Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.

Recent clinical studies have revealed that increased serum triglyceride (TG) levels are closely related to atherosclerosis, independently of serum levels of high-density lipoproteins (HDL) and low-density lipoproteins (LDL). Among triglyceride-rich lipoproteins (TRLs), remnant lipoproteins (RLPs) are considered to be atherogenic and an independent coronary risk factor. We previously reported that monocytes cultured in the presence of RLPs increased their adhesion to vascular endothelial cells. The underlying mechanism involved activation of RhoA, a member of small GTP binding proteins, resulting in activation of focal adhesion kinase (FAK) and s1-integrin. It is also known that RLPs enter vessel walls. In another study, we reported that RLPs induced smooth muscle cell (SMC) proliferation, independently of oxidative stress. Recently, we identified the molecular mechanisms, in which RLPs from hypertriglyceridemic patients stimulated SMC proliferation via epidermal growth factor (EGF) receptor transactivation and heparin-binding EGF-like growth factor (HB-EGF) shedding. More recently, we reported that apoB48 receptor was involved in RLP-induced foam cell formation in macrophages. The current review focused on the molecular mechanisms for the atherogenicity of RLPs.

Several growth factors play an important role in liver regeneration. Once hepatic injury occurs, liver regeneration is stimulated by hepatocyte growth factor (HGF), transforming growth factor (TGF)-alpha, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), whereas TGF-beta1 terminates liver regeneration. In this study, we analyzed the effect of a combination of HGF and epidermal growth factor (EGF) on mitogen-activated protein kinase (MAPK) activity and G1 cyclin expression in primary cultured rat hepatocytes. Treatment with a combination of HGF and EGF, in comparison with that of either HGF or EGF, induced tyrosine phosphorylation of both c-Met and EGF receptor (EGFR) independently and additively stimulated MAPK activity and cyclin D1 expression, resulting in additive stimulation of DNA synthesis. On the other hand, although TGF-beta1 treatment did not affect tyrosine phosphorylation of c-Met and EGFR, MAPK activity, and cyclin D1 expression, which were stimulated by HGF and EGF, DNA synthesis was completely inhibited through a marked decrease in cyclin E expression. These results indicate that potent mitogens, such as HGF, TGF-alpha, and HB-EGF, could induce the additive enhancement of liver regeneration cooperatively through an increase in Ras/MAPK activity followed by cyclin D1 expression, and that TGF-beta1 suppresses the growth factor-induced signals between cyclin D1 and cyclin E, resulting in the inhibition of DNA synthesis.

BACKGROUND

The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.

RESULTS

We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.

CONCLUSIONS

Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic activities. Y-142 may have a potential to be developed into a therapeutic agent for the treatment of HB-EGF-dependent cancers.

OBJECTIVE

It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF.

METHODS

Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration.

RESULTS

Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice.

CONCLUSIONS

HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), an activating ligand for the epidermal growth factor receptor (ErbB1) tyrosine kinase and at least one isoform of the ErbB4 receptor tyrosine kinase, is synthesized by the smooth muscle of the human bladder wall. In this study we tested the hypothesis that HB-EGF plays a role in the bladder-wall thickening that occurs in response to obstructive syndromes affecting the lower urinary tract, possibly by acting as an autocrine smooth muscle cell (SMC) growth factor. HB-EGF was mitogenic for primary culture human bladder SMC, and cell growth in serum-containing medium was inhibited more than 70% by [Glu52]-diphtheria toxin/CRM197, a specific HB-EGF inhibitor, consistent with a physiologic role for HB-EGF as an autocrine bladder SMC mitogen. Human and mouse bladder SMC in vivo and cultured human bladder SMC expressed the primary HB-EGF receptor, ErbB1, but not mRNA for the secondary HB-EGF receptor, ErbB4, thereby identifying ErbB1 as the cognate HB-EGF receptor in the bladder wall. Reverse transcription-polymerase chain reaction analysis also demonstrated ErbB2 and ErbB3 expression in human bladder muscle tissue, suggesting the possibility of receptor cross-talk after ErbB1 activation. Urethral ligation in mice resulted in an increase in steady-state HB-EGF mRNA expression up to 24 hours in whole bladder tissue in comparison with ErbB1 and glyceraldehyde 3-phosphate dehydrogenase mRNA levels, which did not change in a demonstrable pattern. HB-EGF protein increased coordinately with HB-EGF mRNA levels. Dissection of bladder tissue into muscle and mucosal layers demonstrated that the increase in HB-EGF mRNA occurred predominantly in the muscle layer, with peak levels (13-fold higher than sham controls) occurring 12 hours after obstruction. These data support a physiologic role for HB-EGF as a mediator of hypertrophic bladder tissue growth.

BACKGROUND

All-trans retinoic acid (RA) is known to regulate keratinocyte proliferation and differentiation, and retinoids are used as therapeutic agents in certain dermatological disorders, such as psoriasis and acne. Epidermal expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced by RA treatment and HB-EGF is responsible for RA-mediated epidermal hyperplasia in vivo. RA also induces HB-EGF expression in cultured keratinocytes and alters their differentiating phenotype. R115866 is a specific inhibitor of the cytochrome P450 isoform CYP26, which is involved in the metabolic inactivation pathway of RA. Thereby, R115866 is thought to be able to increase the intracellular levels of endogenous RA.

OBJECTIVE

To determine whether or not R115866 potentiates the effect of low concentrations of RA on keratinocytes.

METHODS

We analysed HB-EGF, involucrin and keratin 10 mRNA and protein levels in autocrine human keratinocyte cultures incubated for 18 h with RA or R115866 alone and with RA and R115866 combinations.

RESULTS

RA induced HB-EGF and involucrin expression in a concentration-dependent manner, whereas it inhibited keratin 10 expression. R115866 alone had no effect on the expression of these genes. However, when R115866 was combined with low concentrations of RA, HB-EGF and involucrin expression was induced.

CONCLUSIONS

These results strongly suggest that R115866 potentiates the effects of RA on epidermal keratinocytes when RA is present at low concentrations.

During pregnancy, up-regulation of heparin-binding (HB-) EGF and cyclooxygenase-2 (COX-2) in the uterine epithelium contributes to decidualization, a series of uterine morphological changes required for placental formation and fetal development. Here, we report a key role for the lipid mediator lysophosphatidic acid (LPA) in decidualization, acting through its G-protein-coupled receptor LPA3 in the uterine epithelium. Knockout of Lpar3 or inhibition of the LPA-producing enzyme autotaxin (ATX) in pregnant mice leads to HB-EGF and COX-2 down-regulation near embryos and attenuates decidual reactions. Conversely, selective pharmacological activation of LPA3 induces decidualization via up-regulation of HB-EGF and COX-2. ATX and its substrate lysophosphatidylcholine can be detected in the uterine epithelium and in pre-implantation-stage embryos, respectively. Our results indicate that ATX-LPA-LPA3 signaling at the embryo-epithelial boundary induces decidualization via the canonical HB-EGF and COX-2 pathways.

Increased expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) is frequently observed in certain cancers such as ovarian and breast cancers, and this protein is a desirable target for drug delivery by a drug delivery system (DDS). In the present study, we developed novel immunoliposomes targeting HB-EGF for cancer therapy. The immunoliposomes significantly associated with Vero-H cells overexpressing HB-EGF compared with their binding to wild-type Vero cells, whereas liposomes without modification by the antibody did not associate with either type of cells. Moreover, enhanced uptake of the immunoliposomes into Vero-H cells was observed as well as that into MDA-MB-231 human breast cancer cells, which are known to highly express HB-EGF. These results suggest that HB-EGF mediates the binding and uptake of the immunoliposomes in HB-EGF-expressing cells. Next, we determined the therapeutic effect of these immunoliposomes encapsulating an anticancer drug on tumor-bearing mice. For this purpose, we prepared doxorubicin (DOX)-encapsulated immunoliposomes and injected them intravenously into mice bearing MDA-MB-231 cancer cells. As a result, these DOX-encapsulated immunoliposomes suppressed not only tumor progression but also tumor regression. In conclusion, our results indicate that anti-HB-EGF antibody-modified liposomes could be a useful DDS carrier for the treatment of HB-EGF-expressing cancers.

OBJECTIVE

To test the hypothesis that the epoxyeicosatrienoic acid (EET)-induced transactivation of EGF-R depends on the activation of metalloproteinases and the subsequent release of HB-EGF in cancer cells.

METHODS

Exogenous 14,15-EET were added to four human-derived cancer cell lines Tca-8113, A549, HepG2, and MDA-MB-231, or these same cell lines were transfected with a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase). The effects of elevated EET levels on the phosphorylation of tyrosine residues in the EGF receptor and on ERK1/2 activation were then assessed.

RESULTS

Both the addition of 14,15-EET and the transfection of cells with CYP102 F87V markedly increased the phosphorylation of the tyrosine residues of EGF-R and ERK1/2, an effect that was blocked by a selective EGF-R tyrosine kinase inhibitor (tyrphostin AG1478), a broad-spectrum metalloproteinase inhibitor (1,10-phenanthroline), and an inhibitor of HB-EGF release (CRM197) in Tca-8113 cells. In addition, AG1478, 1,10-phenanthroline, and CRM197 also inhibited the tyrosine phosphorylation of EGF-R and ERK1/2 that was induced by 14,15-EET in the A549, HepG2, and MDA-MB-231 cell lines.

CONCLUSIONS

These results suggest that the EET-induced transactivation of EGF-R depends on activation of metalloproteinases and the subsequent release of HB-EGF in cancer cell lines.

Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

We show a novel drug delivery system (DDS) for improved all-trans retinoic acid (atRA) therapy for external treatments of photo-damaged skin. We prepared inorganic-coated atRA nanoparticles, in turn an egg-like structure in nano-scale (Nano-atRA), using boundary-organized reaction droplets. The interfacial properties of organic architectures, in atRA micelles, were used to template the nucleation of inorganic minerals. As a result, irritation and inflammation associated with atRA therapy were substantially reduced due to the complete encapsulation of the carboxylic function. Both irritative symptoms and physicochemical instability of the atRA micelle were improved. Since Nano-atRA which is prepared following to this new DDS system developmentally improved the permeability to the stratum corneum, the remarkable pharmacological effects were resulted in comparison with atRA as such as follows: (1) thicker epidermis than classical atRA treatment and (2) the overexpression of mRNA for heparin-binding epidermal growth factor (HB-EGF) as the provocation epidermal hyperplasia. Furthermore, we found a surprising boost in production of hyaluronan (HA) among the intercellular spaces of the basal and spinous cell layers in epidermis. Nano-atRA technology for atRA therapy could not only efficiently regulate keratinocyte cell proliferation and differentiation, but also markedly produce the additional benefit. Severely injured human skin by chronic ultraviolet irradiation will completely repair due to the accelerated turnover of skin tissue, which is induced by Nano-atRA.

Neisseria meningitidis, the causative agent of meningitis and septicemia, attaches to and invades various cell types. Both steps induce and/or require tyrosine phosphorylation of host cell proteins. Here, we used a phospho array platform to identify active receptor tyrosine kinases (RTKs) and key signaling nodes in N. meningitidis-infected brain endothelial cells to decipher RTK-dependent signaling pathways necessary for bacterial uptake. We detected several activated RTKs, including the ErbB family receptors epidermal growth factor receptor (EGFR), ErbB2, and ErbB4. We found that pharmacological inhibition and genetic ablation of ErbB receptor tyrosine phosphorylation and expression resulted in decreased bacterial uptake and heterologous expression of EGFR, ErbB2, or ErbB4 in Chinese ovary hamster (CHO-K1) cells, which do not express of EGFR and ErbB4; the decrease caused a significant increase in meningococcal invasion. Activation of EGFR and ErbB4 was mediated by transactivation via the common ligand HB-EGF (heparin-binding EGF-like ligand), which was significantly elevated in infected cell culture supernatants. We furthermore determined that N. meningitidis induced phosphorylation of EGFR at Tyr845 independent of ligand binding, which required c-Src activation and was involved in mediating uptake of N. meningitidis into eukaryotic cells. Increased uptake was repressed by expression of EGFR Y845F, which harbored a point mutation in the kinase domain. In addition, activation of ErbB4 at its autophosphorylation site, Tyr1284, and phosphorylation of ErbB2 Thr686 were observed. Altogether, our results provide evidence that EGFR, ErbB2, and ErbB4 are activated in response to N. meningitidis infection and shed new light on the role of ErbB signaling in meningococcal infection biology.

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

The interaction between cancer cells and their microenvironment is a paradoxical cycle that exacerbates cancer progression and results in metastasis. Our study investigated the mechanism underlying the synergistic enhancement of release of soluble factors from tumor-associated dendritic cells and its effect on cancer development. The combination of HB-EGF (heparin-binding EGF-like growth factor) and CXCL5 (CXCL5/epithelial neutrophil-activating peptide-78) produced a strong synergistic effect on cancer proliferation, epithelial-mesenchymal transition, migration and invasion. CXCL5 not only potentiated the classical EGFR pathway and the AKT and ERK/RSK1/2 signaling pathways but also increased the phosphorylation of heat shock protein 27 (HSP27), which was slightly increased in A549 cells treated with either HB-EGF or CXCL5 only. Phosphorylated HSP27 stabilized sustained AKT activity by direct interaction, leading to enhanced tumor spheroid formation. Knockdown of HSP27 by shRNA decreased HB-EGF plus CXCL5-mediated tumor spheroid formation in a three-dimensional culture system, suggesting that AKT/HSP27 was required for HB-EGF/CXCL5-mediated cancer progression. Inhibiting RSK also reduces the modulation of c-Fos phosphorylation, Snail upregulation and cell migration by HB-EGF plus CXCL5, suggesting a synergistic effect of ERK/RSK and HB-EGF plus CXCL5 on cell migration. In mice, CXCL5 antibody synergistically enhances the efficiency of the tyrosine kinase inhibitor, gefitinib, without increasing its toxicity. These results provide evidence that elucidates potential cross-points between extracellular signals affecting lung cancer progression. Targeting CXCL5 may provide therapeutic benefits for lung cancer chemotherapy or immunotherapy.

In this study, the role of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK1/2), heparin-binding EGF-like growth factor (HB-EGF), general metalloproteinases, matrix metalloproteinases-2 (MMP-2) in mediating the mitogenic action of thrombin in rat vascular smooth muscle cells (VSMC) was investigated. The incubation of rat VSMC with thrombin (1 U/ml) for 5 min resulted in significant (p < 0.001) increase of ERK1/2 phosphorylation by 8.7 ± 0.9-fold, EGFR phosphorylation by 8.5 ± 1.3-fold (p < 0.001) and DNA synthesis by 3.6 ± 0.4-fold (p < 0.001). Separate 30-min pretreatments with EGFR tyrosine kinase irreversible inhibitor, 10 µM PD169540 (PD), and 20 µM anti-HB-EGF antibody significantly reduced thrombin-stimulated EGFR and ERK1/2 phosphorylation by 81, 72 % and by 48 and 61 %, respectively. Furthermore, the same pretreatments with PD or anti-HB-EGF antibody reduced thrombin-induced VSMC proliferation by 44 and 45 %, respectively. In addition, 30-min pretreatments with 10 µM specific MMP-2 inhibitor significantly reduced thrombin-stimulated phosphorylation of both EGFR and ERK1/2 by 25 %. Moreover, the same pretreatment with MMP-2 inhibitor reduced thrombin-induced VSMC proliferation by 45 %. These results show that the thrombin-induced DNA synthesis correlates with the level of ERK1/2 activation rather than EGFR activation. These results further suggest that thrombin acts through EGFR and ERK 1/2 signaling pathways involving MMP-2 to upregulate proliferation of VSMC.

BACKGROUND

Necrotizing enterocolitis (NEC) is associated with loss of neurons and glial cells in the enteric nervous system (ENS). Our goal was to determine whether enteric neural stem cell (NSC) transplantation, in conjunction with heparin-binding epidermal growth factor-like growth factor (HB-EGF), could protect against experimental NEC.

METHODS

In vitro, HB-EGF on NSC proliferation and migration, and the effects of receptors utilized by HB-EGF to exert these effects, were determined. In vivo, mouse pups were exposed to experimental NEC and treated with NSC alone, HB-EGF alone, NSC+HB-EGF, or HB-EGF overexpressing NSC. NSC engraftment and differentiation into neurons in the ENS, intestinal injury, intestinal permeability, and intestinal motility were determined.

RESULTS

HB-EGF promoted NSC proliferation via ErbB-1 receptors and enhanced NSC migration via ErbB-1, ErbB-4, and Nardilysin receptors. HB-EGF significantly enhanced the engraftment of transplanted NSC into the ENS during NEC. NSC transplantation significantly reduced NEC incidence and improved gut barrier function and intestinal motility, and these effects were augmented by simultaneous administration of HB-EGF or by transplantation of HB-EGF overexpressing NSC.

CONCLUSIONS

HB-EGF promotes NSC proliferation and migration. HB-EGF and NSC reduce intestinal injury and improve gut barrier function and intestinal motility in experimental NEC. Combined HB-EGF and NSC transplantation may represent a potential future therapy to prevent NEC.

The epidermal growth factor (EGF) receptor (EGFR) has a key role in normal embryonic development, adult tissue homeostasis and many pathological processes, in particular tumour formation. Aberrant EGFR activation occurs in many cancer types, and inhibition of this receptor is a promising anti-tumour strategy. Besides overexpression of the wild-type receptor, mutated oncogenic EGFR variants are often associated with malignant transformation. In human non-small-cell lung cancers, kinase mutants of the EGFR are rather common. Human glioblastoma often express the truncated EGFRvIII version as well as other dimerized and permanently activated mutants of the receptor, which are considered as tumour drivers. Similarly, the mutated and dimerized EGFR variant Xiphophorus melanoma receptor kinase (Xmrk) is causative for the development of malignant pigment cell tumours in medaka and Xiphophorus melanoma models. It is generally believed that oncogenic receptors that are active due to dimerizing mutations are ligand independent. Here, we show that different EGFR variants from fish and human efficiently induce autocrine loops by inducing EGFR ligands such as amphiregulin and HB-EGF. Importantly, the pre-dimerized oncogenic EGFR versions Xmrk from Xiphophorus and human EGFR(C600F), though already active in absence of ligands, respond to ligand stimulation with enhanced oncogenic signalling. In summary, our data show that autocrine or paracrine loops are still acting on pre-dimerized oncogenic EGFRs and contribute to their pro-tumorigenic signalling.

The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion.

ADAM19, an enzyme from the ADAM (a disintegrin and metalloproteinase) family, is involved in various cell-cell and cell-matrix interactions. It can cleave epidermal growth factor (EGF)-like growth factors, such as heparin-binding (HB)-EGF and neuregulin (NRG), from the cell membrane. ADAM-mediated EGF receptor activation is crucial in the development of renal pathology. Based on these data, we studied ADAM19 in human nephrogenesis and renal disease. We collected 20 fetal kidneys and 56 biopsies from patients with various renal diseases. The unaffected part of kidneys from eight patients with renal cell carcinoma served as control. RNA in situ hybridization revealed widespread ADAM19 mRNA expression in the nephrogenic zone of human fetal kidneys. Normal human kidneys showed constitutive ADAM19 expression in distal tubules and endothelial cells, whereas proximal tubules were negative. In renal disease, ADAM19 was de novo expressed in proximal tubules and glomerular mesangium and upregulated in distal tubules and endothelial cells. ADAM19 colocalized with tubular and interstitial NRG, however, not with HB-EGF. Independent of renal disorder, mesangial ADAM19 expression was associated with glomerular damage as assessed by mesangial matrix expansion, focal glomerulosclerosis, and glomerular macrophage influx (all P<0.001). ADAM19 in proximal tubules and in peritubular capillaries was associated with interstitial fibrosis (P<0.05). Finally, increasing tubular ADAM19 was associated with declining renal function (P<0.05). The abundant ADAM19 expression during nephrogenesis points to a role in growth promotion and regulation. The high ADAM19 expression in renal disease suggests involvement in profibrotic and proinflammatory processes leading to renal deterioration.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family of growth factors, is synthesized as a membrane-an-chored precursor (proHB-EGF) that is capable of stimulating adjacent cells in a juxtacrine manner. ProHB-EGF is cleaved in a protein kinase C-dependent process, to yield the soluble form. It was observed that HB-EGF acts as a morphogen for the collecting duct system in developing kidneys. HB-EGF protein was expressed in the ureteric bud of embryonic kidneys. Cultured mouse ureteric bud cells (UBC) produced HB-EGF via protein kinase C activation. After stimulation with phorbol ester (12-O-tetradecanoylphorbol-13-acetate) or recombinant soluble HB-EGF, UBC cultured in three-dimensional collagen gels formed short tubules with varied abundant branches. When proHB-EGF-transfected UBC were stimulated with 12-O-tetradecanoylphorbol-13-acetate and cultured in collagen gels, they exhibited linear growth, forming long tubular structures with few branches at the time of appearance of proHB-EGF on the cell surface. The structures exhibited a strong resemblance to the early branching ureteric bud of embryonic kidneys. When UBC were cultured in the presence of transforming growth factor-beta and soluble HB-EGF, they formed long tubules and few branches, similar to the structures observed in proHB-EGF-transfected UBC. These cells exhibited apical-basolateral polarization and expression of the water channel aquaporin-2. These findings indicate that soluble HB-EGF and proHB-EGF induce branching tubulogenesis in UBC in different ways. Juxtacrine activation by proHB-EGF or the synergic action of soluble HB-EGF with transforming growth factor-beta is important for well balanced morphogenesis of the collecting duct system.

BACKGROUND

The factors driving the onset and progression of ovarian cancer are not well understood. Recent reports have identified cell lines that are representative of the genomic pattern of high-grade serous ovarian cancer (HGSOC), in which greater than 90 % of tumors have a mutation in TP53. However, many of these representative cell lines have not been widely used so it is unclear if these cell lines capture the variability that is characteristic of the disease.

METHODS

We investigated six TP53-mutant HGSOC cell lines (Caov3, Caov4, OV90, OVCA432, OVCAR3, and OVCAR4) for migration, MMP2 expression, proliferation, and VEGF secretion, behaviors that play critical roles in tumor progression. In addition to comparing baseline variation between the cell lines, we determined how these behaviors changed in response to four growth factors implicated in ovarian cancer progression: HB-EGF, NRG1β, IGF1, and HGF.

RESULTS

Baseline levels of each behavior varied across the cell lines and this variation was comparable to that seen in tumors. All four growth factors impacted cell proliferation or VEGF secretion, and HB-EGF, NRG1β, and HGF impacted wound closure or MMP2 expression in at least two cell lines. Growth factor-induced responses demonstrated substantial heterogeneity, with cell lines sensitive to all four growth factors, a subset of the growth factors, or none of the growth factors, depending on the response of interest. Principal component analysis demonstrated that the data clustered together based on cell line rather than growth factor identity, suggesting that response is dependent on intrinsic qualities of the tumor cell rather than the growth factor.

CONCLUSIONS

Significant variation was seen among the cell lines, consistent with the heterogeneity of HGSOC.