Animal models of acute renal injury suggest that the epidermal growth factor receptor (EGFR) axis may have a beneficial role in the recovery from acute renal injury, but recent reports describe detrimental effects of EGFR activation in chronic renal injury. Expression of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) increases following renal injury, but the effects of this sustained upregulation have not been well studied. Here, stable overexpression of soluble HB-EGF (sHB-EGF) in mouse inner medullary collecting duct (IMCD) cells led to marked phenotypic changes: sHB-EGF-expressing cells demonstrated a fibroblast-like morphology, did not form epithelial sheets, exhibited cytoplasmic projections, decreased expression of epithelial markers, and increased expression of fibroblast-specific protein-1. They also demonstrated anchorage-independent growth and formed tumors when injected subcutaneously into nude mice. Quantitative RT-PCR and a luciferase reporter assay suggested that sHB-EGF repressed transcription of E-cadherin, and a concomitant TGF-beta-independent upregulation of the E-cadherin repressor Snail-2 was observed. Stable downregulation of Snail-2 in sHB-EGF-overexpressing cells restored epithelial characteristics (E-cadherin and cytokeratin expression) but did not alter their anchorage-independent growth. In summary, sustained exposure to sHB-EGF induces epithelial-to-mesenchymal transition of IMCD cells, in part by upregulating the E-cadherin transcriptional repressor Snail-2.

A novel pyranoside mimetic compound, DMBO (2-(2,6-difluorophenyl)-5-(4-methoxyphenyl)-1-oxa-3-azaspiro[5.5]undecane), was designed and synthesized. The sugar mimicking behavior of DMBO was addressed by its ability to bind several growth factors/cytokines such as vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and tumor necrosis factor (TNF)-α as demonstrated by the recently developed surface plasmon resonance assay. DMBO exhibited strong anti-proliferation activity in vitro against tumor cells including a highly metastatic murine osteosarcoma cell line LM8G7 that secretes VEGF as well as two human ovarian cell lines, OVSAHO and SKOV-3, which secrete TNF-α and HB-EGF respectively. Furthermore, DMBO inhibited the metastatic activity to the mouse liver of LM8G7 cells injected from a lateral tail vein, and affected the heparan-degrading activity of LM8G7 cells. Here, we report that DMBO acts as a human heparanase inhibitor in vitro possibly as a substrate mimetic. DMBO also inhibited the migration and invasion of LM8G7 cells and angiogenic events such as endothelial cell proliferation, migration and capillary tube-like formation in vitro. More prominently, the administration of DMBO with heparin resulted in synergistic anti-tumor effects in mouse modelofosteosarcoma. These preclinical data shows the potential anti-cancer effects of DMBO.

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Expression of endogenous heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a proven intestinal cytoprotective molecule, was examined in intestinal epithelial cells (IEC) in vitro, and in intestine undergoing ischemia/reperfusion (I/R) injury in vivo. In vitro, cells were exposed to anoxia for 90 min followed by reoxygenation for 1-3 h (A/R). In vivo, total midgut I/R injury was produced in rats by occlusion of the superior mesenteric artery for 30 or 90 min followed by reperfusion for 4 h. In situ hybridization and immunohistochemistry were used to study HB-EGF mRNA expression and protein production. In vitro, normal IEC had no detectable HB-EGF mRNA or protein expression. After anoxia, cells expressed HB-EGF mRNA and protein, with expression reaching a peak 2-3 h after reoxygenation. In vivo, only very low levels of HB-EGF mRNA and no detectable protein were found in normal intestine. Four hours after I/R, HB-EGF protein was detected in villous epithelia subjected to 30 min but not 90 min of ischemia, whereas HB-EGF mRNA was highly expressed after both ischemic intervals. Endogenous HB-EGF is immediately upregulated in IEC after A/R injury and in intestine after I/R injury. Thus, HB-EGF acts as an immediate early gene under these conditions.

Underlying the dynamic regulation of tropoelastin expression and elastin formation in development and disease are transcriptional and post-transcriptional mechanisms that have been the focus of much research. Of particular importance is the cytokine-governed elastin regulatory axis in which the pro-elastogenic activities of transforming growth factor β-1 (TGFβ1) and insulin-like growth factor-I (IGF-I) are opposed by anti-elastogenic activities of basic fibroblast growth factor (bFGF/FGF-2), heparin-binding epidermal growth factor-like growth factor (HB-EGF), EGF, PDGF-BB, TGFα, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β and noncanonical TGFβ1 signaling. A key mechanistic feature of the regulatory axis is that cytokines influence elastin formation through effects on the cell cycle involving control of cyclin-cyclin dependent kinase complexes and activation of the Ras/MEK/ERK signaling pathway. In this article we provide an overview of the major cytokines/growth factors that modulate elastogenesis and describe the underlying molecular mechanisms for their action on elastin production.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the NOTCH3 receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3(R169C) mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3(R169C) mice. This effect was associated with an ∼ 60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3(R169C) mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3(R169C) mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.

OBJECTIVE

To investigate the effects of epiregulin, a newly identified member of the epidermal growth factor (EGF) family, on the proliferation of human corneal epithelial cells (HCECs).

METHODS

The proliferation of HCECs was determined by cell counting and BrdU incorporation assays at specific times after exposure to different concentrations of human recombinant epiregulin (0 to 20 ng/ml). Immunohistochemical staining was used to localize epiregulin in cadaveric corneas. RT-PCR and real-time PCR were used to determine the expression levels of epiregulin in cultured and cadaveric HCECs. To examine the interaction between epiregulin and epidermal growth factor receptors (EGFRs), the phosphorylation of ErbB1 and ERK1/ERK2 (ERK1/2) was estimated by western blot analysis in the presence or absence of AG1478, a specific inhibitor of EGFR kinase activity. To search for cross-induction of epiregulin by other EGF family members, the expressions of EGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR), and transforming growth factor-alpha (TGF-alpha) mRNA were determined by real-time PCR in the presence of 10 ng/ml of epiregulin. Conversely, the expression of epiregulin was also determined following the incubation of HCECs with 10 nM of either of EGF, HB-EGF, TGF-alpha, or AR.

RESULTS

The mRNA of epiregulin was expressed in cultured HCECs and HCECs obtained from cadaveric eyes. Epiregulin was strongly detected in the limbal epithelium and basal epithelium of the peripheral cornea, but it was weakly detected in the central corneal epithelium. HCECs proliferated in the presence of epiregulin in a dose-dependent manner as detected by an increase in cell numbers or in BrdU incorporation. When HCECs were incubated with exogenous epiregulin, the expression of the mRNA of epiregulin was upregulated as detected by real-time PCR, and the phosphorylation of ErbB1 and ERK1/2 was upregulated in a dose-dependent manner as shown by western blot analysis. These upregulations were inhibited by AG1478, a specific inhibitor of EGFR kinase activity. Epiregulin increased the expression of HB-EGF and AR, while TGF-alpha, HB-EGF, AR, and EGF increased the expression of epiregulin in HCECs.

CONCLUSIONS

These findings indicate that epiregulin played an autocrine role in the proliferation of HCECs presumably through cross-induction with other EGF family members.

TWEAK, a member of the TNF superfamily, binds to the Fn14 receptor, eliciting biological responses. EGFR signalling is involved in experimental renal injury. Our aim was to investigate the relationship between TWEAK and EGFR in the kidney. Systemic TWEAK administration into C57BL/6 mice increased renal EGFR phosphorylation, mainly in tubular epithelial cells. In vitro, in these cells TWEAK phosphorylated EGFR via Fn14 binding, ADAM17 activation and subsequent release of the EGFR ligands HB-EGF and TGFα. In vivo the EGFR kinase inhibitor Erlotinib inhibited TWEAK-induced renal EGFR activation and downstream signalling, including ERK activation, up-regulation of proinflammatory factors and inflammatory cell infiltration. Moreover, the ADAM17 inhibitor WTACE-2 also prevented those TWEAK-induced renal effects. In vitro TWEAK induction of proinflammatory factors was prevented by EGFR, ERK or ADAM17 inhibition. In contrast, EGFR transactivation did not modify TWEAK-mediated NF-κB activation. Our data suggest that TWEAK transactivates EGFR in the kidney, leading to modulation of downstream effects, including ERK activation and inflammation, and suggest that inhibition of EGFR signalling could be a novel therapeutic tool for renal inflammation.

OBJECTIVE

The ErbB driven growth pathway has been implicated in most human epithelial malignancies. Therefore, its blockade is a promising therapeutic strategy and several candidate drugs are currently undergoing clinical trials. Paradoxically little is known of the expression pattern or clinical significance of the 4 ErbB receptors and their 11 ligands in TCC of the bladder.

METHODS

To obtain further insight into the molecular pathogenesis of TCC we used quantitative real-time reverse transcriptase-polymerase chain reaction assay to quantify mRNA expression of the 4 ERBB and their 11 known ligand genes, including recently described EPGN/epigen, in 73 tumor samples.

RESULTS

The level of mRNA of 4 ligand genes (EGF, NRG1, NRG2 and NRG3) was extremely low, that is detectable but not quantifiable. Six genes were over expressed (ERBB2, TGFA, HB-EGF, AREG, EREG and EPGN), 3 were under expressed (ERBB1, ERBB4 and NRG4) and 2 were over or under expressed (ERBB3 and BTC). ERBB2 and AREG expression differed between early stage tumors (pTa grade 1) and normal samples. The most marked differences in expression were ERBB3, EREG and NRG4 between superficial and muscle invasive tumors (p = 0.0069, 0.00007 and 0.0000001, respectively), and TGFA and NRG4 between low and high grade superficial tumors, and between pT1 or greater and pTa tumors.

CONCLUSIONS

This study shows the involvement of the ERBB family and ligand genes in TCC. Most receptor and ligand genes are deregulated at different stages of carcinogenesis, implying that they should be studied simultaneously. Quantitative real-time reverse transcriptase-polymerase chain reaction could be used to determine ErbB signaling pathway status in individuals with a view to tailored therapy.

Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits the growth of several types of cancer cells. However, the mechanisms by which this occurs are poorly understood. The goal of the present study was to investigate the effects of PPARgamma on mutated ras-induced cell growth, activation of transcription factors and expression of genes associated with cellular transformation in rat intestinal epithelial cells. A human PPARgamma cDNA was introduced to the activated H-ras-transfected IEC-6 cells (IECras) and 1 clone (IECrasPR82) that stably expresses both activated ras and PPARgamma was obtained. Thiazolidinedione derivatives such as troglitazone and rosiglitazone, selective ligands for PPARgamma, inhibited the cellular growth of IECrasPR82 cells in a time-dependent manner and induced G1 cell cycle arrest. Treatment with troglitazone (20 microM) decreased the expression of cyclin D1, heparin-binding epidermal growth factor-like growth factor (HB-EGF) and amphiregulin and suppressed the promoter activities of cyclin D1 and HB-EGF. Furthermore, a luciferase assay and an electrophoretic mobility shift assay showed that thiazolidinedione derivatives suppressed the transcriptional activities of AP-1 and Ets, both of which play crucial roles in the expression of cyclin D1 and HB-EGF. These findings suggest that reduction of EGF-like growth factors and cyclin D1 through the suppression of AP-1 and Ets may be 1 mechanism whereby PPARgamma inhibits their growth.

Heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, especially antiphospholipid antibody syndrome. Recent studies have suggested that heparin may exert direct effects on placental trophoblast, independently of its anticoagulant activity. We now demonstrate that heparin abrogates apoptosis of primary first trimester villous trophoblast in response to treatment with the pro-inflammatory cytokines interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. This multifunctional glycosaminoglycan also inhibited apoptosis induced by other agents, including staurosporin, broad-spectrum kinase inhibitor and thrombin. Furthermore, heparin attenuated caspase-3 activity, a hallmark of apoptosis, in human first trimester villous and extravillous trophoblast cell lines treated with peptidoglycan, a Toll-like receptor-2 agonist isolated from Staphylococcus aureus. The ability of heparin to antagonize cell death induced by such diverse apoptotic signals suggested that it acts as a survival factor for human trophoblast. We demonstrate that heparin, like epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), elicits phosphorylation of the EGF receptor and activation of the phosphatidyl inositol 3-kinase (PI3K)-, the extracellular signal-related kinase 1/2 (ERK1/2)- and the c-Jun NH2 terminal kinase (JNK)-signal transduction pathways in primary villous trophoblast. In summary, we have demonstrated that heparin activates multiple anti-apoptotic pathways in human trophoblast. Our results suggest that heparin may be useful in the management of at-risk patients, even in the absence of an identifiable thrombophilic disorder.

Smoking is an established risk factor for pancreatic cancer (PC), but late diagnosis limits the evaluation of its mechanistic role in the progression of PC. We used a well-established genetically engineered mouse model (LSL-K-ras(G12D)) of PC to elucidate the role of smoking during initiation and development of pancreatic intraepithelial neoplasia (PanIN). The 10-week-old floxed mice (K-ras(G12D); Pdx-1cre) and their control unfloxed (LSL-K-ras(G12D)) littermates were exposed to cigarette smoke (total suspended particles: 150 mg/m(3)) for 20 weeks. Smoke exposure significantly accelerated the development of PanIN lesions in the floxed mice, which correlated with tenfold increase in the expression of cytokeratin19. The systemic accumulation of myeloid-derived suppressor cells (MDSCs) decreased significantly in floxed mice compared with unfloxed controls (P<0.01) after the smoke exposure with the concurrent increase in the macrophage (P<0.05) and dendritic cell (DCs) (P<0.01) population. Further, smoking-induced inflammation (IFN-γ, CXCL2; P<0.05) was accompanied by enhanced activation of pancreatic stellate cells and elevated levels of serum retinoic acid-binding protein 4, indicating increased bioavailability of retinoic acid which contributes to differentiation of MDSCs to tumor-associated macrophages (TAMs) and DCs. TAMs predominantly contribute to the increased expression of heparin-binding epidermal growth factor-like growth factor (EGFR ligand) in pre-neoplastic lesions in smoke-exposed floxed mice that facilitate acinar-to-ductal metaplasia (ADM). Further, smoke exposure also resulted in partial suppression of the immune system early during PC progression. Overall, the present study provides a novel mechanism of smoking-induced increase in ADM in the presence of constitutively active K-ras mutation.

M2-type tumor-associated macrophages (TAMs) infiltration contributes to cancer malignant progression. However, the mechanisms for controlling recruitment and M2 polarization of macrophages by cancer cells are largely unclear. NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and mediates cancer progression. NOXs are in close relation with cancer-related inflammation, nevertheless, whether tumoral NOXs influence microenvironmental macrophages remains undentified. This study found that there was a close association between NOX4 expression and macrophage chemotaxis in patients with NSCLC analyzed using TCGA RNA-sequencing data. NOX4 in NSCLC cells (A549 and Calu-1 cell lines) efficiently enhanced murine peritoneal macrophage migration and induces M2 polarization. Immunohistochemical analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206. The mechanical study revealed that tumoral NOX4-induced reactive oxygen species (ROS) stimulated various cytokine production, including CCL7, IL8, CSF-1 and VEGF-C, via PI3K/Akt signaling-dependent manner. Blockade of the function of these cytokines reversed NOX4 effect on macrophages. Specifically, the results showed that tumoral NOX4-educated M2 macrophages exhibited elevated JNK activity, expressed and released HB-EGF, thus facilitating NSCLC proliferation in vitro. Pretreatment of macrophages with JNK inhibitor blocked tumoral NOX4-induced HB-EGF production in M2 macrophages. Finally, in a xenograft mouse model, overexpression of NOX4 in A549 cells enhanced the tumor growth. Elimination of ROS by NAC or inhibition of NOX4 activity by GKT137831 suppressed tumor growth accompanied by reduction in macrophage infiltration and the percentage of M2 macrophages. In conclusion, our study indicates that tumoral NOX4 recruits M2 TAMs via ROS/PI3K signaling-dependent various cytokine production, thus contributing NSCLC cell growth.

BACKGROUND

Despite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer.

METHODS

PG545's anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models.

RESULTS

PG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response.

CONCLUSIONS

Our results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.

Neuronal differentiation of PC12 cells in response to NGF is a prototypical model in which signal duration determines a biological response. Sustained ERK activity induced by NGF, as compared with transient activity induced by EGF, is critical to the differentiation of these cells. To characterize the transcriptional program activated preferentially by NGF, we compared global gene expression profiles between cells treated with NGF and EGF for 2-4 h, when sustained ERK signaling in response to NGF is most distinct from the transient signal elicited by EGF. This analysis identified 69 genes that were preferentially up-regulated in response to NGF. As expected, up-regulation of these genes was mediated by sustained ERK signaling. In addition, they were up-regulated in response to other neuritogenic treatments (pituitary adenylate cyclase-activating polypeptide and 12-O-tetradecanoylphorbol-13-acetate plus dbcAMP) and were enriched for genes related to neuronal differentiation/function. Computational analysis and chromatin immunoprecipitation identified binding of CREB and AP-1 family members (Fos, FosB, Fra1, JunB, JunD) upstream of >30 and 50%, respectively, of the preferentially NGF-induced genes. Expression of several AP-1 family members was induced by both EGF and NGF, but their induction was more robust and sustained in response to NGF. The binding of Fos family members to their target genes was similarly sustained in response to NGF and was reduced upon MEK inhibition, suggesting that AP-1 contributes significantly to the NGF transcriptional program. Interestingly, Fra1 as well as two other NGF-induced AP-1 targets (HB-EGF and miR-21) function in positive feedback loops that may contribute to sustained AP-1 activity.

Stromal fibroblasts actively participate in normal mammary gland homeostasis and in breast carcinoma growth and progression by secreting paracrine factors; however, little is known about the identity of paracrine mediators in individual patients. The purpose of this study was to characterize paracrine signaling pathways between breast carcinoma cells and breast carcinoma-associated fibroblasts (CAF) or normal mammary fibroblasts (NF), respectively. CAF and NF were isolated from breast carcinoma tissue samples and adjacent normal mammary gland tissue of 28 patients. The fibroblasts were grown in 3D collagen gel co-culture with T47D human breast carcinoma cells and T47D cell growth was measured. CAF stimulated T47D cell growth to a significantly greater degree than NF. We detected a considerable inter-individual heterogeneity of paracrine interactions but identified FGF2, HB-EGF, heparanase-1 and SDF1 as factors that were consistently responsible for the activity of carcinoma-associated fibroblasts. CAF from low-grade but not high-grade carcinomas required insulin-like growth factor 1 and transforming growth factor beta 1 to stimulate carcinoma growth. Paradoxically, blocking of membrane-type 1 matrix metalloprotease stimulated T47D cell growth in co-culture with NF. The results were largely mirrored by treating the fibroblasts with siRNA oligonucleotides prior to co-culture, implicating the fibroblasts as principal production site for the secreted mediators. In summary, we identify a paracrine signaling network with inter-individual commonalities and differences. These findings have significant implications for the design of stroma-targeted therapies.

Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.

BCL6 is a transcriptional repressor that has important functions in lymphocyte differentiation and lymphomagenesis, but there have been no reports of BCL6 expression in gastric cancers. In the present study, we investigated the BCL6 function in gastric cancers. Treatment with TPA resulted in BCL6 degradation and cyclin D2 upregulation. This phenomenon was inhibited by the suppression of the nuclear translocation of HB-EGF-CTF (C-terminal fragment of pro-HB-EGF). The HB-EGF-CTF nuclear translocation leads to the interaction of BCL6 with HB-EGF-CTF and the nuclear export of BCL6, and after that BCL6 degradation was mediated by ubiquitin/proteasome pathway. Real-time RT-PCR and siRNA targeting BCL6 revealed that BCL6 suppresses cyclin D2 expression. Our data indicate that BCL6 interacts with nuclear-translocated HB-EGF-CTF and that the nuclear export and degradation of BCL6 induces cyclin D2 upregulation. We performed immunohistochemical analyses of BCL6, HB-EGF and cyclin D2 in human gastric cancers. The inverse correlation between BCL6 and cyclin D2 was also found in HB-EGF-positive human gastric cancers. BCL6 degradation caused by the HB-EGF-CTF also might induce cyclin D2 expression in human gastric cancers. Inhibition of HB-EGF-CTF nuclear translocation and maintenance of BCL6 function are important for the regulation of gastric cancer progression.

The role of retinoic acid receptors (RARs) in intercellular regulation of cell growth was assessed by targeting a dominant-negative RARalpha mutant (dnRARalpha) to differentiated suprabasal cells of mouse epidermis. dnRARalpha lacks transcriptional activation but not DNA-binding and receptor dimerization functions. Analysis of transgenic mice revealed that dnRARalpha dose-dependently impaired induction of basal cell proliferation and epidermal hyperplasia by all-trans RA (tRA). dnRARalpha formed heterodimers with endogenous retinoid X receptor-alpha (RXRalpha) over RA response elements in competition with remaining endogenous RARgamma-RXRalpha heterodimers, and dose-dependently impaired retinoid-dependent gene transcription. To identify genes regulated by retinoid receptors and involved in cell growth control, we analyzed the retinoid effects on expression of the epidermal growth factor (EGF) receptor, EGF, transforming growth factor-alpha, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin genes. In normal epidermis, tRA rapidly and selectively induced expression of HB-EGF but not the others. This induction occurred exclusively in suprabasal cells. In transgenic epidermis, dnRARalpha dose-dependently inhibited tRA induction of suprabasal HB-EGF and subsequent basal cell hyperproliferation. Together, our observations suggest that retinoid receptor heterodimers located in differentiated suprabasal cells mediate retinoid induction of HB-EGF, which in turn stimulates basal cell growth via intercellular signaling. These events may underlie retinoid action in epidermal regeneration during wound healing.

Epidermal growth factor receptor (EGFR) has been implicated in tumour growth and extension of ovarian cancer. Peritoneal fluid in ovarian cancer patients contains various growth factors that can promote tumour growth and extension. In order to investigate the clinical significance of EGFR ligands as activating factors of ovarian cancer, we examined the cell proliferation-promoting activity and the level of EGFR ligands in peritoneal fluid obtained from 99 patients. Proliferation-promoting activity in peritoneal fluid from 63 ovarian cancer patients (OVCA) was much higher than peritoneal fluid from 18 ovarian cyst patients (OVC) and 18 normal ovary patients (NO), and the activity was suppressed only by antibodies against EGFR or heparin-binding epidermal growth factor (HB-EGF). A large difference was observed in the level of EGFR ligands between HB-EGF and TGF-alpha or amphiregulin. The concentration of HB-EGF in OVCA significantly increased compared to that in OVC or NO (P<0.01). No significant difference in the concentration of TGF-alpha and amphiregulin was found between the OVCA and NO or OVC groups. In peritoneal fluid, HB-EGF is sufficiently elevated to activate cancer cells even at an early stage of OVCA. These results suggested that HB-EGF in peritoneal fluid might play a key role in cell survival and in the proliferation of OVCA.

BACKGROUND

We have shown previously that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is cytoprotective for intestinal epithelial cells exposed to hypoxia in vitro. We now examine the effects of HB-EGF on the recovery of small intestine from ischemic injury in vivo.

METHODS

Segmental intestinal ischemia of 60-min duration was produced in adult rats by occlusion of a first-order branch of the superior mesenteric artery. Recombinant HB-EGF (100 microg) was injected intraluminally into the proximal small bowel after 45 min of ischemia in experimental animals, and buffered saline was injected in control animals. Animals were sacrificed after 48 h, and the affected bowel was resected, processed, and examined microscopically, with histologic grading of the ischemic injury. Additional animals were allowed to recover for up to 1 month to evaluate mortality differences.

RESULTS

Intraluminal administration of HB-EGF resulted in significantly decreased extent and severity of ischemia/reperfusion injury, with significantly decreased grade of injury in the HB-EGF-treated compared with nontreated animals (average injury grade 0.66 compared with 2.44, respectively). Moreover, the mortality rate was significantly lower in the HB-EGF-treated animals compared with nontreated animals (0% vs 25%, respectively). HB-EGF-treated animals had increased weight gain in the postischemia recovery period.

CONCLUSIONS

We conclude that HB-EGF, given intraluminally, reduces both the amount and the severity of ischemia/reperfusion injury in the small bowel, reduces the mortality associated with intestinal ischemia, and may enhance intestinal recovery. The in vitro and in vivo cytoprotective effects of this growth factor suggest that it may, in the future, be clinically useful in treating patients with intestinal ischemia.

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.

In order to identify metastasis-associated and promoting genes of pancreatic carcinoma we investigated the transcriptional profile of rat pancreatic carcinoma cell lines BSp73-AS (non-metastatic) and BSp73-ASML (highly metastatic) with Affymetrix GeneChip Array technology. We analyzed the expression profile of 7000 genes. Two hundred and ten genes (3%) were up-regulated and 247 genes (3.5%) were down-regulated in the metastatic cell line based on a fold change of expression of at least 3 and a change factor quality of>> or = 2. In order to classify the de-regulated genes we defined the following categories: proteases and protease-related genes, cytokines, receptor tyrosine kinases, other transmembrane proteins/receptors, transcription, cell cycle/apoptosis, signaling, adhesion/extracellular matrix, metabolism, detoxification, protein modification, trafficking, immune response and other genes. We identified de-regulated AP1, FRA-1 and c-myc-mediated transcription in cell line BSp73-ASML. Up-regulation of transmembrane tyrosine kinase receptors c-met, IGFR1, IGFR2 and EGFR family-related ligands such as HB-EGF, TGFa, amphiregulin and neuregulin as well as c-met ligand HGF point to a possible role of this system in metastasis. We identified 56 non-tyrosine kinase transmembrane receptors as new target candidates for inhibition of metastasis, four of them representing already validated targets. In addition, we identified MMP9, uPA, uPAR, cyclin D1 and S100A4 (mts1) as possible contributors of the metastatic phenotype.

OBJECTIVE

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent mitogen for smooth muscle cells and has been implicated in atherosclerosis, tissue regeneration after ischemia, vascular development, and tumor angiogenesis. We examined the hypothesis that HB-EGF participates in angiogenesis and collateral growth in ischemia.

RESULTS

During 3 weeks after femoral artery ligation, no attenuation occurred in recovery of hindlimb perfusion or distal saphenous artery flow in HB-EGF-null (HB-EGF(-/-)) versus wild-type mice. Lumen diameters of remodeled collaterals in gracilis muscle were similar by morphometry (87+/-8 versus 94+/-6 microm) and angiography, although medial thickening was reduced. Gastrocnemius muscle underwent comparable angiogenesis (41% and 33% increase in capillary-to-muscle fiber ratio). Renal renin mRNA, arterial pressure, and heart rate during anesthesia or conscious unrestrained conditions were similar between groups. These latter findings validate comparisons of perfusion data and also suggest that differences in arterial pressure and/or renin-angiotensin activity are not masking an otherwise inhibitory effect of HB-EGF absence. Four days after ligation, EGF receptor phosphorylation increased in muscle by 104% in wild-type but by only 30% in HB-EGF(-/-) mice. This argues against compensation by other EGF receptor ligands.

CONCLUSIONS

Our results suggest that HB-EGF is not required for arteriogenesis or angiogenesis in hindlimb ischemia.