CM. HB-EGF messenger RNA (mRNA) expression in NHBEC was detected by reverse transcriptase/polymerase chain reaction and Northern blot analysis. HB-EGF protein was detected by enzyme-linked immunosorbent assay. Vanadium pentoxide (V2O5), a fibrogenic metal associated with occupational asthma, caused a several-fold increase in HB-EGF mRNA expression and protein, whereas the inert metal titanium dioxide had no effect on HB-EGF expression. V2O5-induced HB-EGF mRNA expression was inhibited by the EGF-R tyrosine kinase inhibitor AG1478, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580, and the MAP kinase kinase inhibitor PD98059. Finally, HB-EGF induced the production of fibroblast growth factor (FGF)-2 by human lung fibroblasts and anti-FGF-2 antibody partially blocked the mitogenic activity of NHBEC-CM on fibroblasts. These data suggest that HB-EGF is a fibroblast mitogen produced by NHBECs and that induction of an FGF-2 autocrine loop in fibroblasts by HB-EGF accounts for part of this mitogenic activity.

BACKGROUND

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurely born infants. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) have protective effects against intestinal injury. The aim of this study was to compare the effect of oral administration of HB-EGF, EGF, or both on the incidence of NEC in a neonatal rat model.

METHODS

Premature rats were fed by hand and exposed to asphyxia and cold stress to develop NEC. Four diets were used: formula (NEC), formula supplemented with 500 ng/mL HB-EGF (HB), 500 ng/mL EGF (EGF), or a combination of both (E+HB). Ileal injury, endogenous HB-EGF production, expression of EGF receptors, goblet cell density, and expression of apoptotic proteins were evaluated.

RESULTS

Oral administration of either EGF or HB-EGF significantly reduced the incidence of NEC; however, EGF provided better protection in physiologically relevant doses. Simultaneous administration of both growth factors did not result in any synergistic protective effect against NEC. There were no significant differences between treatment groups in ileal gene expression of EGF receptors or HB-EGF. However, the balance of apoptotic proteins in the ileum was shifted in favor of cell survival in EGF-treated rats. This mechanism may be responsible for the higher efficiency of EGF protection against NEC.

CONCLUSIONS

These data suggest that a physiological dosage of EGF or a pharmacological dosage of HB-EGF could be used for prevention of NEC.

We previously reported that pharmacological preconditioning of rabbit hearts with acetylcholine involves activation of phosphatidylinositol 3-kinase (PI3-K) through transactivation of the epidermal growth factor receptor (EGFR). Transactivation is thought to be initiated by cleavage of membrane-bound pro-heparin-binding EGF-like growth factor (HB-EGF) by a membrane metalloproteinase thus releasing HB-EGF which binds to the EGFR. This pathway leads to redox signaling with the generation of reactive oxygen species (ROS) by mitochondria. We tested whether preconditioning's physiological triggers, bradykinin and opioid, also signal through the EGFR. Both bradykinin and the synthetic delta-opioid agonist DADLE increased ROS production in isolated cardiomyocytes by approximately 50%. DADLE's effect was abrogated by either metalloproteinase inhibitor III (MPI) or the diphtheria toxin mutant CRM-197 which blocks heparin-binding EGF shedding indicating that DADLE signals through EGFR transactivation. MPI also blocked DADLE's infarct-sparing effect in whole hearts. Additionally, blocking Src kinase (a component of the EGFR's signaling complex) with PP2 or PI3-K with wortmannin blocked DADLE's effect on cardiomyocyte ROS production and PP2 blocked DADLE's salvage of ischemic myocardium. Finally, DADLE increased phosphorylation of Akt and extracellular signal-regulated protein kinases (ERK) 1/2 in left ventricular myocardium, and this increase was blocked by the EGFR antagonist AG1478. On the other hand, neither MPI nor CRM-197 prevented bradykinin from increasing ROS production, and MPI did not affect bradykinin's infarct-sparing effect in intact hearts. Conversely, both PP2 and wortmannin blocked bradykinin's effect on ROS generation and also aborted bradykinin's cardioprotective effect in intact hearts. While bradykinin also increased phosphorylation of Akt and ERK in myocardium, that increase was not affected by AG1478. Hence bradykinin, unlike acetylcholine or opioid, does not transactivate EGFR, although all 3 agonists do signal through Src and PI3-K.

OBJECTIVE

The aim of this study was to explore meprinα-mediated transactivation of the epidermal growth factor receptor (EGFR) and reactive oxygen species (ROS) production in macrophages.

RESULTS

Accelerated atherosclerotic lesions were established by administration of a high-fat diet in apolipoprotein E-deficient (apoE(-/-)) mice. Lentiviral overexpression of meprinα in the thoracic aortic artery during plaque formation enhanced intra-plaque macrophage induction of ROS as well as formation of atherosclerotic plaques, whereas AG1478 (specific inhibitor of the EGFR) treatment exerted the opposite effect. A meprinα inhibitor abrogated EGFR activation in mice. In cultured J774a.1 macrophages, oxidized low-density lipoprotein (OxLDL) increased ROS formation and EGFR activation through a ligand [heparin-binding epidermal growth factor-like growth factor (HB-EGF)]-dependent pathway. However, a meprinα inhibitor or specific siRNA inhibited ROS production and EGFR activation. Recombinant mouse meprinα enhanced OxLDL-stimulated production of ROS and induced HB-EGF. Inhibition of p38 mitogen-activated protein kinase by SB203580 decreased OxLDL-stimulated production of ROS. Conversely, inhibition of meprinα or PI3K-Rac1 inhibitors also decreased p38 activity in OxLDL-stimulated macrophages. In addition, inhibition of meprinα reversed OxLDL-stimulated activation of PI3K.

CONCLUSIONS

Meprinα promotes OxLDL-induced plaque formation and ROS release by transactivation of the EGFR, followed by activation of the PI3K/Rac1/p38 pathway.

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis-associated cancer in mice. AOM/DSS-induced tumor formation was reduced in CCL3- or its specific receptor, CCR5-deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS-induced type I collagen-positive fibroblast accumulation in the colon was reduced in CCL3- or CCR5-deficient mice. This was associated with depressed expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB-EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB-EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3-CCR5-mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full-blown colitis-associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis-associated cancer.

The epidermal growth factor (EGF) family of polypeptides is regulators for tissue development and repair, and is characterized by the fact that their mature forms are proteolytically derived from their integral membrane precursors. This article reviews roles of the prominent members of the EGF family (EGF, transforming growth factor-alpha [TGF-alpha] and heparin-binding EGF [HB-EGF]) and the related neuregulin family in the nerve system. These polypeptides, produced by neurons and glial cells, play an important role in the development of the nervous system, stimulating proliferation, migration, and differentiation of neuronal, glial, and Schwann precursor cells. These peptides are also neurotrophic, enhancing survival and inhibiting apoptosis of post-mitotic neurons, probably acting directly through receptors on neurons, or indirectly via stimulating glial proliferation and glial synthesis of other molecules such as neurotrophic factors. TGF-alpha, EGF, and neuregulins are involved in mediating glial-neuronal and axonal-glial interactions, regulating nerve injury responses, and participating in injury-associated astrocytic gliosis, brain tumors, and other disorders of the nerve system. Although the collective roles of the EGF family (as well as those of the neuregulins) are shown to be essential for the nervous system, redundancy may exist among members of the EGF family.

Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-alpha, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-alpha signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-alpha. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-alpha gradient serving as a morphogen of eyelid morphogenesis.

Disruption of intestinal epithelial homeostasis, including enhanced apoptosis, is a hallmark of inflammatory bowel disease (IBD). We have recently shown that tumor necrosis factor (TNF) increases the kinase activity of ErbB4, a member of the epidermal growth factor receptor family that is elevated in mucosa of IBD patients and that promotes colon epithelial cell survival. In this study, we tested the hypothesis that TNF transactivates ErbB4 through TNF-α converting enzyme (TACE)-mediated ligand release and that this transactivation is necessary to protect colonic epithelial cells from cytokine-induced apoptosis. Using neutralizing antibodies, we show that heparin-binding EGF-like growth factor (HB-EGF) is required for ErbB4 phosphorylation in response to TNF. Pharmacological or genetic inhibition of the metalloprotease TACE, which mediates HB-EGF release from cells, blocked TNF-induced ErbB4 activation. MEK, but not Src or p38, was also required for transactivation. TACE activity and ligand binding were required for ErbB4-mediated antiapoptotic signaling; whereas mouse colon epithelial cells expressing ErbB4 were resistant to TNF-induced apoptosis, TACE inhibition or blockade of ErbB4 ligand binding reversed the survival advantage. We conclude that TNF transactivates ErbB4 through TACE-dependent HB-EGF release, thus protecting colon epithelial cells from cytokine-induced apoptosis. These findings have important implications for understanding how ErbB4 protects the colon from apoptosis-induced tissue injury in inflammatory conditions such as IBD.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a critical growth factor for a number of physiological and pathological processes. HB-EGF is synthesized as a membrane-anchored form (pro-HB-EGF), and pro-HB-EGF is cleaved at the cell surface to yield soluble HB-EGF by a mechanism called "ectodomain shedding." We show here that the ectodomain shedding of pro-HB-EGF in Vero cells is induced by various stress-inducing stimuli, including UV light, osmotic pressure, hyperoxidation, and translation inhibitors. The pro-inflammatory cytokine interleukin-1beta also stimulated the ectodomain shedding of pro-HB-EGF. An inhibitor of p38 MAPK (SB203580) or the expression of a dominant-negative (dn) form of p38 MAPK inhibited the stress-induced ectodomain shedding of pro-HB-EGF, whereas an inhibitor of JNK (SP600125) or the expression of dnJNK1 did not. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and lysophosphatidic acid (LPA) are also potent inducers of pro-HB-EGF shedding in Vero cells. Stress-induced pro-HB-EGF shedding was not inhibited by the inhibitors of TPA- or LPA-induced pro-HB-EGF shedding or by dn forms of molecules involved in the TPA- or LPA-induced pro-HB-EGF shedding pathway. Reciprocally, SB203580 or dnp38 MAPK did not inhibit TPA- or LPA-induced pro-HB-EGF shedding. These results indicate that stress-induced pro-HB-EGF shedding is mediated by p38 MAPK and that the signaling pathway induced by stress is distinct from the TPA- or LPA-induced pro-HB-EGF shedding pathway.