The epidermal growth factor receptor (EGFR) is the prototypical member of a family of membrane-associated intrinsic tyrosine kinase receptors, the ErbB family. EGFR is activated by multiple ligands, including EGF, transforming growth factor (TGF)-α, HB-EGF, betacellulin, amphiregulin, epiregulin, and epigen. EGFR is expressed in multiple organs and plays important roles in proliferation, survival, and differentiation in both development and normal physiology, as well as in pathophysiological conditions. In addition, EGFR transactivation underlies some important biologic consequences in response to many G protein-coupled receptor (GPCR) agonists. Aberrant EGFR activation is a significant factor in development and progression of multiple cancers, which has led to development of mechanism-based therapies with specific receptor antibodies and tyrosine kinase inhibitors. This review highlights the current knowledge about mechanisms and roles of EGFR in physiology and disease.

Aspirin causes gastroduodenal ulcers and complications. Food bioactive compounds could exert beneficial effects in the gastrointestinal tract. We evaluated whether apple polyphenol extract (APE) reduced aspirin-induced injury to the rat gastric mucosa. Rats were treated with APE (10(-4) m catechin equivalent) before oral aspirin (200 mg/kg). Cyclo-oxygenase-2 (COX-2), transforming growth factor-alpha (TGF alpha) and heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA and protein expression were assessed by RT-PCR and Western blot analysis, respectively; malondialdehyde (MDA) was determined by HPLC; gastric secretion was evaluated in pylorus-ligated rats. APE decreased acute and chronic aspirin injury both macroscopically and microscopically (approximately 50 % decrease in lesion score; P < 0.05). Aspirin up-regulated mRNA and protein expression of COX-2 and HB-EGF, but not of TGF alpha; APE reduced aspirin-induced mRNA and protein over-expression of COX-2 and HB-EGF; aspirin significantly increased gastric MDA and this effect was counteracted by APE pre-treatment. APE did not significantly affect gastric acid secretion. In conclusion, APE reduces aspirin-induced gastric injury independently of acid inhibition. We speculate that APE might be of therapeutic use in the prophylaxis of aspirin-related gastropathy.

In mammals the embryo must implant in the uterus and develop a placenta to gain nutrition and facilitate gas exchange. In this article, the earliest events in this process are reviewed. The embryo can implant only when it has reached the blastocyst stage. The blastocyst is composed of an inner clump of cells, the inner cell mass, that gives rise to the fetus and an outer layer of trophectoderm (TE), the precursor of the placenta. Both blastocyst and uterus must differentiate in parallel to reach the appropriate state of maturity (activated blastocyst and receptive uterus) at which implantation can occur. Interaction between TE and the luminal epithelium (LE) lining the uterus initiates implantation, and both soluble signals and association between molecules on apposed surfaces appear to be involved. A number of cell surface molecules have been implicated in the initial attachment between TE and LE. These include HSPG, Le-y and the H-type-1 antigen, HB-EGF, trophinin-tastin-bystin complex, integrins, and extracellular matrix molecules such as osteopontin and laminin. Others, such as mucins, may need to be removed or modified to allow adhesion to proceed. Evidence for the role of these components is discussed.

Amiloride-sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate-limiting step for Na(+) absorption. Epidermal growth factor (EGF) is involved in the regulation of Na(+) transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na(+) absorption. The aim of the present study was to characterize the EGF regulation of Na(+) transport in cultured mouse renal collecting duct principal mpkCCD(c14) cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na(+) transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF-alpha, HB-EGF, and amphiregulin which also belong to the EGF-related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na(+) reabsorption. Inhibition of PI3-kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD(c14) cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague-Dawley rat kidneys. Both acute and long-term effects of EGF were abolished when cells were treated with tyrphostin AG-825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na(+) transport and that EGF biphasically modulates sodium transport in mpkCCD(c14) cells via the ErbB2 receptor.

OBJECTIVE

Patients with long-standing diabetes commonly develop diabetic encephalopathy, which is characterized by cognitive impairment and dementia. To identify potential treatments for diabetic encephalopathy, we focused on the protective action of glucagon-like peptide-1 (GLP-1) against neural cell apoptosis. In this study, we evaluated whether exposure of cells to GLP-1 leads to epidermal growth factor receptor (EGFR) transactivation and signaling through the PI3K/Akt/mTOR/GCLc/redox pathway, which we previously reported.

METHODS

We monitored the phosphorylation of EGFR and Akt in PC12 cells exposed to MG and GLP-1 that had been first incubated in the presence or absence of various inhibitors of EGFR transactivation.

RESULTS

DAPI staining revealed that pretreatment of cells with BiPS, HB-EGF and anti-TGF-α neutralization antibodies or AG1478 abrogated the ability of GLP-1 to rescue cells from MG-induced apoptosis. We show that exposure of PC12 cells to GLP-1 induces EGFR phosphorylation and that this effect was inhibited by prior exposure of the cells to BiPS, HB-EGF and anti-TGF-α neutralization antibodies or AG1478. Interestingly, these agents also diminished the capacity of GLP-1 to protect cells from MG-induced apoptosis. Moreover, these agents reduced GLP-1-induced phosphorylation of Akt. EGF itself also protected the cells from MG-induced apoptosis and induced phosphorylation of Akt, which was inhibited by LY294002.

CONCLUSIONS

The neuroprotective effects of GLP-1 against MG-induced apoptosis are mediated by EGFR transactivation, which signals through the PI3K/Akt/mTOR/GCLc/redox pathway in PC12 cells.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family, is produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (sHB-EGF). It is known that high expression of pro-HB-EGF occurs in hepatoma tissues, although its biological meaning remains unknown. We established two types of hepatoma cell lines (AH66tc), which stably produce pro-HB-EGF and sHB-EGF, respectively. While sHB-EGF-producing cells (sHB-AH) showed rapid growth, pro-HB-EGF-producing cells (pHB-AH) showed markedly suppressed cell growth as compared with the parental cells. Transforming growth factor beta or serum-starved conditions induced apoptosis of mock and sHB-AH as well as the parental cells, but not of pHB-AH. The resistance to apoptosis upon serum-starved treatment was correlated with an increase in the rate of the G1 phase in the cell cycle due to up-regulation of the cyclin-dependent kinase inhibitor p21. The mechanism underlying this resistance of pHB-AH to apoptosis was thought to be related to the prolonged half-life of the EGF receptor followed by continuous phosphorylation of the tyrosine residues. These observations demonstrate a unique function of pro-HB-EGF that is not observed for the mature form and show that pro-HB-EGF may act as a tumor survival factor in hepatoma cells.

BACKGROUND

The aim of the current study was to determine the role of heparin-binding (HB) epidermal growth factor (EGF)-like growth factor as a mediator of gut barrier function after hemorrhagic shock and resuscitation (HS/R) in mice.

METHODS

HS/R was induced in HB-EGF knockout (KO) and wild-type (WT) mice. Intestinal histologic injury scores, intestinal epithelial cell apoptosis, and gut barrier function were determined. Statistical analyses were performed using linear mixed models with P<.05 considered significant.

RESULTS

All mice subjected to HS/R had significantly increased intestinal histologic injury scores, apoptosis indices, and intestinal permeability compared with mice subjected to sham operation. Compared with WT mice, HB-EGF KO mice subjected to HS/R had significantly increased histologic injury (mean injury grade, 4.5 +/- 1 vs 2.75 +/- 0.5 at 3 hours of resuscitation; P<.05), increased apoptosis indices (mean apoptosis index, 6.84 +/- 1.95 vs 3.24 +/- 1.00 at 3 hours of resuscitation; P < .05), and increased mucosal permeability (FD4 clearance 78 +/- 18.91 vs 47.75 +/- 8.06 nL/min/cm(2) at 3 hours of resuscitation; P<.05).

CONCLUSIONS

HB-EGF is essential for the preservation of gut barrier function after HS/R. These findings support the clinical use of HB-EGF in protection of the intestines from disease states associated with intestinal hypoperfusion injury.

In pulmonary hypertension, induced in rats breathing high oxygen at normobaric pressure, vascular cell hypertrophy and hyperplasia thicken the walls of lung microvessels (15-100 microns in diameter). Over a 28-day time course, new contractile cells develop from intimal precursor smooth muscle cells, which include intermediate cells and interstitial fibroblasts. Cell labeling studies in vivo have shown that these cells proliferate more than other vascular cells and that most of this activity occurs between 4 and 7 days of hyperoxia. The growth factors responsible for this proliferation are unknown. In the present study, we investigate the expression of mRNA for the epidermal growth factor (EGF)-related protein, heparin-binding EGF-like growth factor (HB-EGF), a newly discovered mitogen for fibroblasts and smooth muscle cells. Northern analysis shows HB-EGF mRNA levels to be low in normal lung but increased 100-fold by day 7 of hyperoxia. In situ hybridization identifies a select group of cells expressing HB-EGF mRNA. In normal lung, hybridizing cells are randomly distributed in the alveolar wall and space. By day 7, they increase in number and cluster around the microvessels. Histochemical techniques identify cells expressing HB-EGF mRNA as eosinophils.

Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.

BACKGROUND

To detect the presence of multiple mediators and growth factors in tears of vernal keratoconjunctivitis (VKC) patients with active disease using stationary phase antibody arrays.

METHODS

Tears were collected from 12 normal subjects (CT) and 24 active VKC patients. Tears were centrifuged and successively probed using three microwell plate arrays specific for: (i) cytokines: interleukin (IL)-2, IL-4, IL-5, IL-8, IL-10, IL-12, IL-13, interferon-gamma and tumour necrosis factor-alpha; (ii) growth factors: basic fibroblast growth factor (bFGF), platelet-derived growth factor, thrombopoietin, angiopoietin-2, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), keratocyte growth factor, tissue inhibitor of metalloprotease (TIMP)-1 and heparin-binding epithelial growth factor (HB-EGF) and (iii) matrix metalloprotease (MMP)-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1 and TIMP-2.

RESULTS

Interleukin-8 signals were detected in all CT and highly detected in all VKC samples. The Th2-type cytokines, IL-4, IL-5 and IL-10 were detected only in tears of VKC patients. Signals for bFGF, HB-EGF, VEGF and HGF were detected in 41-87% of VKC samples and in few CT samples. Only TIMP-1 and TIMP-2 were found in all normal and patient tear samples, whereas MMP-1, MMP-2, MMP-3, MMP-9 and MMP-10 were highly present in all VKC samples.

CONCLUSIONS

Stationary phase antibody array methodology was useful for the screening of various cytokines, growth factors and MMPs in tears. These analyses identified in tears of VKC patients previously unreported factors including MMP-3 and MMP-10 and multiple proteases, growth factors and cytokines, which may all play an important role in the pathogenesis of conjunctival inflammation.

Vanadium pentoxide (V(2)O(5)) is a transition metal derived from the burning of petrochemicals that causes airway fibrosis and remodeling. Vanadium compounds activate many intracellular signaling pathways via the generation of hydrogen peroxide (H(2)O(2)) or other reactive oxygen species. In this study, we investigated the regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in human lung fibroblasts after V(2)O(5) treatment. V(2)O(5)-induced HB-EGF mRNA expression was abolished by N-acetyl-l-cysteine, suggesting an oxidant-mediated effect. Exogenous H(2)O(2) (>10 microM) mimicked the effect of V(2)O(5) in upregulating HB-EGF expression. Fibroblasts spontaneously released low levels of H(2)O(2) (1-2 microM), and the addition of V(2)O(5) depleted the endogenous H(2)O(2) pool within minutes. V(2)O(5) caused a subsequent increase of H(2)O(2) into the culture medium at 12 h. However, the burst of V(2)O(5)-induced H(2)O(2) occurred after V(2)O(5)-induced HB-EGF mRNA expression at 3 h, indicating that the V(2)O(5)-stimulated H(2)O(2) burst did not mediate HB-EGF expression. Either V(2)O(5) or H(2)O(2) activated ERK-1/2 and p38 MAP kinase. Inhibitors of the ERK-1/2 pathway (PD-98059) or p38 MAP kinase (SB-203580) significantly reduced either V(2)O(5)- or H(2)O(2)-induced HB-EGF expression. These data indicate that vanadium upregulates HB-EGF via ERK and p38 MAP kinases. The induction of HB-EGF is not related to a burst of H(2)O(2) in V(2)O(5) treated cells, yet the action of V(2)O(5) in upregulating HB-EGF is oxidant dependent and could be due to the reaction of V(2)O(5) with endogenous H(2)O(2).

Agonist stimulation of certain G protein-coupled receptors (GPCRs) causes shedding of heparin-binding epidermal growth factor (HB-EGF) through activation of matrix metalloproteinases (MMPs), with subsequent transactivation of the EGF receptor. MMPs are widely expressed, and their dysregulated expression is crucial in cancer, inflammation, and cardiovascular remodeling. Recent studies in hypertensive animals have shown enhanced expression and activation of MMPs and EGF receptors, and their inhibition attenuates cardiac hypertrophy, vasoconstriction and hypertension induced by GPCR agonists such as angiotensin II, endothelin-1 and phenylepherine. These findings suggest that selective inhibition of MMPs might have therapeutic potential in hypertension and other cardiovascular diseases.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) stimulates cell proliferation in the adult mammalian brain, but the mechanism involved is unknown. To address this issue we treated mouse brain cerebral cortical cultures enriched in neuronal precursors with full-length HB-EGF, its HB or EGF-like domain alone, or both domains in combination. Labeling of cultures with bromodeoxyuridine (BrdU), a marker of cell proliferation, was increased approximately 10% by the HB domain and approximately 20% by the EGF-like domain, and the effects of the two domains were additive. Full-length HB-EGF was most effective (approximately 50% increase) in stimulating BrdU incorporation. Preincubation with heparinase III or with Na-chlorate abolished cell proliferation induced by HB-EGF, consistent with dependence on cell-surface heparan sulfate proteoglycans. The effect of HB-EGF was also blocked by the EGF receptor (EGFR/ErbB1) inhibitors PD153035 and PD158780, implicating EGFR in HB-EGF-induced cell proliferation. The phosphatidylinositol 3'-kinase (PI3K) inhibitors LY294002 and wortmannin, and the MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors U0126 and PD98059, reduced HB-EGF-induced BrdU incorporation into cultures, and HB-EGF enhanced phosphorylation of Akt and ERK, implying a role for PI3K/Akt and MEK/ERK signaling in HB-EGF-stimulated cell proliferation. These findings help to clarify the molecular mechanisms through which HB-EGF operates.

The amount of mucin on the ocular surface is regulated by the rate of mucin synthesis, mucin secretion, and the number of goblet cells. We have previously shown that cholinergic agonists are potent stimuli of mucin secretion. In contrast, there have been no studies on the control of goblet cell proliferation. In this study we investigate the presence of the EGF family of growth factors and their receptors in rat conjunctiva and cultured rat conjunctival goblet cells as well as their effects on activation of signaling intermediates and goblet cell proliferation. Rat conjunctival goblet cells were grown in organ culture and identified as goblet cells by their morphology and positive staining for the lectin UEA-1 and cytokeratin 7. In the rat conjunctiva, the presence of the EGF family members epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), heparin binding EGF (HB-EGF), and heregulin was determined by RT-PCR. The receptors for these ligands, EGF receptor (EGFR), erbB2, erbB3, and erbB4 were detected in both rat conjunctiva and goblet cells by Western blot analysis. Immunofluorescence microscopy of conjunctival tissue determined that EGFR was present as punctate staining in the cytoplasm of conjunctival goblet cells while ErbB2 was present in the basolateral and lateral membranes of goblet cells. ErbB3 was localized to the cytosol of rat conjunctival goblet cells. In cultured goblet cells, EGFR and ErbB2 were present in the perinuclear area of the cells. ErbB3 was widely distributed throughout the cytoplasm of the cells. ErbB4 was not detected in either the conjunctiva or goblet cells by immunofluorescence microscopy. Using a multiplex assay system we measured phosphorylation (activation) of p44/p42 mitogen-activated protein kinase (MAPK), also known as ERK, Jun N-terminal kinase (JNK), p38 MAPK and AKT (also known as protein kinase B), molecules known to be activated by EGF receptor members. EGF, TGF-alpha and HB-EGF activated the signaling intermediate proteins whereas heregulin did not. No EGF family member significantly activated AKT. Consistent with these findings, EGF, TGF-alpha and HB-EGF each stimulated goblet cell proliferation as measured by WST-1 assay or immunofluorescence microscopy using an antibody against Ki-67, a protein expressed in dividing cells. Heregulin did not cause goblet cell proliferation. We conclude that multiple members of the EGF family, EGF, TGF-alpha and HB-EGF, and heregulin are present with three of the four erbB receptor subtypes. EGF, TGF-alpha and HB-EGF all stimulated the activation of signaling intermediates and caused goblet cell proliferation.

OBJECTIVE

The etiology of interstitial cystitis is unknown. Urine from patients with interstitial cystitis has been shown to inhibit urothelial proliferation through a putative antiproliferative factor and to contain decreased levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared to controls. Stretch of detrusor smooth muscle cells is known to stimulate HB-EGF production. Because bladder hydrodistention sometimes alleviates the symptoms of interstitial cystitis, we determined whether the stretch stimulus of hydrodistention alters antiproliferative factor activity and/or HB-EGF in interstitial cystitis urine specimens.

METHODS

Urine was collected immediately before, and 2 to 4 hours and 2 weeks after hydrodistention from 15 patients with symptoms and cystoscopic findings compatible with interstitial cystitis and 13 controls. Hydrodistention was performed with the subject under general or regional anesthesia and bladders were distended to 80 cm. water 3 times. Urinary HB-EGF was measured by enzyme-linked immunosorbent assay and urinary antiproliferative factor activity was determined by measuring 3H-thymidine uptake by normal human bladder urothelial cells.

RESULTS

Hydrodistention significantly increased urinary HB-EGF in patients with interstitial cystitis toward normal control values (before distention p = 0.003, 2 weeks after distention p = 0.67). Urine antiproliferative factor activity decreased significantly after hydrodistention in patients with interstitial cystitis. However, antiproliferative factor activity in interstitial cystitis and control specimens was still statistically different 2 weeks after distention (before distention p = 0.0000004, 2 weeks after distention p = 0.04).

CONCLUSIONS

Bladder stretch increased HB-EGF and conversely reduced antiproliferative factor activity in urine from patients with interstitial cystitis but not controls up to 2 weeks after distention. These results provide additional evidence for the possible role of antiproliferative factor and decreased HB-EGF in the pathophysiology of interstitial cystitis. To our knowledge this is also the first human study to show that in vivo bladder stretch can alter urinary factors that regulate cell growth.

Proliferation of neural stem cells in the embryonic cerebral cortex is regulated by many growth factors and their receptors. Among the key molecules stimulating stem cell proliferation are FGF-2 and the FGF receptor-1. This ligand-receptor system is highly dependent on the surrounding heparan sulfates. We have found that heparin-binding growth-associated molecule (HB-GAM, also designated as pleiotrophin) regulates neural stem cell proliferation in vivo and in vitro. Deficiency of HB-GAM results in a pronounced, up to 50% increase in neuronal density in the adult mouse cerebral cortex. This phenotype arises during cortical neurogenesis, when HB-GAM knockout embryos display an enhanced proliferation rate as compared to wild-type embryos. Further, our in vitro studies show that exogenously added HB-GAM inhibits formation and growth of FGF-2, but not EGF, stimulated neurospheres, restricts the number of nestin-positive neural stem cells, and inhibits FGF receptor phosphorylation. We propose that HB-GAM functions as an endogenous inhibitor of FGF-2 in stem cell proliferation in the developing cortex.

Hyaluronic acid (HA), a glycosaminoglycan located between keratinocytes in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat keratinocyte organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to 24 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by 24 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive factor. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble factor. From earlier evidence, we hypothesized that an EGF-like growth factor might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (2) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like growth factor (HB-EGF) blocked wounding-induced HA synthesis by>> or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor>> 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen and migration factor for vascular smooth muscle cells (SMC), promoted neovascularization in vivo in the rabbit cornea. MRI demonstrated quantitatively the angiogenic effect of HB-EGF when introduced subcutaneously into nude mice. HB-EGF is not directly mitogenic to endothelial cells but it induced the migration of bovine endothelial cells and release of endothelial cell mitogenic activity from bovine vascular SMC. This mitogenic activity was specifically blocked by neutralizing anti-vascular endothelial growth factor (VEGF) antibodies. In contrast, EGF or transforming growth factor-alpha (TGF-alpha) had almost no effect on release of endothelial mitogenicity from SMC. In addition, RT-PCR analysis demonstrated that VEGFHB-EGF, respectively. Our data suggest that HB-EGF, as a mediator of intercellular communication, may play a new important role in supporting wound healing, tumor progression and atherosclerosis by stimulating angiogenesis.

Insulin and insulin-like growth factor-1 (IGF-1) act through highly homologous receptors that engage similar intracellular signaling pathways, yet these hormones serve largely distinct physiological roles in the control of metabolism and growth, respectively. In an attempt to uncover the molecular mechanisms underlying their divergent functions, we compared insulin receptor (IR) and IGF-1 receptor (IGF-1R) regulation of gene expression by microarray analysis, using 3T3-L1 cells expressing either TrkC/IR or TrkC/IGF-1R chimeric receptors to ensure the highly selective activation of each receptor tyrosine kinase. Following stimulation of the chimeric receptors for 4 h, we detected 11 genes to be differentially regulated, of which 10 were up-regulated to a greater extent by the IGF-1R. These included genes involved in adhesion, transcription, transport, and proliferation. The expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen, was markedly increased by IGF-1R but not IR activation. This effect was dependent on MAPK, but not phosphatidylinositol 3-kinase, and did not require an autocrine loop through the epidermal growth factor receptor. HB-EGF mitogenic activity was detectable in the medium of 3T3-L1 preadipocytes expressing activated IGF-1R but not IR, indicating that the transcriptional response is accompanied by a parallel increase in mature HB-EGF protein. The differential abilities of the IR and IGF-1R tyrosine kinases to stimulate the synthesis and release of a growth factor may provide, at least in part, an explanation for the greater role of the IGF-1R in the control of cellular proliferation.

OBJECTIVE

To delineate the mechanism underlying the potential functional relationship between interstitial cystitis antiproliferative factor (APF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF), as APF has previously been shown to decrease the proliferation rate of normal bladder epithelial cells and the amount of HB-EGF produced by these cells.

METHODS

APF-responsive T24 transitional carcinoma bladder cells were treated with high-pressure liquid chromatography-purified native APF with or without HB-EGF to determine the involvement of signalling pathways and proliferation by Western blot analysis, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (Erk)/MAPK assays, and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay.

RESULTS

Cyclic stretch induced the secretion of HB-EGF from T24 cells overexpressing the HB-EGF precursor, resulting in enhanced proliferation. T24 cells treated with APF had increased p38MAPK activity and suppressed cell growth, events that were both reversed by treatment with a p38MAPK-selective inhibitor. Activation of Erk/MAPK by HB-EGF was inhibited by APF, and APF did not stimulate p38MAPK in the presence of soluble HB-EGF or when cells overexpressed constitutively secreted HB-EGF. Lastly, APF inhibitory effects on cell growth were attenuated by HB-EGF.

CONCLUSIONS

These results indicate that HB-EGF and APF are functionally antagonistic and signal through parallel MAPK signalling pathways in bladder cells.

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D₄ (LTD₄) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD₄ on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD₄. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT₁ cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD₄, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT₁ receptor antagonist, blocked the LTD₄-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD₄-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD₄ and exogenous HB-EGF. These findings suggest LTD₄ induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.

Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting that Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src-transformed Abl(-/-)/Arg(-/-) fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib-treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with inhibitors of the EGFR-invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively, our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating a EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib.

The purpose of this study was to evaluate the role of the epidermal growth factor receptor (EGFR) in parathyroid hormone-related protein (PTHrP) expression and humoral hypercalcaemia of malignancy (HHM), using two different human squamous-cell carcinoma (SCC) xenograft models. A randomised controlled study in which nude mice with RWGT2 and HARA xenografts received either placebo or gefitinib 200 mg kg(-1) for 3 days after developing HHM. Effectiveness of therapy was evaluated by measuring plasma calcium and PTHrP, urine cyclic AMP/creatinine ratios, and tumour volumes. The study end point was at 78 h. The lung SCC lines, RWGT2 and HARA, expressed high levels of PTHrP mRNA as well as abundant EGFR protein, but very little erbB2 or erbB3. Both lines expressed high transcript levels for the EGFR ligand, amphiregulin (AREG), as well as, substantially lower levels of transforming growth factor-alpha (TGF-alpha), and heparin binding-epidermal growth factor (HB-EGF) mRNA. Parathyroid hormone-related protein gene expression in both lines was reduced 40-80% after treatment with 1 muM of EGFR tyrosine kinase inhibitor PD153035 and precipitating antibodies to AREG. Gefitinib treatment of hypercalcaemic mice with RWGT2 and HARA xenografts resulted in a significant reduction of plasma total calcium concentrations by 78 h. Autocrine AREG stimulated the EGFR and increased PTHrP gene expression in the RWGT2 and HARA lung SCC lines. Inhibition of the EGFR pathway in two human SCC models of HHM by an anilinoquinazoline demonstrated that the EGFR tyrosine kinase is a potential target for antihypercalcaemic therapy.