TRIM11 (tripartite motif-containing protein 11), an E3 ubiquitin ligase, is known to be involved in the development of the central nervous system. However, very little is known regarding the role of TRIM11 in cancer biology. Here, we examined the expression profile of TRIM11, along with two stem cell markers CD133 and nestin, in multiple glioma patient specimens, glioma primary cultures derived from tumors taken at surgery and normal neural stem/progenitor cells (NSCs). The oncogenic function of TRIM11 in glioma biology was investigated by knockdown and/or overexpression in vitro and in vivo experiments. Our results showed that TRIM11 expression levels were upregulated in malignant glioma specimens and in high-grade glioma-derived primary cultures, whereas remaining low in glioblastoma multiforme (GBM) stable cell lines, low-grade glioma-derived primary cultures and NSCs. The expression pattern of TRIM11 strongly correlated with that of CD133 and nestin and differentiation status of malignant glioma cells. Knock down of TRIM11 inhibited proliferation, migration and invasion of GBM cells, significantly decreased epidermal growth factor receptor (EGFR) levels and mitogen-activated protein kinase activity, and downregulated HB-EGF (heparin-binding EGF-like growth factor) mRNA levels. Meanwhile, TRIM11 overexpression promoted a stem-like phenotype in vitro (tumorsphere formation) and enhanced glial tumor growth in immunocompromised mice. These findings suggest that TRIM11 might be an indicator of glioma malignancy and has an oncogenic function mediated through the EGFR signaling pathway. TRIM11 overexpression potentially leads to a more aggressive glioma phenotype, along with increased malignant tumor growth and poor survival. Taken together, clarification of the biological function of TRIM11 and pathways it affects may provide novel therapeutic strategies for treating malignant glioma patients.

Growth factor binding to transmembrane protein receptors is generally understood to initiate cell signaling. Receptor binding of heparin-binding growth factors (HB-GFs), such as fibroblast growth factor-2 (FGF-2), is regulated by interactions with heparan sulfate proteoglycans. While there is some specificity for binding to heparan sulfate, overlap in sites for different growth factors may allow for cross regulation. Here we demonstrate, using experiments and computer simulations, that the HB-GFs FGF-2 and heparin-binding EGF-like growth factor (HB-EGF) can cross regulate receptor binding of the other despite having unique receptors. The ability of HSPG to stabilize HB-GF receptor binding is critical for competing growth factors to modulate receptor binding with both enhanced and reduced binding possible depending on this stabilization process. HSPG density and affinity for HB-GF are also critical factors for HB-GF cross regulation. Simulations further reveal that HB-GF can regulate receptor binding of non-HB-GFs such as EGF even when the two proteins share no binding sites when other HB-GF are present within the network. Proliferation studies demonstrate potentiation of HB-EGF-induced growth by FGF-2 indicating that competition networks can alter biological response. Exogenous manipulation of cellular responses to growth factors in complex living systems will require understanding the HSPG-controlled network.

Acetylcholine (ACh) mimics ischemic preconditioning by a mechanism dependent on phosphatidylinositol 3-kinase (PI3-kinase) and reactive oxygen species (ROS). In other tissues muscarinic receptors activate a metalloproteinase, which liberates surface-associated heparin-binding epidermal growth factor (HB-EGF) and causes transactivation of epidermal growth factor receptors (EGFRs) with activation of PI3-kinase. We tested whether this pathway is operative in myocardium. Adult rabbit cardiomyocytes were incubated in reduced MitoTracker Red, which fluoresces after ROS exposure. ACh caused a 36 +/- 6% increase in fluorescence (P<0.001) and metalloproteinase inhibitor III (MPI) abolished this increase. Both exogenous EGF as well as HB-EGF caused similar increases in the ROS signal (41 +/- 12%, P=0.005 and 40 +/- 7%, P<0.001, respectively). The ROS burst from HB-EGF was unaffected by MPI (37 +/- 6%, P=0.002), confirming that inhibition of metalloproteinase activity blocked ACh's effect at a site upstream of EGFR. CRM-197, which inhibits HB-EGF activity, also blocked ACh-induced ROS generation, again implicating release of HB-EGF as a necessary step for ROS generation. An HB-EGF-neutralizing antibody also prevented ACh-induced increase in ROS. In isolated, perfused rabbit hearts ACh increased phosphorylation of EGFR by 127.4 +/- 43.7%, and this increase was abolished by MPI. Finally, ACh decreased infarct size from 30.1 +/- 2.9% of the risk zone in control hearts to 13.7 +/- 3.0% (P=0.002), and this protection could be abolished by co-treatment with MPI (28.7 +/- 2.6%, P=n.s. vs. control). Stimulation of a second Gi-protein-coupled receptor by the delta-opioid agonist [D-Ala2, D-Leu5]-enkephalin acetate (DADLE) also protected the heart (9.1 +/- 2.0% infarction, P<0.005 vs. control), and this protection was similarly blocked by MPI (28.9 +/- 2.3% infarction). We conclude that ACh-induced ROS generation in myocytes is mediated via transactivation of EGFR through metalloproteinase-dependent release of HB-EGF, and that this pathway is also operative in the intact heart and is required for ACh's cardioprotection.

Wound healing is a dynamic process that relies on coordinated signaling molecules to succeed. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is proven to accelerate healing, however precise control over its application is necessary to reduce side effects and achieve desired therapeutic benefit. To achieve effective growth factor delivery we designed a bioactive heparin-based coacervate. In vitro, HB-EGF released from the coacervate delivery system displayed enhanced bioactivity and promoted human keratinocyte migration while preserving cell proliferative capability. In a mouse excisional full-thickness wound model, controlled release of HB-EGF within the wound significantly accelerated wound closure more effectively than an equal dosage of free HB-EGF. Healing was induced by rapid re-epithelialization, granulation tissue formation, and accompanied by angiogenesis. Consistent with in vitro results, wounds treated with HB-EGF coacervate exhibited enhanced migration of keratinocytes with retained proliferative potential, forming a confluent layer for regained barrier function within 7 days. Collectively, these results suggest that coacervate-based controlled release of HB-EGF may serve as a new therapy to accelerate healing of cutaneous wounds.

ADAM 17 (TNF-α converting enzyme, TACE) is a potential target for cancer therapy, but the small molecule inhibitors reported to date are not specific to this ADAM family member. This membrane-bound metalloproteinase is responsible for ectodomain shedding of pathologically significant substrates including TNF-α and EGFR ligands. The aim of this study was to evaluate the pharmacokinetics, pharmacodynamics and anti-tumour efficacy of the first specific inhibitor, an anti-human ADAM17 IgG antibody, clone D1(A12). We used intraperitoneal xenografts of the human ovarian cancer cell line IGROV1-Luc in Balb/c nude mice, chosen because it was previously reported that growth of these xenografts is inhibited by knock-down of TNF-α. In vitro, 200 nM D1(A12) inhibited shedding of ADAM17 substrates TNF-α, TNFR1-α, TGF-α, amphiregulin (AREG), HB-EGF and IL-6Rα, from IGROV1-Luc cells, (4.7 nM IC(50) for TNF-α shedding). In IGROV1-Luc xenografts in vivo, D1(A12) IgG showed pharmacokinetic properties suitable for efficacy studies, with a single i.p. dose of 10 mg/kg D1(A12) sufficient to maintain IgG plasma and ascites fluid concentrations above 100 nM for more than 7 days. The plasma half life was 8.6 days. Next, an efficacy study was performed, dosing D1(A12) or anti-human TNF-α antibody infliximab at 10 mg/kg q7d, quantifying IGROV1-Luc tumour burden by bioluminescence. D1(A12) IgG showed a significant reduction in tumour growth (p = 0.005), 56% of vehicle control. Surprisingly, D1(A12) did not reduce the concentration of circulating human TNF-α, suggesting that another enzyme may compensate for inhibition of ADAM17 in vivo (but not in vitro). However, D1(A12) did show clear pharmacodynamic effects in the mice, with significant inhibition of shedding from tumour of ADAM17 substrates TNFR1-α, AREG, and TGF-α (4-15-fold reductions, p<0.0001 for all three). Thus, D1(A12) has anti-ADAM17 activity in vivo, inhibits shedding of EGFR ligands and has potential for use in EGF ligand-dependent tumours.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestinal epithelial cells from anoxia/reoxygenation in vitro, and to protect the intestines from ischemia/reperfusion (I/R) injury in vivo. The goal of the present study was to determine whether the cytoprotective effects of HB-EGF were due, in part, to its ability to decrease reactive oxygen species (ROS) production. Human whole blood, polymorphonuclear leukocytes, and monocytes, as well as rat intestinal epithelial cells, were exposed to stimuli designed to produce an oxidative burst in these cells. Treatment of the cells with HB-EGF led to a significant decrease in oxidative burst production. In vivo, total midgut I/R injury in rats led to increased ROS production, which was markedly decreased by HB-EGF treatment. Histochemically, I/R injury led to increased ROS production, which was significantly decreased with HB-EGF treatment. HB-EGF cytoprotection is due, in part, to its ability to decrease ROS production. Future studies will determine the mechanisms by which HB-EGF exerts these effects.

Human keratinocytes (KCs) express multiple EGF receptor (EGFR) ligands; however, their functions in specific cellular contexts remain largely undefined. To address this issue, first we measured mRNA and protein levels for multiple EGFR ligands in KCs and skin. Amphiregulin (AREG) was by far the most abundant EGFR ligand in cultured KCs, with >19 times more mRNA and >7.5 times more shed protein than any other family member. EGFR ligand expression in normal skin was low (<8 per thousand of RPLP0/36B4); however, HB-EGF and AREG mRNAs were strongly induced in human skin organ culture. KC migration in scratch wound assays was highly metalloproteinase (MP)- and EGFR dependent, and was markedly inhibited by EGFR ligand antibodies. However, lentivirus-mediated expression of soluble HB-EGF, but not soluble AREG, strongly enhanced KC migration, even in the presence of MP inhibitors. Lysophosphatidic acid (LPA)-induced ERK phosphorylation was also strongly EGFR and MP dependent and markedly inhibited by neutralization of HB-EGF. In contrast, autocrine KC proliferation and ERK phosphorylation were selectively blocked by neutralization of AREG. These data show that distinct EGFR ligands stimulate KC behavior in different cellular contexts, and in an MP-dependent fashion.

Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor(165) (VEGF(165)), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF(165)-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 microl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization.

BACKGROUND

Angiotensin II (Ang II) has been implicated in the development of glomerulosclerosis by stimulating fibronectin (FN) synthesis. The processing and release of heparin binding-endothelin growth factor (HB-EGF) are activated by protein kinase C (PKC) and Ca2+ signaling. We studied the roles of HB-EGF and endothelial growth factor (EGF) receptor (EGFR) in Ang II-induced FN expression using mesangial cells.

METHODS

Mesangial cells were prepared from mouse kidneys by the explant method and cells were used at passages 4 and 5.

RESULTS

Ang II stimulated FN mRNA levels dose-dependently with a maximal increase (3.4-fold) after 12 hours of incubation. This action was completely inhibited by PKC inhibitors and slightly blocked by Ca2+ chelating agents. FN mRNA accumulation by Ang II was abolished by tyrosine kinase inhibitors, a specific inhibitor for EGFR (AG1478) and extracellular signal-regulated kinase (ERK) inactivation. Addition of neutralizing anti-HB-EGF antibody, as well as pretreatment with heparin or the metalloproteinase inhibitor batimastat abolished induction of FN expression by Ang II. In mesangial cells stably transfected with a chimeric construct containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly increased by Ang II (1.7-fold at 0.5 min) and reached a 4.1-fold increase at two minutes. Ang II phosphorylated EGFR (maximal at 2 min) and ERK (maximal at 8 min) in a PKC- and metalloproteinase-dependent manner. Ang II stimulated the expression and release of transforming growth factor-beta (TGF-beta) via EGFR-mediated signaling, and the released TGF-beta also contributed to Ang II-mediated FN expression via EGFR transactivation.

CONCLUSIONS

Ang II-mediated FN expression was regulated by autocrine effects of HB-EGF and TGF-beta, suggesting a novel paradigm for cross-talk between Ang II and growth factor receptor signaling pathways.

Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR(1)), and by PAR(1) inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR(1)-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

Heparin-binding EGF-like growth factor (HB-EGF) is a 22 kDa, O-glycosylated protein that is mitogenic for fibroblasts, smooth muscle cells (SMC) and epithelial cells. This review describes the primary structure of HB-EGF, as well as its processing. The structure of the mouse and human HB-EGF genes is also discussed. Finally, this review summarizes HB-EGF expression patterns, receptor-mediated signaling, and role in several important biological systems.

Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different extracellular stimuli into ectodomain cleavage.

Transient elevation of intracellular calcium (Ca2+(i)) by various means accelerates murine preimplantation development and trophoblast differentiation. Several G-protein-coupled receptors (GPCRs), including the lysophosphatidic acid (LPA) receptor (LPAR), induce Ca2+(i) transients and transactivate the EGF receptor (ErbB1) through mobilization of EGF family members, including heparin-binding EGF-like growth factor (HB-EGF). Because HB-EGF accelerates blastocyst differentiation in vitro, we examined whether crosstalk between LPA and HB-EGF regulates peri-implantation development. During mouse blastocyst differentiation, embryos expressed LPAR1 mRNA constitutively, LPAR2 only in late stage blastocysts and no LPAR3. Consistent with a mechanism based on Ca2+(i) signaling, LPA rapidly accelerated the rate of trophoblast outgrowth, an index of blastocyst differentiation, and chelation of Ca2+(i) with BAPTA-AM blocked LPA stimulation. Interfering with HB-EGF signaling through ErbB1 or ErbB4 also attenuated LPA stimulation. We established that mouse blastocysts indeed express HB-EGF and that LPA induces the transient accumulation of HB-EGF on the embryo surface, which was blocked by treatment with either BAPTA-AM or the protein trafficking inhibitor, brefeldin A. We conclude that LPA accelerates blastocyst differentiation through its ability to induce Ca2+(i) transients and HB-EGF autocrine signaling. Transactivation of ErbB1 or ErbB4 by HB-EGF could represent a convergent signaling pathway accessed in the trophoblast by stimuli that mobilize Ca2+(i).

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.

In 6 HCC cell lines, clear expressions of EGFR and TGF-alpha were found in flow cytometry, while expressions of EGF, HB-EGF and AR were quite low. TGF-alpha secretion into culture supernatants became measurable when TPA 0.5 microM was added. TPA accelerated the proliferation of KYN-3 cells, and anti-TGF-alpha neutralizing antibody suppressed this proliferation in a dose-dependent manner. Addition of exogenous TGF-alpha, EGF, AR, or HB-EGF with heparin accelerated cell proliferation. In non-stimulated cultures, cell proliferation was suppressed by anti-EGFR neutralizing antibody, but not by the antibodies for EGF, TGF-alpha, AR and HB-EGF. HCC may possess a paracrine system regulated by these 4 ligands, and an autocrine system, under a certain condition, via TGF-alpha and EGFR.

Thrombin and factor Xa (FXa) are agonists for G protein-coupled receptors (GPRCs) and may contribute to vascular lesion formation by stimulating proliferation of vascular smooth muscle cells (SMCs). Mitogenic signaling of GPCRs requires transactivation of receptor tyrosine kinases (RTKs). In rat SMCs, thrombin transactivates the epidermal growth factor receptor (EGFR) via a pathway that involves heparin-binding EGF-like growth factor (HB-EGF) as ligand for EGFR. The purpose of this study was to investigate in human SMCs the role of receptor transactivation in the mitogenic response to thrombin and FXa. Thrombin (10 nmol/L) and FXa (100 nmol/L) cause a 3.3- and 2.6-fold increase in DNA synthesis, respectively. In human SMCs, neither thrombin nor FXa causes EGFR phosphorylation, and blockade of EGFR kinase does not inhibit DNA synthesis. However, DNA synthesis and phosphorylation of fibroblast growth factor receptor-1 (FGFR-1) induced by thrombin or FXa are inhibited by antibodies neutralizing basic fibroblast growth factor (bFGF) or by heparin. Hirudin inhibits thrombin-, but not FXa-induced mitogenesis, indicating that FXa acts independently of thrombin. We further demonstrate by ELISA that upon thrombin and FXa stimulation, bFGF is released and binds to the extracellular matrix. Our data suggest that in human vascular SMCs, both thrombin and FXa rapidly release bFGF into the pericellular matrix. This is followed by transactivation of the FGFR-1 and increased proliferation. Heparin may inhibit the mitogenic effects of thrombin and FXa in human SMCs by preventing bFGF binding to FGFR-1.

Neuropilin-1 (NRP1) is expressed by endothelial cells and neurons and serves as a receptor for both vascular endothelial growth factor (V<em>EGF</em>), an angiogenesis factor, and semaphorin 3A (Sema3A), a mediator of axonal guidance. We show here that NRP1 is also expressed in keratinocytes in vitro and in vivo. However, nothing has been reported about the regulation or function of keratinocyte NRP1. Using NRP1 promoter constructs in HaCaT cells, a keratinocyte cell line, we could demonstrate that a neuron restrictive silencer element (NRSE) was implicated in transcriptional repression of the NRP1 gene. Electrophoretic mobility shift assays demonstrated that the neuron restrictive silencer factor (NRSF) binds to NRSE. Overexpression of NRSF in HaCaT cells decreased NRP1 RNA and protein, whereas a dominant negative NRSF increased NRP1. Furthermore, the histone deacetylase inhibitor trichostatin A, an inhibitor of NRSF silencing activity, also increased NRP1 levels. NRP2 expression was not affected. Epidermal growth factor (<em>EGF</em>) and heparin-binding <em>EGF</em>-like growth factor (<em>HB</em>-<em>EGF</em>) strongly up-regulated NRP1 expression, concomitant with down-regulation of NRSF. Other keratinocyte mitogens such as keratinocyte growth factor (KGF) had no effect. To address function, HaCaT cells were exposed to two NRP1 ligands, V<em>EGF</em>165 and Sema3A. Neither had an effect on proliferation, whereas Sema3A, but not V<em>EGF</em>165, inhibited cell migration. Down-regulation of NRP1 by NRSF overexpression reduced Sema3A activity. It was concluded that NRSF is a transcription factor that silences NRP1 expression and thereby diminishes the Sema3A mediated inhibition of HaCaT keratinocyte migration.

Oxyhemoglobin (OxyHb) can suppress voltage-dependent K(+) channel (K(V)) currents through protein tyrosine kinase activation, which may contribute to cerebral vasospasm following subarachnoid hemorrhage. Here we have tested the hypothesis that shedding of heparin-binding EGF-like growth factor (HB-EGF) and the resulting activation of the tyrosine kinase EGF receptor (EGFR) underlie OxyHb-induced K(V) channel suppression in the cerebral vasculature. With the use of the conventional whole cell patch-clamp technique, two EGFR ligands, EGF and HB-EGF, were found to mimic OxyHb-induced K(V) suppression in rabbit cerebral artery myocytes. K(V) current suppression by OxyHb or EGF ligands was eliminated by a specific EGFR inhibitor, AG-1478, but was unaffected by PKC inhibition. Compounds (heparin and CRM-197) that specifically interfere with HB-EGF signaling eliminated OxyHb-induced K(V) suppression, suggesting that HB-EGF is the EGFR ligand involved in this pathway. HB-EGF exists as a precursor protein that, when cleaved by matrix metalloproteases (MMPs), causes EGFR activation. MMP activation was detected in OxyHb-treated arteries by gelatin zymography. Furthermore, the MMP inhibitor (GM-6001) abolished OxyHb-induced K(V) current suppression. We also observed K(V) current suppression due to EGFR activation in human cerebral artery myocytes. In conclusion, these data demonstrate that OxyHb induces MMP activation, causing HB-EGF shedding and enhanced EGFR activity, ultimately leading to K(V) channel suppression. We propose that EGFR-mediated K(V) suppression contributes to vascular pathologies, such as cerebral vasospasm, and may play a more widespread role in the regulation of regional blood flow and peripheral resistance.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF growth factor family. Initially synthesized as a membrane-bound precursor (pro-HB-EGF), it is cleaved at the juxtamembrane domain to release the soluble form of HB-EGF (s-HB-EGF) by sheddases, including matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases. This is a process referred to as ectodomain shedding and is implicated in the process of all ligands of the EGF receptor (EGFR) family. The tumorigenic potential of s-HB-EGF has been studied extensively; however, the role of pro-HB-EGF in tumor progression is unknown, despite the fact that a considerable amount of pro-HB-EGF remains on the cell membrane. Our data here clearly indicated the distinct role of pro-HB-EGF in the regulation of E-cadherin expression and the epithelial-mesenchymal transition. We showed here that the expression of pro-HB-EGF was associated with the differentiation status in pancreatic tumors and cell lines. Expression of noncleaved pro-HB-EGF in pancreatic cells resulted in the up-regulation of E-cadherin through suppression of ZEB1, which is a transcriptional repressor of E-cadherin. Inhibition of HB-EGF shedding using a MMP inhibitor, GM6001, also dramatically augmented the E-cadherin expression while suppressing the EGFR activation. Moreover, up-regulation of E-cadherin by pro-HB-EGF not only resulted in cellular morphologic change but also decreased cell motility and enhanced apoptotic sensitivity in response to gemcitabine-erlotinib treatment. Collectively, our data defined a distinct role of pro-HB-EGF in the regulation of E-cadherin, suggesting that inhibition of shedding may be a novel approach to suppress pancreatic metastasis and sensitize cells to cancer therapy.

Transforming growth factor-alpha (TGF-alpha) is abnormally expressed in autosomal recessive polycystic kidney disease (ARPKD). Tumor necrosis factor-alpha converting enzyme (TACE), a metalloproteinase, mediates TGF-alpha processing. In this study, we sought to determine whether TGF-alpha was an absolute requirement for renal cystogenesis and whether its absence would modulate disease severity or related growth factors/receptors expression. Bpk heterozygotes were bred with TGF-alpha null mice to produce cystic and noncystic offspring with or without TGF-alpha. Assessments included kidney weight (KW), body weight (BW), blood urea nitrogen (BUN), and kidney and liver immunohistology. Western analysis assessed kidney expression of amphiregulin (AR), epidermal growth factor (EGF), heparin-binding EGF (HB-EGF), and their receptors, EGFR and ErbB4. A PCR-based methodology for genotyping bpk mice was also developed. No significant differences in KW, BW, KW/BW%, or BUN were seen in cystic mice with versus without TGF-alpha. Cystic kidney disease and liver disease histology were similar. AR, EGF, HB-EGF, EGFR, and ErbB4 were abnormally expressed to an equal degree in kidneys of mice with versus without TGF-alpha. Although previous data suggest a critical role of TGF-alpha in murine PKD, these data show that TGF-alpha is not required for renal cyst formation or kidney or liver disease progression. We speculate that the therapeutic effect of WTACE2 could have been due to effects on several TACE targets, including TGF-alpha, AR, and ErbB4, as well as metalloproteinases other than TACE.

OBJECTIVE

Interleukin (IL)-1beta-induced matrix metalloproteinase (MMP-9) expression is regulated by mitogen activated protein kinases (MAPKs) and NF-kappaB. IL-1beta also stimulates transactivation of growth factor receptors and phosphatidylinositol 3-kinase (PI3K)/Akt., leading to the expression of inflammatory proteins. Here, we investigated whether these transactivation mechanisms participated in IL-1beta-induced MMP-9 expression in A549 cells.

METHODS

A549 cells were treated with/without pharmacological inhibitors and neutralizing antibody or transfected with dominant negative mutants and siRNA of particular protein kinases before stimulation with IL-1beta. Cell migration was measured by in vitro scratch assay. Expression and enzymatic activity of MMP-9 were analysed by Western blot and gelatin zymography. Transcriptional activity of MMP-9 was analysed by RT-PCR, chromatin immunoprecipitation and promoter assays.

RESULTS

Inhibition of MMP-9 expression by inhibitors of Src (PP1), platelet-derived growth factor (PDGF) receptor and epithelial growth factor (EGF) receptor or transfection with siRNA for Src and Akt prevented IL-1beta-induced migration of A549 cells. These tyrosine kinases were involved through phosphorylation of Src, PDGF, or EGF receptors (EGFRs) via the formation of Src/PDGFR or Src/EGFR complexes, attenuated by PP1. IL-1beta-induced MMP-9 expression through EGFR transactivation was diminished by inhibitors of MMPs and heparin-binding EGF-like factor (HB-EGF), or a neutralizing HB-EGF antibody. IL-1beta-stimulated activation and translocation of Akt and NF-kappaB (p65); the recruitment of activated NF-kappaB (p65) to the MMP-9 promoter region was attenuated by LY294002.

CONCLUSIONS

IL-1beta-induced MMP-9 expression and cell migration was mediated through c-Src-dependent transactivation of EGFR/PDGFR/PI3K/Akt linking to the NF-kappaB pathway in A549 cells.

BACKGROUND

We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo.

METHODS

In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined.

RESULTS

Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05).

CONCLUSIONS

Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.

OBJECTIVE

Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance.

METHODS

HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis.

RESULTS

A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance.

CONCLUSIONS

Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles.

Various kinds of growth factors are involved in the regulation of human keratinocyte function. Among them, the epidermal growth factor (EGF) family and the transforming growth factor-beta (TGF-beta) family play central roles, providing dual-mode regulation of keratinocyte growth through the proliferation-stimulating effect of EGF and the proliferation-inhibiting effect of TGF-beta. Human keratinocytes synthesize four EGF family growth factors, TGF-alpha, amphiregulin, HB-EGF and epiregulin, which efficiently regulate keratinocyte growth and differentiation via auto- and cross-induction pathways. Human keratinocytes also synthesize TGF-beta1 and TGF-beta2, which are potent inhibitors of human keratinocytes. These intrinsic EGF family growth factors and TGF-betas are responsible for the autonomous growth regulation of keratinocytes.