We have used the reverse transcription-polymerase chain reaction (RT-PCR) to determine whether transcripts for the epidermal growth factor (EGF) receptor and its four known ligands--EGF, transforming growth factor alpha (TGF alpha), amphiregulin (Ar), and heparin-binding EGF (HB-EGF)--are expressed in porcine oviduct and endometrium. We were able to detect mRNA for the EGF receptor, EGF, TGF alpha, and Ar in both the oviduct and endometrium, whereas HB-EGF mRNA was not detectable in either tissue. Through use of an antiserum raised against recombinant pig EGF, expression of EGF was found to be localized to the columnar epithelial cells of the oviduct and to the glandular epithelial cells of the endometrium. The possible physiological roles of the EGF family in the reproductive tract are discussed.

Recent studies demonstrated that chitinase 3-like-1 (Chi3l1) binds to and signals via IL-13Rα2. However, the mechanism that IL-13Rα2 uses to mediate the effects of Chi3l1 has not been defined. Here, we demonstrate that the membrane protein, TMEM219, is a binding partner of IL-13Rα2 using yeast two-hybrid, co-immunoprecipitation, co-localization and bimolecular fluorescence complementation assays. Furthermore, fluorescence anisotropy nanodisc assays revealed a direct physical interaction between TMEM219 and IL-13Rα2-Chi3l1 complexes. Null mutations or siRNA silencing of TMEM219 or IL-13Rα2 similarly decreased Chi3l1-stimulated epithelial cell HB-EGF production and macrophage MAPK/Erk and PKB/Akt activation. Null mutations of TMEM219 or IL-13Rα2 also phenocopied one another as regards the ability of Chi3l1 to inhibit oxidant-induced apoptosis and lung injury, promote melanoma metastasis and stimulate TGF-β1. TMEM219 also contributed to the decoy function of IL-13Rα2. These studies demonstrate that TMEM219 plays a critical role in Chi3l1-induced IL-13Rα2 mediated signalling and tissue responses.

In ovarian cancer, a high ratio of anti-inflammatory M2 to pro-inflammatory M1 macrophages correlates with poor patient prognosis. The mechanisms driving poor tumor outcome as a result of the presence of M2 macrophages in the tumor microenvironment remain unclear and are challenging to study with current techniques. Therefore, in this study we utilized a micro-culture device previously developed by our lab to model concentrated paracrine signaling in order to address our hypothesis that interactions between M2 macrophages and ovarian cancer cells induce tumor cell proliferation. Using the micro-culture device, we determined that co-culture with M2-differentiated primary macrophages or THP-1 increased OVCA433 proliferation by 10-12%. This effect was eliminated with epidermal growth factor receptor (EGFR) or heparin-bound epidermal growth factor (HB-EGF) neutralizing antibodies and HBEGF expression in peripheral blood mononuclear cells from ovarian cancer patients was 9-fold higher than healthy individuals, suggesting a role for HB-EGF in tumor progression. However, addition of HB-EGF at levels secreted by macrophages or macrophage-conditioned media did not induce proliferation to the same extent, indicating a role for other factors in this process. Matrix metalloproteinase-9, MMP-9, which cleaves membrane-bound HB-EGF, was elevated in co-culture and its inhibition decreased proliferation. Utilizing inhibitors and siRNA against MMP9 in each population, we determined that macrophage-secreted MMP-9 released HB-EGF from macrophages, which increased MMP9 in OVCA433, resulting in a positive feedback loop to drive HB-EGF release and increase proliferation in co-culture. Identification of multi-cellular interactions such as this may provide insight into how to most effectively control ovarian cancer progression.

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while-based on our previous data-PIBF might control trophoblast invasion by suppressing proinvasive genes.

OBJECTIVE

The G protein-coupled oestrogen receptor-1 (GPER-1) agonist G1 induces endothelium-dependent relaxation. Activation of the epidermal growth factor (EGF) receptor leads to transduction of signals from the plasma membrane for the release of nitric oxide. We tested the hypothesis that G1 induces endothelium-dependent vasorelaxation through activation of the EGF receptor.

METHODS

Rat aortic rings were mounted in organ baths. After pretreatment with various inhibitors, aortic rings contracted with 11,9-epoxymethano-prostaglandin F2α or KCl were subjected to relaxation by G1.

RESULTS

G1 induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L -N(ω) -nitroarginine methyl ester (L -NAME), an inhibitor of nitric oxide synthase, or (3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline HB-EGF, heparin-binding EGF-like growth factor, a GPER-1 antagonist. Neither a general oestrogen receptor antagonist, ICI 182 780, nor a selective oestrogen receptor-α antagonist, methyl-piperidino-pyrazole dihydrochloride (MPP), had an effect on G1-induced vasorelaxation. However, pretreatment with EGF receptor blockers, AG1478 or DAPH, resulted in attenuated G1-induced vasorelaxation. In addition, pretreatment with Src inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Akt inhibitor VIII also resulted in attenuated vascular relaxation induced by the cumulative addition of G1. However, neither phosphatidylinositol-3 kinase inhibitors LY294002 and wortmannin nor an extracellular signal-regulated kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene monoethanolate had effect on vascular relaxation induced by the cumulative addition of G1.

CONCLUSIONS

G1 induces endothelium-dependent vasorelaxation through Src-mediated activation of the EGF receptor and the Akt pathway in rat aorta.

In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor alpha (TGFalpha), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFalpha treatment decreased the Bmax of [3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFalpha treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFalpha effect. Moreover, TGFalpha counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFalpha with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFalpha, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.

Heparin-binding EGF-like growth factor (HB-EGF) exists as a membrane-anchored form (proHB-EGF) and as its soluble cleaved product (sHB-EGF). The conversion (ectodomain shedding) of proHB-EGF to sHB-EGF is tightly regulated by specific metalloproteinases. Ectodomain shedding plays a central role in GPCR-mediated EGFR transactivation. Antagonizing metalloproteinases can inhibit EGFR transactivation and might be of therapeutic value, for example in cardiac hypertrophy, skin remodeling and tumor growth.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is known to induce cell growth in various cell types via transactivation of epidermal growth factor receptor (EGFR). To investigate the involvement of HB-EGF and EGFR in cardiac remodeling after myocardial infarction (MI), we examined the expressions of mRNA and protein in rat hearts 6 weeks after MI-induction. Where increased expressions of HB-EGF mRNA and protein were observed, infarcted myocardium was replaced by extracellular matrix and interstitial fibroblasts. EGFR mRNA and protein expression did not show significant changes in sham-operated heart tissues, non-infarcted region, and infarcted region. In vitro study demonstrated that HB-EGF mRNA was expressed mainly in cultured fibroblasts rather than in myocytes. We suggest that the interaction between HB-EGF and EGFR transactivation is closely related to the proliferation of cardiac fibroblasts and cardiac remodeling after MI in an autocrine, paracrine, and juxtacrine manner.

Vascular endothelial growth factor (VEGF) is a potent growth factor playing diverse roles in vasculogenesis and angiogenesis. In the brain, VEGF mediates angiogenesis, neural migration and neuroprotection. As a permeability factor, excessive VEGF disrupts intracellular barriers, increases leakage of the choroid plexus endothelia, evokes edema, and activates the inflammatory pathway. Recently, we discovered that a heparin binding epidermal growth factor like growth factor (HB-EGF)-a class of EGF receptor (EGFR) family ligands-contributes to the development of hydrocephalus with subarachnoid hemorrhage through activation of VEGF signaling. The objective of this review is to entail a recent update on causes of death due to neurological disorders involving cerebrovascular and age-related neurological conditions and to understand the mechanism by which angiogenesis-dependent pathological events can be treated with VEGF antagonisms. The Global Burden of Disease study indicates that cancer and cardiovascular disease including ischemic and hemorrhagic stroke are two leading causes of death worldwide. The literature suggests that VEGF signaling in ischemic brains highlights the importance of concentration, timing, and alternate route of modulating VEGF signaling pathway. Molecular targets distinguishing two distinct pathways of VEGF signaling may provide novel therapies for the treatment of neurological disorders and for maintaining lower mortality due to these conditions.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates EGF receptor, and is expressed in a variety of tissues, predominantly in the lung, heart, brain and skeletal muscle. HB-EGF is known to induce vascular smooth muscle cell (VSMC) proliferation by activating PI3K-Akt and MAPK pathway. However, our preliminary data showed that Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway was also involved in HB-EGF induced VSMC proliferation. More interestingly, HB-EGF (10 ng/ml) induced a biphasic activation of STAT3 (early at 5 min and late at 60-120 min). Therefore, we tried to elucidate the underlying mechanism of this delayed STAT3 activation by HB-EGF in VSMCs. First, we examined the effect of HB-EGF on interleukin-6 (IL-6) mRNA expressions, since IL-6 have been implicated in the regulation of STAT3 activation. According to our data, HB-EGF increased transcription of IL-6, cardiotrophin-1 (CT-1), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). The secretion of IL-6 was also increased by HB-EGF. Furthermore, these HB-EGF-mediated up-regulation of IL-6 mRNA expression and secretion were inhibited by NF-kappaB inhibitor Bay117082 (2.5 microM) treatment suggesting involvement of NF-kappaB pathway. Again, the late activation of STAT3 by HB-EGF was abolished by both Bay117082 and IL-6 neutralizing antibody (1 microg/ml) indicating IL-6 is a key molecule in the delayed activation of STAT3 by HB-EGF. In addition, IL-6 neutralizing antibody inhibited both HB-EGF conditioned media induced STAT3 activation and HB-EGF induced VSMC proliferation. In conclusion, IL-6 plays an important role in the delayed activation of STAT3 and VSMC proliferation induced by HB-EGF.

Vascular deficits are a fundamental contributing factor of diabetes-associated diseases. Although previous studies have demonstrated that the pro-angiogenic phase of wound healing is blunted in diabetes, a comprehensive understanding of the mechanisms that regulate skin revascularization and capillary stabilization in diabetic wounds is lacking. Using a mouse model of diabetic wound healing, we performed microCT analysis of the 3-dimensional architecture of the capillary bed. As compared to wild type, vessel surface area, branch junction number, total vessel length, and total branch number were significantly decreased in wounds of diabetic mice as compared to WT mice. Diabetic mouse wounds also had significantly increased capillary permeability and decreased pericyte coverage of capillaries. Diabetic wounds exhibited significant perturbations in the expression of factors that affect vascular regrowth, maturation and stability. Specifically, the expression of VEGF-A, Sprouty2, PEDF, LRP6, Thrombospondin 1, CXCL10, CXCR3, PDGFR-β, HB-EGF, EGFR, TGF-β1, Semaphorin3a, Neuropilin 1, angiopoietin 2, NG2, and RGS5 were down-regulated in diabetic wounds. Together, these studies provide novel information about the complexity of the perturbation of angiogenesis in diabetic wounds. Targeting factors responsible for wound resolution and vascular pruning, as well those that affect pericyte recruitment, maturation, and stability may have the potential to improve diabetic skin wound healing.

TNF-α plays a crucial role in psoriasis; therefore, TNF inhibition has become a gold standard for the treatment of psoriasis. TNF-α is processed from a membrane-bound form by TNF-α converting enzyme (TACE) to soluble form, which exerts a number of biological activities. EGF receptor (EGFR) ligands, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor (TGF)-α are also TACE substrates and are psoriasis-associated growth factors. Vascular endothelial growth factor (VEGF), one of the downstream molecules of EGFR and TNF signaling, plays a key role in angiogenesis for developing psoriasis. In the present study, to assess the possible role of TACE in the pathogenesis of psoriasis, we investigated the involvement of TACE in TPA-induced psoriasis-like lesions in K5.Stat3C mice, which represent a mouse model of psoriasis. In this mouse model, TNF-α, amphiregulin, HB-EGF and TGF-α were significantly up-regulated in the skin lesions, similar to human psoriasis. Treatment of K5.Stat3C mice with TNF-α or EGFR inhibitors attenuated the skin lesions, suggesting the roles of TACE substrates in psoriasis. Furthermore, the skin lesions of K5.Stat3C mice showed down-regulation of tissue inhibitor of metalloproteinase-3, an endogenous inhibitor of TACE, and an increase in soluble TNF-α. A TACE inhibitor abrogated EGFR ligand-dependent keratinocyte proliferation and VEGF production in vitro, suggesting that TACE was involved in both epidermal hyperplasia and angiogenesis during psoriasis development. These results strongly suggest that TACE contributes to the development of psoriatic lesions through releasing two kinds of psoriasis mediators, TNF-α and EGFR ligands. Therefore, TACE could be a potential therapeutic target for the treatment of psoriasis.

Glioblastoma (GBM) usually develops in adult brain white matter. Even after complete resection, GBM recurs around the tumor removal cavity, where GBM cells acquire chemo-radioresistance. Characterization of the tumor border microenvironment is critical for improving prognosis in patients with GBM. Here, we compared microRNA (miRNA) expression in samples from the tumor, tumor border, and periphery by miRNA microarray. The top three of miRNAs showing higher expression in the tumor border were related to oligodendrocyte differentiation, and pathologically oligodendrocyte lineage cells were increased in the border, where macrophages and microglia also colocalized. Medium cultured with oligodendrocyte progenitor cells (OPCs) and macrophages induced stemness and chemo-radioresistance in GBM cells, similar to that produced by FGF1, EGF and HB-EGF, IL-1β, corresponding to OPCs and macrophages, respectively. Thus, OPCs and macrophages/microglia may form a glioma stem cell niche at the tumor border, representing a promising target for prevention of recurrence.

Familial dilated cardiomyopathy (FDCM) is caused by defective genes and specific medicines are not currently available to treat this. Ginsenoside-Rb1 provides cardioprotection in the experimental models of myocardial ischemia-reperfusion injury. Here we investigate Rb1's effect on DCM in cTnT(R141W) transgenic mouse. The transgene-positive mice aged 2 months were randomized into the model group and Rb1 [70 mg/(kg.day)] group; transgene-negative mice were used as a control. After 4-month treatment, cardiac function was assessed by echocardiography; cardiac tissues were prepared for histology and electron microscopy. Expression levels of molecular markers of cardiac hypertrophy, fibrosis, and intercalated disc proteins were detected by RT-PCR. Rb1 significantly decreased mortality, chamber dilation, and contractile dysfunction in cTnT(R141W) mice. Rb1 attenuated cardiac hypertrophy, interstitial fibrosis, ultrastructural degeneration, and intercalated disc remodeling in DCM hearts. Western blotting showed that Rb1 significantly decreased heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression and signal transduction and activators of transcription 3 (STAT3) activation, which were gradually increased in DCM hearts. Our results showed that Rb1 clearly alleviated cardiac dysfunction and remodeling in the cTnT(R141W) transgenic mouse, indicating its potential utility in the treatment of FDCM.

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood-brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics.

We have previously shown that the presence of trophoblast cells enhances invasiveness of decidualizing human endometrial stromal cells. The metastasis suppressor CD82, which has antimigratory function in tumor cells, is up-regulated in decidualizing endometrial stromal cells. CEACAM1 is expressed in trophoblast cells at the invasion front in early placenta and is considered proinvasive. Here, we investigate the role of CD82 and CEACAM1 in cocultures of the endometrial stromal cell line T-HESC and AC-1M88 trophoblast cells. In transwell migration assays, chemotaxis of AC-1M88 cells was stimulated by coplated T-HESC in the lower compartment or by the combination of heparin-binding EGF-like growth factor (HB-EGF), interleukin-1 beta (IL-1beta), and leukemia inhibitory factor (LIF), local factors present at the time of implantation. In an implantation model of AC-1M88 trophoblast spheroids on a monolayer of T-HESC, spheroid expansion was enhanced in the presence of HB-EGF/IL-1beta/LIF. Silencing of CEACAM1 in AC-1M88 blunted this response. Chemotactic migration of T-HESC was stimulated by trophoblast secretions or HB-EGF/IL-1beta/LIF. These responses were suppressed by CD82 depletion in T-HESC. Proteome profiling revealed the presence of platelet-derived growth factor (PDGF)-AA in trophoblast supernatant. Chemotaxis of T-HESC toward PDGF-AA was significantly inhibited by CD82 silencing. Neutralization of PDGF-AA in AC-1M88 conditioned media reduced the chemotactic effect on T-HESC. In summary, we demonstrate a mutual stimulation of chemotactic migration between trophoblast and endometrial stromal cells and promigratory roles for the cell surface molecules CEACAM1 and CD82, which may serve to support tissue remodeling at the implantation site.

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.

Helicobacter pylori (H. pylori) is one of the most common human pathogens, affecting half of the world's population. Approximately 20% of the infected patients develop gastric ulcers or neoplastic changes in the gastric stroma. An infection also leads to the progression of epithelial-mesenchymal transition within gastric tissue, increasing the probability of gastric cancer development. This paper aims to review the role of H. pylori and its virulence factors in epithelial-mesenchymal transition associated with malignant transformation within the gastric stroma. The reviewed factors included: CagA (cytotoxin-associated gene A) along with induction of cancer stem-cell properties and interaction with YAP (Yes-associated protein pathway), tumor necrosis factor α-inducing protein, Lpp20 lipoprotein, Afadin protein, penicillin-binding protein 1A, microRNA-29a-3p, programmed cell death protein 4, lysosomal-associated protein transmembrane 4β, cancer-associated fibroblasts, heparin-binding epidermal growth factor (HB-EGF), matrix metalloproteinase-7 (MMP-7), and cancer stem cells (CSCs). The review summarizes the most recent findings, providing insight into potential molecular targets and new treatment strategies for gastric cancer.

The airway smooth muscle (ASM) cells' proliferation, migration, and their progenitor's migration are currently regarded as causative factors for ASM remodeling in asthma. Heparin-binding epidermal growth factor (HB-EGF), a potent mitogen and chemotactic factor, could promote ASM cell proliferation through MAPK pathways. In this study, we obtained primary ASM cells and their progenitors from C57BL/6 mice and went on to explore the role of HB-EGF in these cells migration and the underlying mechanisms. We found that recombinant HB-EGF (rHB-EGF) intratracheal instillation accelerated ASM layer thickening in an OVA-induced asthmatic mouse. Modified Boyden chamber assay revealed that rHB-EGF facilitate ASM cell migration in a dose-dependent manner and ASM cells from asthmatic mice had a greater migration ability than that from normal counterparts. rHB-EGF could stimulate the phosphorylation of ERK1/2 and p38 in ASM cells but further migration assay showed that only epidermal growth factor receptor inhibitor (AG1478) or p38 inhibitor (SB203580), but not ERK1/2 inhibitor (PD98059), could inhibit rHB-EGF-mediated ASM cells migration. Actin cytoskeleton experiments exhibited that rHB-EGF could cause actin stress fibers disassembly and focal adhesions formation of ASM cells through the activation of p38. Finally, airway instillation of rHB-EGF promoted the recruitment of bone marrow-derived smooth muscle progenitor cells, which were transferred via caudal vein, migrating into the airway from the circulation. These observations demonstrated that ASM remodeling in asthma might have resulted from HB-EGF-mediated ASM cells and their progenitor cells migration, via p38 MAPK-dependent actin cytoskeleton remodeling.

To investigate the role of ERK signaling in human skin responses to wounding, organ cultures of human skin were maintained for 0.5-24 h in the presence of various inhibitors, followed by measurement of ERK phosphorylation or mRNA levels. The MEK inhibitor PD98059 produced near-complete (97-98%) inhibition of ERK phosphorylation, whereas inhibition of c-Fos, c-Jun, HB-EGF, AR, and VEGF mRNA by this compound was incomplete (41-65%). PD98059 was significantly more effective than either PD158780 or BB2516 as an inhibitor of ERK phosphorylation and of the rapid rise in c-Fos and c-Jun mRNA expression. In contrast, all three compounds inhibited the more delayed rise in HB-EGF mRNA to the same extent. Exogenous epidermal growth factor abrogated the inhibition of ERK phosphorylation caused by BB2516. These data indicate that one or more metalloproteinases activate ErbB signaling in skin organ culture, that ErbB signaling plays an important but not exclusive role in the activation of ERK, and that non-ERK pathways contribute to gene expression in this system. Because metalloproteinase-mediated cleavage of the HB-EGF transmembrane precursor is known to be ERK-dependent, our data suggest that ERK activation resulting from initial trauma leads to metalloproteinase-mediated cleavage of HB-EGF, thereby triggering the ErbB signaling cascade.

In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1) Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2) Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3) In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4) HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that has a high affinity for heparin and heparan sulfate. While interactions with heparin are thought to modulate the biological activity of HB-EGF, the precise role of the heparin-binding domain has remained unclear. We analyzed the activity of wild-type HB-EGF and a mutant form lacking the heparin-binding domain (DeltaHB) in the presence or absence of heparin. The activity of the EGF-like domain of HB-EGF was determined by measuring binding to diphtheria toxin (DT) as well as the growth factor activity in EGF receptor-expressing cells. The binding affinity of DeltaHB for DT was much higher than that of wild-type HB-EGF in the absence of heparin. The binding affinity of HB-EGF for DT was increased by addition of exogenous heparin and reached the level close to the affinity of DeltaHB, whereas that of DeltaHB was not affected. Moreover, the growth factor activity of DeltaHB was much higher than that of wild-type HB-EGF in the absence of heparin but was not affected by addition of exogenous heparin, whereas HB-EGF had increased growth factor activity with added heparin. These results indicate that the heparin-binding domain suppresses the activity of the EGF-like domain of HB-EGF and that association of heparin with HB-EGF via this domain removes the suppressive effect. Thus, we conclude that the heparin-binding domain serves as a negative regulator of this growth factor.

As a part of synthetic studies on MMP (matrix metalloproteinase)/ADAM (a disintegrin and metalloproteinase) inhibitors, we have preliminarily communicated that azasugar-based compound 1a exhibited a potential inhibitory activity on some metalloprotease-catalyzed proteolytic reactions. To find promising candidates for the topical treatment of psoriasis, we investigated stability in aqueous solution of compound 1a and its derivative 1b and then optimized the P1' substuent (2-5). In the present study, we synthesized novel derivatives of compound 1a and evaluated their inhibitory activity toward MMP-1, -3, and -9, TACE, and HB-EGF shedding, from a viewpoint of versatility of azasugars as a functional scaffold. As a result, it was found that compound 1b demonstrated desirable inhibitory activity as an antipsoriatic agent, and some of the derivatives showed selective inhibitory activity. In addition, it was found that compound 1b exhibited a significant therapeutic effect on a mouse TPA-induced epidermal hyperplasia model. Therefore, compound 1b could become a promising candidate as a practical antipsoriatic agent.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family that binds to and activates the EGF receptor. Transactivated by angiotensin II, ET-1, and various growth factors in cardiomyocytes, HB-EGF is known to induce cardiac hypertrophy via the PI3K-Akt, MAP kinase, and JAK-STAT pathways. However, little is known about the potential involvement of the ERK5 pathway in HB-EGF-induced cardiac hypertrophy. In the present report, we identify and characterize a novel MEK5-ERK5 pathway that is involved in HB-EGF-induced cardiomyocyte hypertrophy. HB-EGF (10ng/ml) significantly increased [(3)H]-leucine incorporation and atrial natriuretic factor (ANF) mRNA expression in H9c2 cells. In addition, HB-EGF activated a MEK5-ERK5 pathway. Pretreatment with the EGFR inhibitor AG1478 attenuated the activation of ERK5. Blockade of MEK5-ERK5 signaling using MEK5 siRNA reduced the ability of HB-EGF to increase cell size and the expression of ANF mRNA, suggesting the involvement of an EGFR-ERK5 pathway in HB-EGF-induced cardiomyocyte hypertrophy. We further analyzed cyclooxygenase-2 (COX-2). HB-EGF enhanced the expression of COX-2, a response mediated by MEK5-ERK5 signaling, while the COX-2 inhibitor rofecoxib attenuated HB-EGF-induced ANF mRNA expression, suggesting that COX-2 is also associated with HB-EGF-induced cardiomyocyte hypertrophy. It has been known that ERK5 activates the myocyte enhancer factor (MEF) 2 family of transcription factor, we next tested whether activation of MEF2A contributes to HB-EGF-induced COX-2 expression. Inhibition of MEF2A using siRNA attenuated HB-EGF-induced COX-2, ANF expression and cell size. In conclusion, HB-EGF induces cardiomyocyte hypertrophy through an EGFR-ERK5-MEF2A-COX-2 pathway. Our findings will help us to better understand the molecular mechanisms behind HB-EGF-induced cardiomyocyte hypertrophy.