The AP-1 transcription factor c-Jun is essential for cellular proliferation in many cell types, but the molecular link between growth factors and c-Jun activation has been enigmatic. In this study we identify a previously uncharacterized RING-domain-containing protein, RACO-1 (RING domain AP-1 co-activator-1), as a c-Jun co-activator that is regulated by growth factor signalling. RACO-1 interacted with c-Jun independently of amino-terminal phosphorylation, and was both necessary and sufficient for c-Jun/AP-1 activation. Growth factor-mediated stimulation of AP-1 was attributable to MEK/ERK-dependent stabilization of RACO-1 protein. Stimulation of the MEK/ERK pathway strongly promoted Lys 63-linked ubiquitylation of RACO-1, which antagonized Lys 48-linked degradative auto-ubiquitylation of the same Lys residues. RACO-1 depletion reduced cellular proliferation and decreased expression of several growth-associated AP-1 target genes, such as cdc2, cyclinD1 and hb-egf. Moreover, transgenic overexpression of RACO-1 augmented intestinal tumour formation triggered by aberrant Wnt signalling and cooperated with oncogenic Ras in colonic hyperproliferation. Thus RACO-1 is a co-activator that links c-Jun to growth factor signalling and is essential for AP-1 function in proliferation.

BACKGROUND

Pancreatic cancer still has one of the worst prognoses of all cancers with a 5-year survival rate of 5%, making it necessary to find markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens. Especially heparanase (HPSE) has recently been discussed as a key factor in pancreatic cancer.

METHODS

Paraffin-embedded tissue samples were obtained from 41 patients with pancreatic adenocarcinoma who were scheduled for primary surgical resection. Direct quantitative real-time reverse transcriptase polymerase chain reaction (TaqMan) assays were performed in triplicates to determine HPSE, hypoxia inducible factor-1 alpha (HIF1a), platelet-derived growth factor alpha (PDGFA), heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) gene expression levels.

RESULTS

HPSE was significantly correlated to PDGFA (p = 0.04) and HIF1a (p = 0.04). The correlation of HIF1a to bFGF and HB-EGF was significant (p = 0.04, p = 0.02). Stepwise multiple linear regression models showed a significant independent association of HPSE with lymph node metastasis (p = 0.025) and with dedifferentiation (p = 0.042).

CONCLUSIONS

Heparanase seems to be significantly associated with lymph node metastasis (p = 0.025) as well as dedifferentiation (p = 0.042). We assume that HPSE plays a crucial role for the aggressiveness of pancreatic cancer. Larger studies including more patients seem to be warranted.

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.

Although the mechanism is unknown, infiltration anesthetics are believed to have membrane-stabilizing action. We report here that such a most commonly used anesthetic, lidocaine, effectively inhibited the invasive ability of human cancer (HT1080, HOS, and RPMI-7951) cells at concentrations used in surgical operations (5-20 mM). Ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) from the cell surface plays an important role in invasion by HT1080 cells. Lidocaine reduced the invasion ability of these cells by partly inhibiting the shedding of HB-EGF from the cell surface and modulation of intracellular Ca2+ concentration contributed to this action. The anesthetic action of lidocaine (sodium channel blocking ability) did not contribute to this anti-invasive action. In addition, lidocaine (5-30 mM), infiltrated around the inoculation site, inhibited pulmonary metastases of murine osteosarcoma (LM 8) cells in vivo. These data point to previously unrecognized beneficial actions of lidocaine and suggest that lidocaine might be an ideal infiltration anesthetic for surgical cancer operations.

EGF receptor (EGFR) and its signaling have been investigated for many years, but how its different ligands regulate signaling has not been thoroughly explored. When investigating EGFR activation and downstream signaling in HeLa cells using a panel of ligands, we found a ligand-dependent differential activation of EGFR and the signaling pathways Akt, PLCγ and STAT with HB-EGF and BTC being the most potent ligands. All the tested ligands induced full activation of Erk signaling at 1 nM, whereas only HB-EGF and partly BTC and EGF induced strong activation of Akt, STAT3 and PLCγ at this concentration. Interestingly, we also found that the high activation potencies of HB-EGF and BTC could only partially be explained by their binding affinities, and are therefore likely to be regulated by other mechanisms. We thus suggest that the signaling pathways initiated from the EGFR vary depending on the ligands bound in a cell specific manner.

Human peripheral blood monocytes responded to stimulation of platelet-activating factor (PAF) with up-regulation of the transcript for heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells. This function of PAF was observed at nanomolar concentrations of the ligand, starting at 30 min after stimulation. The PAF-induced up-regulation of HB-EGF mRNA was accompanied by an increase in kappa B binding activity. These functions of PAF appeared to be mediated through the cell surface PAF receptors, as two PAF receptor antagonists, WEB 2086 and L-659,989, blocked both the up-regulation of HB-EGF mRNA and kappa B binding activity induced by PAF. The antagonists, however, had no effect on phorbol ester-induced up-regulation of HB-EGF mRNA and kappa B binding activity. Pretreatment of monocytes with pertussis toxin inhibited these functions of PAF, whereas cholera toxin had no inhibitory effect. Pyrrolidine dithiocarbamate, an inhibitor for NF-kappa B activation, markedly reduced PAF-stimulated kappa B binding activity as well as up-regulation of HB-EGF mRNA. These results suggest a potential role of PAF in HB-EGF expression and provide evidence that this stimulation may occur through increased kappa B binding activity.

BACKGROUND

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a newly identified member of the EGF family. Our previous in vitro studies showed that HB-EGF is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs), suggesting the role of HB-EGF in the pathogenesis of atherosclerosis. The purposes of the present study were to investigate the localization of HB-EGF in both normal and atherosclerotic human coronary arteries and to elucidate the possible roles of this growth factor in the formation of atherosclerotic lesions.

RESULTS

The immunohistochemical localization of HB-EGF, SMCs, macrophages, and EGF receptors (EGFRs) was examined in human coronary arteries obtained at autopsy. The medial SMCs of coronary arteries in neonates, infants, and children consistently synthesized HB-EGF protein. In normal adults, however, the relative number of HB-EGF-positive medial SMCs decreased gradually with age after about 30 years of age. In nonatherosclerotic coronary arteries with diffuse intimal thickening, SMCs of the intima, especially those located in the area of the medial side of the intima, were strongly positive for HB-EGF protein. In atherosclerotic plaques of coronary arteries with eccentric intimal thickening, both SMCs and macrophages in and around the core lesions, in addition to the intimal and medial SMCs located adjacent to the plaque, produced HB-EGF protein. A strong immunostaining of EGFRs was observed in these SMCs, suggesting a close association of HB-EGF and EGFR expression.

CONCLUSIONS

These data suggest that HB-EGF might play important roles in the migration of SMCs from the media to the intima, the proliferation of intimal SMCs, and the interaction between SMCs and macrophages in the process of coronary atherogenesis.

BACKGROUND

Tissue plasminogen activator (tPA) is the major activator of plasminogen in plasma. This serine protease is overexpressed by exocrine pancreas tumour cells, where it promotes tumour cell proliferation, growth, and invasion. Here we have explored the signalling pathways used by tPA to activate the proliferation of pancreatic cancer cells.

METHODS

Transcriptional profiling on cDNA micro arrays was used to analyse the pattern of gene expression in response to tPA compared to the response to epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Results were confirmed using different biochemical assays in which specific kinase inhibitors or RNA interference were used.

RESULTS

Transcriptional profiling showed that tPA modulates the expression of a set of genes commonly regulated by EGF, but distinct from the major set of genes modulated by PDGF. This suggested that tPA and EGF share common signalling pathways, a conclusion supported by further experimental evidence. Firstly, we found that tPA induced a rapid and transient phosphorylation of the EGFR. Secondly, specific EGFR kinase inhibitors, but not PDGFR kinase inhibitors, abolished the tPA induced phosphorylation of the ERK1/2 kinases and cell proliferation. The mitogenic activity of tPA was also inhibited by siRNA depletion of EGFR, thus confirming the involvement of this receptor in tPA triggered signalling. Thirdly, we show that the signalling and mitogenic effects of tPA require its proteolytic activity, the activity of the metalloprotease-9 and active hb-EGF.

CONCLUSIONS

Our results suggest that tPA induces proliferation by triggering a proteolytic cascade that sequentially activates plasmin, metalloprotease-9 (MMP-9) and hb-EGF. These events are required to activate the EGFR signalling pathway and cell proliferation.

The neuronal expression of mRNA of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated in immature rat brains. Two rat models were used in this study. One was a hypoxic/ischemic (HI) brain injury model, and the other was an N-methyl-d-aspartate (NMDA) intracerebral injection model. The former model was made by permanent ligation of the left carotid artery and subsequent exposure to 2 h of hypoxia. After the HI insult, the HB-EGF mRNA was assessed by a Northern blot analysis. The levels of transcripts for HB-EGF in the cerebral cortex and the hippocampus of the ligated side were significantly higher than those of non-treated rats from 3 to 24 h after the insult. The spatial distribution of the mRNA of HB-EGF was also studied using in situ hybridization. Three to 24 h after the hypoxia, hybridization signals were intense in neuronal cytoplasm on the ligated side, but a focally decreased signal was seen in infarcted areas. Strongly increased mRNA expression was observed in the neurons surrounding the infarct. These results indicate that a neonatal HI insult induces a neuronal upregulation of HB-EGF immediately after hypoxia. In the latter model, the intracerebral NMDA injection also induced an immediate, strong upregulation of HB-EGF transcripts. Our results indicate that HB-EGF may act as a neuroprotective factor in the immature brain with HI injury by modulating the neurotoxic process which is mediated by overactivation of the NMDA receptor.

To determine whether the gene encoding the recently identified heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle cell (SMC) mitogen of macrophage origin, is transcribed and regulated in vascular SMC, we isolated cDNA clones encoding rat HB-EGF from a macrophage library. Using the rat HB-EGF cDNA as a probe for RNA blot analysis, we detected low levels of HB-EGF mRNA in rat aortic SMC in culture. However, 20 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 10(-6) M angiotensin II (AII) induced a marked increase in HB-EGF mRNA levels in rat aortic SMC (11- and 4.6-fold, respectively) that was both dose- and time-dependent. In response to TPA and AII, HB-EGF mRNA levels increased rapidly, peaked at 2 h, and returned to base line at 7 h. This effect of AII on HB-EGF induction was specific, as evidenced by the fact that it could be completely blocked by the AII antagonist saralasin. This is the first demonstration that HB-EGF is transcribed and regulated in SMC. The inducible transcription of this potent SMC mitogen gene in vascular SMC suggests that HB-EGF may have an important autocrine role in the proliferation of SMC in vascular diseases such as atherosclerosis and hypertension.

Bronchoconstriction applies compressive stress to airway epithelial cells. We show that the application of compressive stress to cultured murine tracheal epithelial cells elicits the increased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt through an epidermal growth factor receptor (EGFR)-dependent process, consistent with previous observations of the bronchoconstriction-induced activation of EGFR in both human and murine airways. Mechanotransduction requires metalloprotease activity, indicating a pivotal role for proteolytic EGF-family ligand shedding. However, cells derived from mice with targeted deletions of the EGFR ligands Tgfα and Hb-egf showed only modest decreases in responses, even when combined with neutralizing antibodies to the EGFR ligands epiregulin and amphiregulin, suggesting redundant or compensatory roles for individual EGF family members in mechanotransduction. In contrast, cells harvested from mice with a conditional deletion of the gene encoding the TNF-α-converting enzyme (TACE/ADAM17), a sheddase for multiple EGF-family proligands, displayed a near-complete attenuation of ERK and Akt phosphorylation responses and compressive stress-induced gene regulation. Our data provide strong evidence that TACE plays a critical central role in the transduction of compressive stress.

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of G(i), which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

Heparin-binding EGF-like growth factor (HB-EGF), a newly discovered potent mitogen and chemoattractant for smooth muscle cells, is a member of the EGF superfamily and binds to EGF receptors. To investigate the role of HB-EGF in the kidney, we determined the distribution of HB-EGF immunohistochemically in normal rat kidneys. The localization of mRNA expression was also studied by in situ hybridization, using a synthesized digoxigenin-labeled anti-sense riboprobe of HB-EGF. Immunohistochemical and in situ hybridization studies revealed that the tubular epithelial cells of the S3 segment of the outer stripe in the outer medulla were the predominant renal source of HB-EGF. In addition, in the immunohistochemical analysis, HB-EGF was ubiquitously present in the epithelial cells of the proximal tubules and the arterial smooth muscle cells, while HB-EGF expression was not detected in other parts of the kidney, including the glomeruli. Although EGF receptors were found to be present in the proximal tubules as well as in the distal tubules and collecting ducts, EGF has not been found to be expressed in the proximal tubules. Therefore, the present results indicate that HB-EGF might be a ligand for EGF receptors in the proximal tubules and might play a role in the functions of proximal tubules.

The structural complexity within heparan sulfate has suggested that it contains multiple protein-specific binding sites. To evaluate the selectivity of growth factor binding to heparan sulfate, we conducted a detailed study of the intercompetition of fibroblast growth factor-2 (FGF-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) binding to heparan sulfate (HS) on bovine aortic smooth muscle cells. Radioligand binding assays were conducted, and an analytical method was developed for determining the apparent binding constants and numbers of specific and shared binding sites within HS. These studies revealed the presence of two general classes of HS-binding sites for FGF-2 and HB-EGF. The major class (approximately 10(6) sites per cell) was able to bind to either growth factor with relatively low affinity (K(d) = 12 and 44 nM for FGF-2 and HB-EGF, respectively) and was termed "common" binding sites. However, both FGF-2 and HB-EGF also showed specific high affinity (0.6 and 6.1 nM for FGF-2 and HB-EGF, respectively) binding to a minor subset (118,000 and 28,000 sites per cell for FGF-2 and HB-EGF, respectively) of "unique" binding sites, which were unable to bind the other growth factor. These studies indicate that growth factor binding to HS involves multiple binding sites of variable affinity, density, and selectivity. The approach outlined in this study could be applied to aid in the evaluation of the relative biological roles of these selective and nonselective growth factor binding domains within HS.

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R(-) cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R(+) cells), stimulation of R(+) cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R(-) cells. The R(-) cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R(+) cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner.

Retinoic acid (RA) has profound effects on epidermal homeostasis; however, the molecular mechanisms by which retinoids regulate keratinocyte cell proliferation and differentiation are not well understood. Here we report that mRNA expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family of growth factors, is induced by RA in human keratinocytes and skin, and is overexpressed in the context of epidermal hyperplasia in vivo. Treatment of normal adult human keratinocytes with micromolar concentrations of RA significantly induced the expression of HB-EGF. The response was efficiently blocked by specific inhibitors of ErbB tyrosine kinase activity, MAP kinase kinase (MEK), or p38 stress-activated protein kinase. RA also enhanced the induction of HB-EGF mRNA in human skin organ culture, an ex vivo model system displaying many similarities to wound healing in vivo. HB-EGF transcripts were markedly increased in human skin by topical treatment with RA under conditions known to provoke epidermal hyperplasia. HB-EGF transcripts were also markedly overexpressed in the hyperplastic epidermis of psoriatic lesions, relative to normal skin. These results support the hypothesis that the effects of RA on epidermal hyperplasia are mediated at least in part by HB-EGF, and suggest that signal transduction mechanisms other than or in addition to nuclear RA receptors contribute to this effect.

The extracellular environment and tissue architecture contribute to proper cell function and growth control. Cells growing in monolayers on standard polystyrene tissue culture plates differ in their shape, growth rate and response to external stimuli, compared with cells growing in vivo. Here, we showed that the EGFR (epidermal growth factor receptor) ligand heparin-binding EGF-like growth factor (HB-EGF) strongly stimulated cell growth in nude mice, but not in cells cultured in vitro. We explored the effects of HB-EGF on cell growth under various cell culture conditions and found that growth promotion by HB-EGF was needed in three-dimensional (3D) or two-dimensional (2D) culture systems in which cell-matrix adhesion was reduced. Under such conditions, cell growth was extremely suppressed in the absence of HB-EGF, but markedly potentiated in the presence of HB-EGF. When the integrin signal was reduced using antibodies or knockout of either integrin beta1 or focal adhesion kinase (FAK), cells showed HB-EGF-dependent growth. We also showed that EGF, transforming growth factor-alpha (TGFalpha) or ligands of other receptor tyrosine kinases (RTKs) stimulated cell growth in 3D culture, but not in tissue culture plates. These results indicate that the integrin signal was sufficient to support cell growth in 2D tissue culture plates without addition of the growth factor, whereas stimulation by growth factors was clearly demonstrated in culture systems in which integrin signals were attenuated.

Many cells upon injury mount extensive, compensatory responses that increase cell survival; however, the intracellular signals that regulate these responses are not completely understood. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been implicated as a cytoprotective agent. We have previously demonstrated that pretreatment of human intestinal epithelial cells with HB-EGF significantly decreased cytokine-induced activation of inducible NO synthase mRNA expression and NO production and protected the cells from apoptosis and necrosis. However, the mechanisms by which HB-EGF exerts these effects are not known. Here we show that cytokine exposure (IL-1beta and IFN-gamma) induced NF-kappaB activation and IL-8 and NO production in DLD-1 cells. Transient expression of a dominant negative form of IkappaBalpha decreased NO production, suggesting that the cytokines stimulated NO production in part through activation of NF-kappaB. HB-EGF dramatically suppressed NF-kappaB activity and IL-8 release and decreased NO production in cells pretreated with HB-EGF. HB-EGF blocked NF-kappaB activation by inhibiting IkappaB kinase activation and IkappaB phosphorylation and degradation, thus interfering with NF-kappaB nuclear translocation, DNA-binding activity, and NF-kappaB-dependent transcriptional activity. The data demonstrate that HB-EGF decreases inflammatory cytokine and NO production by interfering with the NF-kappaB signaling pathway. Inhibition of NF-kappaB may represent one of the mechanisms by which HB-EGF exerts its potent anti-inflammatory and cytoprotective effects.

OBJECTIVE

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction and is closely associated with the development of human hepatocellular carcinoma (HCC). Among HCV components, core protein is implicated in cell growth regulation, and we previously demonstrated that HCV core protein interacted with 14-3-3 protein and activated the kinase Raf-1 and mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway. In the present study, we investigated the expression levels and function of downstream molecules in the MAPK/ERK signaling pathway in cells expressing HCV core protein.

METHODS

Heparin-binding EGF-like growth factor (HB-EGF) mRNA, in HepG2 cells stably expressing HCV core protein, was detected by RT-PCR. The soluble HB-EGF in culture media was measured by heparin agarose chromatography/Western blot analysis. Immunodetection of Akt and IKK and IB, in HeLa cells and HepG2 cells expressing HCV core protein, were performed with neutralizing antibody for HB-EGF, phospatidylinositol-3-kinase [PI(3)K] inhibitor and dominant-negative mutant of Ras (DN-Ras).

RESULTS

HB-EGF expression was significantly elevated in cells expressing HCV core protein. HCV core protein activated Akt through the Ras/PI(3)K pathway by autocrine secretion of HB-EGF. Also, HCV core protein activated IKK through Ras/PI(3)K/Akt pathway by autocrine secretion of HB-EGF. As the Ras/PI(3)K/Akt pathway is critical in anti-apoptotic HB-EGF signaling, we examined the possible role of this pathway in cells expressing HCV core protein. In addition, we investigated the relationship between IB kinases (IKK) and Akt in cells expressing HCV core protein, since IKKs are known to be activated by HCV core protein and by Akt in the presence of potent mitogen. We showed that HCV core protein promoted autocrine secretion of HB-EGF and activated Akt through the Ras/PI(3)K pathway. This model indicates a new approach to mechanism of proliferation and anti-apoptosis in HCC.

CONCLUSIONS

HCV core protein is a potent activator of mitogenic and anti-apoptotic signaling involved in hepatocarcinogenesis.

OBJECTIVE

The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane-bound proteins characterized by their multi-domain structure and ADAM-12 expression is elevated in human non-small cell lung cancers. The aim of this study was to investigate the roles played by ADAM-12 in critical steps of bronchial cell transformation during carcinogenesis.

METHODS

To assess the role of ADAM-12 in tumorigenicity, BEAS-2B cells were transfected with a plasmid encoding human full-length ADAM-12 cDNA, and then the effects of ADAM-12 overexpression on cell behaviour were explored. Treatment of clones with heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis.

RESULTS

Overexpression of ADAM-12 in BEAS-2B cells promoted cell proliferation. ADAM-12 overexpressing clones produced higher quantities of HB-EGF in their culture medium which may rely on membrane-bound HB-EGF shedding by ADAM-12. Targeting HB-EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM-12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor-alpha failed to influence cell proliferation; moreover, ADAM-12 transfectants were resistant to etoposide-induced apoptosis and the use of a neutralizing antibody against HB-EGF activity restored rates of apoptosis to be similar to controls.

CONCLUSIONS

ADAM-12 contributes to enhancing HB-EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.

BACKGROUND

Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells.

RESULTS

Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes.

CONCLUSIONS

Hyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-β1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.

Metalloproteinase activities of a disintegrin and metalloproteinases (ADAMs), matrix metalloproteinases (MMPs), and membrane type (MT-)MMPs are involved in many aspects of tumor biology. ADAMs are transmembrane proteins that cleave membrane-anchored proteins to release soluble factors, and thereby mediate important biological phenomena in tumors. The aim of this study was to analyze histopathology, expression and roles of metalloproteinases, especially ADAMs, in gastric gastrointestinal stromal tumor (GIST). Histopathology and immunohistochemical expression of ADAMs were examined in 89 gastric GISTs. In 11 GISTs, ADAM expression was examined at mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting, respectively. RT-PCR analysis showed frequent expression of ADAM9 (91%), ADAM10 (64%), ADAM17 (82%), MMP-2 (82%), and MT1-MMP (73%). However, ADAM17 and MMP-2 were the only metalloproteinases that were up-regulated in GISTs at the protein level compared with non-neoplastic gastric tissues. ADAM17 was immunohistochemically expressed in 93% of GIST versus 16% of normal gastric tissues. Furthermore, CD117-positive interstitial cells of Cajal in normal gastric tissues were all negative for ADAM17 with double immunostaining. Expressions of epidermal growth factor receptor (EGFR) and several EGFR ligands such as amphiregulin, heparin-binding epidermal growth factor (HB-EGF), betacellulin, and epiregulin were also demonstrated in GIST by RT-PCR. Protein expression of EGFR, phosphorylated EGFR, amphiregulin, and HB-EGF, both of which can be shed by ADAM17, was confirmed in tumors coexpressing ADAM17 by immunoblotting. Moreover, proteolytically cleaved soluble forms of amphiregulin were identified in tumor extracts. Considered together, the results suggest that ADAM17 may contribute to the progression and growth of GIST through shedding of EGFR ligands and consequent EGFR stimulation. ADAM17, as a major sheddase in GIST, could be potentially a suitable target in anticancer treatment of imatinib-resistant GISTs.

Sun-protected human skin was maintained in organ culture and treated with all-trans retinoic acid in the presence or absence of reversible or irreversible pharmacologic antagonists of c-erbB receptor tyrosine kinase activity. In the absence of these inhibitors, all-trans retinoic acid induced epidermal hyperplasia comparable to that induced in intact skin by all-trans retinol or all-trans retinoic acid itself. There was a strong correlation between inhibition of epidermal hyperplasia in organ culture and inhibition of epidermal-growth-factor-dependent keratinocyte growth in monolayer culture. In additional studies it was shown that all-trans retinoic acid could overcome the known inhibitory effects of calcium on expression of HB-EGF-like growth factor mRNA in organ-cultured skin. Further, it was shown that an antibody to HB-EGF-like growth factor inhibited retinoid-stimulated epidermal hyperplasia in organ culture and reduced proliferation in cultured keratinocytes. In contrast, the c-erbB receptor tyrosine kinase antagonists and the neutralizing HB-EGF-like growth factor antibody were ineffective in inhibiting all-trans-retinoic-acid-dependent survival and proliferation of human dermal fibroblasts. Taken together, these data indicate (i) that retinoid-induced epidermal hyperplasia in human skin proceeds through c-erbB, and (ii) that HB-EGF-like growth factor is one of the c-erbB ligands mediating this effect.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family. Juxtacrine activity of proHB-EGF (the membrane-anchored form of HB-EGF) has been shown to be significantly potentiated when it is coexpressed with CD9 in vitro. The purpose of our study was to investigate the issue of whether proHB-EGF and CD9 are coexpressed in gastric cancer. HB-EGF gene expression and protein production in human gastric cancers was investigated, and EGF receptor and CD9 expressions were also evaluated. HB-EGF mRNA levels in gastric cancers were elevated, compared with normal gastric tissues, especially in the intestinal type. ProHB-EGF immunoreactivity was detected primarily in the cytoplasm and plasma membrane of gastric cancer cells. Of 66 patients, 40 (60.6%) exhibited proHB-EGF immunoreactivity and the level of its expression was significantly associated with tumor status (p < 0.01) and histological differentiation (p < 0.001). In addition, proHB-EGF mRNA was detected at high levels in the intestinal type by in situ hybridization. CD9 immunoreactivity was found to be preserved in 26 of 36 patients (72.2%) and CD9 protein expression was inversely associated with lymph node status (p < 0.05). A significant correlation between its expression and histological differentiation (p < 0.01) was found, and the association of CD9 with proHB-EGF was increased in the intestinal type, as evidenced by an immunoprecipitation method. These results indicate that the coexpression of proHB-EGF and CD9 may be involved in the tumorigenesis and/or proliferation of gastric cancers in a juxtacrine manner.