OBJECTIVE

Current treatment target toward advanced colorectal cancers is mainly focused on the epidermal growth factor receptor (EGFR) signaling, but its additive effects with chemotherapy are still limited. A disintegrin and metalloproteinase (ADAM) cleaves the proheparin-binding epidermal growth factor like growth factor (proHB-EGF). And soluble HB-EGF activates EGFR. In parallel, the carboxy-terminal fragment of proHB-EGF (HB-EGF-CTF) translocates into the inner nuclear membrane, and subsequently exerts on the regulation of cell proliferation by binding nuclear promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor, thereby causing its nuclear export. We hypothesized that the inhibition of HB-EGF-CTF nuclear translocation may be a new strategy in preventing cell proliferation.

METHODS

12-O-tetradecanoylphorbor-13-acetate (TPA) was treated to activate ADAM. Nine-thousand chemical compounds were screened for their efficacies in blocking the binding of HB-EGF-CTF to promyelocytic leukemia zinc finger (PLZF) with Alphascreen system. The obtained candidates were then used to block the binding of HB-EGF-CTF to PLZF in colon cancer cells, HT29 and HCT116. Cell proliferation was investigated with a growth curve assay. The intracellular localization, and association between HB-EGF-CTF and PLZF, was assessed with immunofluorescent staining, and immunoprecipitation and Western blotting, respectively. The effects of obtained candidates on EGFR phosphorylation and on nuclear translocation of HB-EGF-CTF and export of PLZF during the angiotensin II type1 receptor (AT1R) knockdown were also investigated.

RESULTS

Telmisartan and candesartan were found to be potential candidates. Telmisartan inhibited TPA-induced cell proliferation stronger than candesartan. Telmisartan, but not candesartan blocked the nuclear translocation of HB-EGF-CTF, and binding of HB-EGF-CTF to PLZF, during TPA stimulation. Both telmisartan and candesartan did not inhibit TPA-induced EGFR phosphorylation, and telmisartan, but not candesartan, inhibited TPA-induced nuclear translocation of HB-EGF-CTF after knockdown of AT1R.

CONCLUSIONS

The inhibition of HB-EGF-CTF nuclear translocation with telmisartan may be a novel strategy in preventing cell proliferation.

Molecular mechanisms other than activating KRAS mutations should underlie the occurrence of weaker versus stronger responses to cetuximab (CTX) in EGFR-dependent carcinomas with either an intact KRAS signaling or in which KRAS mutations do not predict CTX efficacy. We hypothesized that KRAS wild-type (WT) tumor cell-line models chronically adapted to grow in the presence of CTX could be interrogated to establish if the positive predictive value of the mRNAs coding for the EGFR ligands amphiregulin (AR) and epiregulin (EPI) could be significantly altered during and/or after treatment with CTX. Gene expression analyses using real-time (kinetic) RT-PCR were performed to monitor the transcriptional evolution of EGFR ligands EGF, TGFα, AR, BTC, EPI, NRG and HB-EGF in experimental modes induced to exhibit acquired resistance to the mono-HER1 inhibitor CTX, the mono-HER2 inhibitor trastuzumab (Tzb) or the dual HER1/HER2 inhibitor lapatinib (LPT). Gene expression signatures for EGFR ligands distinctively related to the occurrence of unresponsiveness to CTX, Tzb or LPT, with minimal overlap between them. CTX's molecular functioning largely depended on the overproduction of the mRNAs coding for the EGFR ligands AR and EPI. Thus, a dramatic down-regulation of AR/EPI mRNA expression occurred upon loss of CTX efficacy in EGFR-positive tumor cells with an intact regulation of RAS signaling. Unlike KRAS mutations, which are informative of unresponsiveness to CTX solely in mCRC, our hypothesis-generating data suggest that expression status of AR and EPI mRNAs might be evaluated as dynamic predictors of response in KRAS WT patients receiving any CTX-based therapy.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interacts with the EGF receptor to exert mitogenic activity for various types of cells. Through its interactions with various molecules, it is involved in diverse biological processes, including wound healing, blast implantation, and tumor formation. At the same time, HB-EGF is widely expressed in the central nervous system, including the hippocampus and cerebral cortex, and is considered to play pivotal roles in the developing and adult nervous system. Because HB-EGF protein levels in the brain are much higher than those of TGF-α and EGF, it is possible that HB-EGF serves as a major physiologic ligand for the EGF receptor (ErbB1) within the central nervous system. Recent studies indicate that HB-EGF contributes to the neuronal survival and proliferation of glial/stem cells. HB-EGF also promotes the survival of dopaminergic neurons, an action mediated by mitogen-activated protein kinase (MAPK) as well as by the Akt signaling pathway. In this review, we discuss recent findings on the implications of HB-EGF in higher brain functions of the central nervous system.

Elastase degradation of elastin within alveolar walls is an important event in the development of pulmonary emphysema. In addition to elastolytic activities, elastases release growth factors from extracellular matrices and interstitial cell surfaces that can regulate elastogenesis and other cellular responses. In the present study, we demonstrate that brief treatment of matrix-laden rat pulmonary fibroblast cultures with pancreatic elastase results in the release of soluble heparin-binding epidermal growth factor-like growth factor (HB-EGF) concomitant with a decrease in HB-EGF binding to both heparan sulfate proteoglycan and receptor sites on the cells. In undigested, matrix-laden fibroblasts, HB-EGF significantly downregulates elastin mRNA via activation of epidermal growth factor receptor. Results from nuclear run-on analyses show that HB-EGF downregulates elastin mRNA via transcriptional suppression. HBEGF treatment stimulates MAP or ERK kinase (MEK)-dependent ERK1/2 phosphorylation and leads to nuclear accumulation of Fra-1. Blocking ERK1/2 activation by MEK1/2 inhibitors (PD-98059 or U-0126) diminishes HB-EGF-induced Fra-1 accumulation and subsequent downregulation of elastin mRNA. Coaddition of two elastase-released growth factors, HB-EGF and FGF-2, results in an additive inhibitory effect on elastin mRNA levels. Furthermore, HB-EGF addition to pulmonary fibroblasts increases FGF-2 mRNA and protein levels. These data suggest that HB-EGF and FGF-2 act in concert to regulate the synthesis of elastin in injury/repair situations.

Human heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) expressed on Chinese hamster ovary (CHO) cells is synthesized as a 19-kDa major, and 22- and 27-kDa minor, membrane-anchored precursors (proHB-EGF). In contrast, the 27-kDa species is major and the 19- and 22-kDa ones are minor in mouse proHB-EGF. The juxtacrine growth factor activities of human and mouse proHB-EGFs on CHO cells toward EP170.7 cells in co-culture are significantly different. To investigate the relationship between the juxtacrine growth factor activities and the molecular species, we prepared human-mouse chimeras. Chimeras that have the human amino-terminal sequence with a mouse EGF-like domain showed approximately 8-fold up-regulation of the juxtacrine growth factor activity and the predominance of a 19-22-kDa major species. In contrast, chimeras that have the mouse amino-terminal sequence with a human EGF-like domain showed approximately 5-fold down-regulation of the juxtacrine activity and the predominance of the 27-kDa major species. A Gly32.HB-EGF (117-amino acid form), which is amino-terminally extended, induced the same mitogenic activity as that of Arg73.HB-EGF (75-amino acid form), which is amino-terminally truncated. These results strongly suggested that amino-terminal processing of human proHB-EGF would be required for up-regulation of its juxtacrine growth factor activity, but not for its paracrine activity.

Gastrin, PGs, and growth factors have important roles in maintaining gastrointestinal mucosal integrity. Cyclooxygenases (COX-1 and COX-2) are the key enzymes involved in PG synthesis. This study aimed to clarify the mechanisms of gastric mucosal protection by gastrin. Fasted rats were administered subcutaneous gastrin 17 with or without gastrin receptor antagonist YM022 pretreatment. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and COX-2 expression were examined using Western blot analysis. Another series of experiments investigated 1) PGE(2) levels in gastric mucosa, 2) the protective action of gastrin against gastric damage by acidified ethanol, 3) the effects of a specific HB-EGF-neutralizing antibody on gastrin-induced COX-2 expression, and 4) the effects of a specific COX-2 inhibitor NS-398 on PGE(2) synthesis and the mucosal protection afforded by gastrin. Gastrin dose-dependently increased HB-EGF, COX-2 expression, and PGE(2) levels and reduced gastric damage. However, pretreatment with YM022 dose-dependently abolished such effects of gastrin. A specific HB-EGF- neutralizing antibody and an EGF receptor inhibitor decreased gastrin-induced COX-2 expression. NS-398 blocked gastrin-induced PGE(2) synthesis and mucosal protection. In conclusion, this study demonstrates that gastrin enhances gastric mucosal integrity through COX-2, which is partially mediated by HB-EGF, and PGE(2) upregulation in rats.

BACKGROUND

Beta-cell neogenesis from pancreatic duct cells has been reported to occur in duct-ligated rat. Nevertheless, detailed process of this phenomenon has not been clarified.

OBJECTIVE

To clarify the mechanism of beta-cell neogenesis, a partial pancreatic duct ligation mouse model was created. Proliferation of duct cells, beta-cell neogenesis, and expression of transcription factors and differentiation/growth factors were studied by immunohistochemistry, cDNA array, and RT-PCR methods.

RESULTS

In the duct-ligated portion of the pancreas, newly formed islet-like cell clusters (ICCs) were observed arising from the ducts on day 7 and afterward. Transcription factors, such as pancreatic and duodenal homeobox gene-1 (PDX-1), paired box factor 6 (Pax6), islet1 and Nkx2.2-positive cells, and protein gene product 9.5 (PGP9.5) were also induced in duct lining cells. By cDNA microarray analysis, expression of insulin-like growth factor-1 (IGF-1) and transforming growth factor beta1 (TGF-beta1) were above control levels on day 5, and RT-PCR showed an increase from day 5 to day 28. IGF-1 and activin A-positive cells were detected in ducts. In addition, expression of betacellulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and TGF-alpha were also increased from day 3 or 5.

CONCLUSIONS

These findings suggest that beta-cell or endocrine precursors are localized among duct lining cells. Induction of several islet cell-associated transcription factors and differentiation and/or growth factors may play important roles during beta-cell neogenesis in this model.

The high-molecular-mass polysaccharide hyaluronan is abundant in the extracellular space between adjacent keratinocytes throughout the vital part of epidermis. It has a rapid turnover, and its content is subject to large fluctuations due to physiological and environmental conditions, with the strongest effects mediated by EGFR signaling. Using an elegant organotypic culture system, Monslow et al. (2009, this issue) demonstrate that heparin-binding (HB)-EGF released from its membrane anchor is the major ligand of EGFR in injured epidermis, accounting for the autocrine and paracrine activation of hyaluronan synthesis by the keratinocytes in the neighborhood, thus facilitating the epidermal wound-healing response.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cardiogenic and cardiohypertrophic growth factor. ProHB-EGF, a product of the Hb-egf gene and the precursor of HB-EGF, is anchored to the plasma membrane. Its ectodomain region is shed by a disintegrin and metalloproteases (ADAMs) when activated by various stimulations. It has been reported that an uncleavable mutant of Hb-egf, uc-Hb-egf, produces uc-proHB-EGF, which is not cleaved by ADAMs and causes dilation of the heart in knock-in mice. This suggests that the shedding of proHB-EGF is essential for the development and survival of cardiomyocytes: however, the molecular mechanism involved has remained unclear. In this study, we investigated the relationship between uc-proHB-EGF expression and cardiomyocyte survival. Human uc-proHB-EGF was adenovirally introduced into the rat cardiomyoblast cell line H9c2, and the cells were cultured under normoxic and hypoxic conditions. Uc-proHB-EGF-expressing H9c2 cells underwent apoptosis under normoxic conditions, which distinctly increased under hypoxic conditions. Furthermore, we observed an increased Caspase-3 activity, reactive oxygen species accumulation, and an increased c-Jun N-terminal kinase (JNK) activity in the uc-proHB-EGF-expressing H9c2 cells. Treatment of the uc-proHB-EGF transfectants with inhibitors of Caspase-3, reactive oxygen species, and JNK, namely, Z-VAD-fmk, N-acetylcysteine, and SP600125, respectively, significantly reduced hypoxic cell death. These data indicate that insufficiency of proHB-EGF shedding under hypoxic stress leads to cardiomyocyte apoptosis via Caspase-3- and JNK-dependent pathways.

A series of phosphonamide-based hydroxamate derivatives were synthesized, and the inhibitory activities were evaluated against various metalloproteinases in order to clarify its selectivity profile. Among the four diastereomeric isomers resulting from the chirality at the C-3 and P atoms, the compound with a (R,R)-configuration both at the C-3 position and the phosphorus atom was found to be potently active, while the other diastereomeric isomers were almost inactive. A number of (R,R)-compounds synthesized here exhibited broad spectrum activities with nanomolar K(i) values against MMP-1, -3, -9, and TACE and also showed nanomolar IC(50) values against HB-EGF shedding in a cell-based inhibition assay. The modeling study using X-ray structure of MMP-3 suggested the possible binding mode of the phosphonamide-based inhibitors.

Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met(flx/flx) and Met(-/-) oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met(flx/flx) cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met(flx/flx) and Met(-/-) oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in normal, untransformed oval cells, c-Met and EGFR represent critical molecular players to control proliferation and survival that function independent of one another.

BACKGROUND

Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC.

METHODS

47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha).

RESULTS

EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01).

CONCLUSIONS

EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.

Peptide growth factors have been proposed as mediators of smooth muscle-epithelial cell interactions in the human prostate; however, the identity of these molecules has not been established. In this study, we compared expression levels of messenger RNAs (mRNAs) encoding the epidermal growth factor (EGF) receptor-related receptor tyrosine kinases (ErbB1 through 4), the six EGF receptor ligands, EGF, transforming growth factor (TGF)-alpha, amphiregulin (ARG), HB-EGF, betacellulin, and epiregulin, and the related molecule heregulin-alpha, in a series of 10 prostate tissue specimens. Only EGF showed a disease-specific association, with increased mRNA levels in four of five PCa specimens in comparison to matched normal tissue from the same subject. In contrast, ARG and HB-EGF mRNAs showed a coordinate pattern of expression in 7/10 specimens that was distinct from all other growth factor or receptor genes examined and from mRNAs for prostate specific antigen, the androgen receptor and GAPDH, a house-keeping enzyme. Analysis of an additional series of benign prostatic hyperplasia and prostate cancer specimens from 60 individuals confirmed that ARG and HB-EGF mRNA levels varied in a highly coordinate manner (r = 0.93; P < 0.0001) but showed no association with disease. ARG was immunolocalized largely to interstitial smooth muscle cells (SMC), previously identified as the site of synthesis of HB-EGF in the prostate, while the cognate ARG and HB-EGF receptor, ErbB1, was localized exclusively to ductal epithelial cells and carcinoma cells. Although ARG was a relatively poor mitogen for Balb/c3T3 cells in comparison to HB-EGF, it was similar in potency to HB-EGF in stimulating human prostate epithelial cell growth, suggesting that prostate epithelia may be a physiologic target for ARG in vivo. Expression of both ARG and HB-EGF mRNAs was induced in cultured prostate SMC by fibroblast growth factor-2, a human prostate SMC mitogen linked to prostate disease. These findings indicate that ARG and HB-EGF are likely to be key mediators of directional signaling between SMC and epithelial cells in the human prostate and appear to be coordinately regulated.

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.

In response to cutaneous injury, expression of collagenase-1 is induced in keratinocytes via alpha2beta1 contact with native type I collagen, and enzyme activity is essential for cell migration over this substratum. However, the cellular mechanism(s) mediating integrin signaling remain poorly understood. We demonstrate here that treatment of keratinocytes cultured on type I collagen with epidermal growth factor receptor (EGFR) blocking antibodies or a specific receptor antagonist inhibited cell migration across type I collagen and the matrix-directed stimulation of collagenase-1 production. Additionally, stimulation of collagenase-1 expression by hepatocyte growth factor, transforming growth factor-beta1, and interferon-gamma was blocked by EGFR inhibitors, suggesting a required EGFR autocrine signaling step for enzyme expression. Collagenase-1 mRNA was not detectable in keratinocytes isolated immediately from normal skin, but increased progressively following 2 h of contact with collagen. In contrast, EGFR mRNA was expressed at high steady-state levels in keratinocytes isolated immediately from intact skin but was absent following 2 h cell contact with collagen, suggesting down-regulation following receptor activation. Indeed, tyrosine phosphorylation of the EGFR was evident as early as 10 min following cell contact with collagen. Treatment of keratinocytes cultured on collagen with EGFR antagonist or heparin-binding (HB)-EGF neutralizing antibodies dramatically inhibited the sustained expression (6-24 h) of collagenase-1 mRNA, whereas initial induction by collagen alone (2 h) was unaffected. Finally, expression of collagenase-1 in ex vivo wounded skin and re-epithelialization of partial thickness porcine burn wounds was blocked following treatment with EGFR inhibitors. These results demonstrate that keratinocyte contact with type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires autocrine EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the re-epithelialization of epidermal wounds.

Genistein, a phytoestrogen present in soybeans, has well established vasodilator properties. The present study examined the mechanisms involved in the rapid vascular effects of genistein. Endothelium-dependent relaxations and contractions, induced by acetylcholine and the calcium ionophore A23187, were obtained in isolated aortic rings from male spontaneously hypertensive rats (SHR). Acute exposure to genistein potentiated relaxations and reduced contractions induced by the two agonists. Both effects of genistein were not affected by transcription- and translation-inhibitors or by tyrosine kinase inhibition. The potentiation of acetylcholine and A23187-induced relaxation by genistein was inhibited by NF023 and GP antagonist-2A, selective G(i) and G(q) α-subunit antagonists, respectively, but not by NF449, a selective G(s) α-subunit antagonist. These G protein antagonists did not alter the inhibitory effect of genistein on acetylcholine and A23187-induced contractions. The potentiation of A23187-induced relaxations by genistein was not inhibited by the conventional estrogen receptor (ER) antagonist, ICI 182,780, but inhibited by the specific ER-α antagonist, MPP, and by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. It was mimicked by heparin-binding epidermal growth factor (HB-EGF). Activation of EGFR and endothelial nitric oxide synthase (eNOS) was detected in genistein-treated rings using Western blotting. These data suggest that the rapid vascular actions of genistein are mediated by non-genomic pathways and are unrelated to its tyrosine kinase inhibitory properties. Furthermore, genistein transactivates EGFR through membrane ERα via G protein-coupled pathways. This in turn enhances eNOS phosphorylation and hence endothelial function in the aorta of the SHR.

OBJECTIVE

We studied the impact of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on inflammation-driven hepatocarcinogenesis.

METHODS

HB-EGF expression was determined by qRT-PCR and immunodetection in hepatocellular adenoma and carcinoma and in mesenchymal (MC) and parenchymal liver cells obtained from different models of inflammation. The functions of HB-EGF in early hepatocarcinogenesis were assessed in co-cultures of unaltered and initiated/premalignant hepatocytes.

RESULTS

In human and rat (pre)malignant liver lesions, HB-EGF levels were comparable to that of the surrounding tissue. In inflamed livers HB-EGF was expressed predominantly in MC and was further increased by pro-inflammatory lipopolysaccharide (LPS) or linoleic acid hydroperoxide (LOOH). In culture, DNA-replication occurred rather in initiated/premalignant than unaltered hepatocytes and was further elevated by LOOH- or LPS-stimulated MC-supernatants. The supernatant effects were abrogated by pre-incubation with HB-EGF-neutralizing antisera. HB-EGF itself induced DNA-replication and mitosis preferentially in the initiated/premalignant cells. When transducing hepatocytes with a dominant-negative ErbB1-construct, HB-EGF-induced DNA-replications were blocked completely in unaltered hepatocytes but incompletely in initiated/premalignant cells, which suggests elevated ErbB-mediated signal transduction in first stages of hepatocarcinogenesis.

CONCLUSIONS

Pro-inflammatory stimuli induce the release of HB-EGF from MC, which stimulates DNA-replication in initiated/premalignant hepatocytes. Similar mechanisms may contribute to carcinogenesis in human inflammatory liver diseases.

To develop a feasible perfusion-type bioartificial liver device, perfusion of hepatocyte-nonparenchymal cell (NPC) cocultures with medium supplemented with hepatocyte growth factor (HGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) was carried out. On day 1 of culture, perfusion at a constant shear stress of 1.3 dyn/cm2 enhanced ammonia metabolic and urea synthetic activities of hepatocytes. These enhanced activities were sustained up to day 7 only when growth factors were present. In contrast, no beneficial effects of growth factors on these activities were observed in static cultures. In perfusion cultures, three-dimensional cell aggregates were formed. On the surface of these aggregates, flattened cell layers composed mainly of NPCs were found, and the central cluster of cell aggregates was composed of round-shaped hepatocytes and reticulin fibrils. These observations strongly suggested that the reconstruction of different types of liver cells and connective tissues formed tissue-mimicking cell aggregates in the perfusion culture that was able to modulate the liver-specific functions of hepatocytes. Thus, perfusion culture conditions of the hepatocyte--NPC coculture system should be appropriately designed to induce suitable reconstruction of the cultured cells for use as a bioartificial liver device.

There have been several reports implying a benefit for heparin therapy in patients with refractory ulcerative colitis. Although this effect has been attributed to the anti-inflammatory properties of heparin, other mechanisms have not been excluded. Heparin is a potent modulator of receptor binding of growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin-binding epidermal growth factor (HB-EGF), that play a role in wound repair. We examined the effect of heparin on the functional levels of FGF and HB-EGF in a model of experimental colitis. Fifty-six Wistar rats were divided into four groups: group 1 was the control group, group 2 received s.c. heparin 50 units/kg/d, group 3 underwent induction of 3% iodoacetamide colitis, and group 4 underwent induction of colitis and heparin treatment. Rats were killed and evaluated for severity of colitis by macroscopic and microscopic colitis scores, area of inflammation, and myeloperoxidase levels. FGF and HB-EGF levels were functionally assessed in colonic tissue in each group. Heparin therapy resulted in significant improvement in macroscopic and microscopic features of colitis (p < 0.05), accompanied by a partial reduction in myeloperoxidase levels. FGF receptor binding activity was identical in groups 1 and 2 but increased more than 3-fold after colitis induction in group 3 (p < 0.05). Treatment with heparin caused a significant decrease in FGF concentration. Levels of HB-EGF binding activity were similar in groups 1 and 2 and decreased in group 3 (p < 0.01). Heparin caused a significant increase in HB-EGF content in group 4 (p < 0.05). Levels of growth factors are altered differently in experimental colitis. Colonic FGF binding activity increases with colitis, whereas HB-EGF binding decreases with colitis. These trends were reversed by heparin, concomitant with a clinical and pathologic improvement in colitis. We suggest that one mechanism of heparin-mediated improvement in colitis may involve tissue healing associated with changes in functional levels of colonic growth factors.

BACKGROUND

Acute respiratory distress syndrome continues to be a major source of morbidity and mortality in critically-ill patients. Heparin binding EGF-like growth factor (HB-EGF) is a biologically active protein that acts as an intestinal cytoprotective agent. We have previously demonstrated that HB-EGF protects the intestines from injury in several different animal models of intestinal injury. In the current study, we investigated the ability of HB-EGF to protect the lungs from remote organ injury after intestinal ischemia/reperfusion (I/R).

METHODS

Mice were randomly assigned to one of the following groups: (1) sham-operated; (2) sham+HB-EGF (1200 microg/kg in 0.6 mL administered by intra-luminal injection at the jejuno-ileal junction immediately after identification of the superior mesenteric artery); (3) superior mesenteric artery occlusion for 45 min followed by reperfusion for 6 h (I/R); or (4) I/R+HB-EGF (1200 microg/kg in 0.6 mL) administered 15 min after vascular occlusion. The severity of acute lung injury was determined by histology, morphometric analysis and invasive pulmonary function testing. Animal survival was evaluated using Kaplan-Meier analysis.

RESULTS

Mice subjected to intestinal I/R injury showed histologic and functional evidence of acute lung injury and decreased survival compared with sham-operated animals. Compared with mice treated with HB-EGF (I/R+HB-EGF), the I/R group had more severe acute lung injury, and decreased survival.

CONCLUSIONS

Our results demonstrate that HB-EGF reduces the severity of acute lung injury after intestinal I/R in mice. These data demonstrate that HB-EGF may be a potential novel systemic anti-inflammatory agent for the prevention of the systemic inflammatory response syndrome (SIRS) after intestinal injury.

BACKGROUND

We have shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) protects the intestines from injury in several different animal models, including hemorrhagic shock and resuscitation (HS/R). The current study was designed to explore the mechanisms underlying the anti-inflammatory role of HB-EGF in preservation of gut barrier function after injury.

METHODS

In vivo, HS/R was induced in wild-type and neutropenic mice, with or without administration of HB-EGF, and intestinal permeability determined by use of the everted gut sac method. In vitro, cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated human peripheral blood mononuclear cells (PMNs) were used to determine the effects of HB-EGF on HUVEC-PMN adhesion, reactive oxygen species production in PMN, adhesion molecule expression in HUVEC and PMN, and the signaling pathways involved.

RESULTS

We found that administration of HB-EGF to healthy mice led to preservation of gut barrier function after HS/R. Likewise, induction of neutropenia in mice also led to preservation of gut barrier function after HS/R. Administration of HB-EGF to neutropenic mice did not lead to further improvement in gut barrier function. In vitro studies showed that HB-EGF decreased neutrophil-endothelial cell (PMN-EC) adherence by down-regulating adhesion molecule expression in EC via the phosphoinositide 3-kinase-Akt pathway, and by inhibiting adhesion molecule surface mobilization and reactive oxygen species production in PMN.

CONCLUSIONS

These results indicate that HB-EGF preserves gut barrier function by inhibiting PMN and EC activation, thereby blocking PMN-EC adherence after HS/R in mice, and support the future use of HB-EGF in disease states manifested by hypoperfusion injury.

OBJECTIVE

That heparin binding epidermal growth factor-like growth factor (HB-EGF) heals chronic tympanic membrane (TM) perforations at higher rates than fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) in an animal model.

BACKGROUND

A nonsurgical treatment for chronic TM perforation would benefit those unable to access surgery or those unable to have surgery, as well as reducing the cost of tympanoplasty. Growth factor (GF) treatments have been reported in the literature with variable success with the lack of a suitable animal providing a major obstacle.

METHODS

The GFs were tested in a validated mouse model of chronic TM perforation. A bioabsorbable hydrogel polymer was used to deliver the GF at a steady concentration as it dissolved over 4 weeks. A control (polymer only, n = 18) was compared to polymer loaded with HB-EGF (5 μg/ml, n = 18), FGF2 (100 μg/ml, n = 19), and EGF (250 μg/ml, n = 19). Perforations were inspected at 4 weeks.

RESULTS

The healing rates, as defined as 100% perforation closure, were control (5/18, 27.8%), HB-EGF (15/18, 83.3%), FGF2 (6/19, 31.6%), and EGF (3/19, 15.8%). There were no differences between FGF2 (p = 0.80) and EGF (p = 0.31) with control healing rates. HB-EGF (p = 0.000001) showed a significant difference for healing. The HB-EGF healed TMs showed layers similar to a normal TM, whereas the other groups showed a lack of epithelial migration.

CONCLUSIONS

This study confirms the advantage of HB-EGF over two other commonly used growth factors and is a promising nonsurgical treatment of chronic TM perforations.

Cisplatin is a key agent in combination chemotherapy for various types of solid tumor. We now show that cisplatin activates signaling by the epidermal growth factor receptor (EGFR) by inducing cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF). Matuzumab, a monoclonal antibody to EGFR, inhibited cisplatin-induced EGFR signaling, likely through competition with the soluble form of HB-EGF for binding to EGFR. Matuzumab enhanced the antitumor effect of cisplatin in nude mice harboring human non-small cell lung cancer xenografts. Our findings shed light on the mechanism by which monoclonal antibodies to EGFR might augment the efficacy of cisplatin.

Growth factors and estrogen receptor (ER) signaling cooperate to play essential roles in cell proliferation, differentiation and tumor progression in mouse reproductive organs. Treatment of neonatal mice with diethylstilbestrol (DES) induces an estrogen-independent persistent proliferation and cornification of the vaginal epithelium, which results in cancerous lesions later in life. However, the mechanisms of the estrogen-dependent and -independent pathways essentially remain unknown. We characterized the expression of epidermal growth factor (EGF)-like growth factors (EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), amphiregulin (APR), epiregulin (EPR) and neuregulin (NRG) 1) and erbB receptors (EGF receptor (EGFR), erbB2/neu, erbB3 and erbB4) in the vaginae of mice treated either neonatally (0-4 day) or as adults (55-59 day) with estrogens. EGFR and erbB2 were activated in the vaginal epithelium of mice by estrogen treatment. This activation was also encountered in vaginae from neonatally DES-exposed mice, along with the expression of EGF, TGF-alpha, HB-EGF, BTC, APR, EPR and NRG1. Immunohistochemical analysis indicated that erbB2 was primarily expressed in vaginal epithelium. Finally, we found that serine 118 and 167 located in the AF-1 domain of ERalpha were phosphorylated in these vaginae. AG825, AG1478 or ICI 182,780 administration blocked proliferation of vaginal epithelium induced by neonatal DES exposure. Thus, signal transduction via EGFR and erbB2 could be related to the estrogen-induced vaginal changes and persistent erbBs phosphorylation and sustained expression of EGF-like growth factors, leading to ERalpha activation that may result in cancerous lesions in vaginae from neonatally DES-exposed mice later in life.