HB-EGF Research Articles

HB-EGF Improves the Hair Regenerative Potential of Adipose-Derived Stem Cells via ROS Generation and Hck Phosphorylation.

Although adipose-derived stem cells (ASCs) have hair regenerative potential, their hair inductive capabilities are limited. The mitogenic and hair inductive effects of heparin binding-epidermal growth factor-like growth factor (HB-EGF) on ASCs were investigated in this study and the underlying mechanism of stimulation was examined. Cell growth, migration, and self-renewal assays, as well as quantitative polymerase chain reactions and immunostaining, were carried out. Telogen-to-anagen transition and organ culture using vibrissa follicles were also conducted. HB-EGF significantly increased ASC motility, including cell proliferation, migration, and self-renewal activity. The preconditioning of ASCs with HB-EGF induced telogen-to-anagen transition more rapidly in vivo, and injected PKH26-ASCs survived for longer periods of time. Conditioned medium obtained from HB-EGF-treated ASCs promoted hair growth in vivo, upregulating growth factors. In particular, thrombopoietin (THPO) also induced hair growth in vivo, stimulating dermal papilla cells (DPCs). Reactive oxygen species (ROS) appeared to play a key role in ASC stimulation as the inhibition of ROS generation and NOX4 knockout attenuated ASC stimulation and THPO upregulation by HB-EGF. In addition, the Hck phosphorylation pathway mediated the stimulation of ASCs by HB-EGF. In summary, HB-EGF increased the motility and paracrine effects of ASCs releasing THPO growth factor and THPO promoted hair growth-stimulating DPCs. ROS generation and Hck phosphorylation are key factors in HB-EGF-induced ASC stimulation. Therefore, combination therapy involving HB-EGF and ASCs may provide a novel solution for hair-loss treatment.

Abstract C31: The glycosaminoglycan syndecan-4 facilitates pancreatic cancer progression and biologic aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a treatment-recalcitrant malignancy that harbors several key driver mutations at a high frequency (e.g., KRAS, TP53, CDKN2A, and SMAD4<), as well numerous driver mutations that occur at a relatively low and variable frequency among patients with PDAC. Heparan-sulfate proteoglycans (HSPGs) constitute a large family of glycanated proteins that modulate numerous cellular processes and interact with components of the extracellular matrix (ECM) and with heparin-binding growth factors such as heparin binding EGF-like growth factor (HBEGF) that activates the EGF receptor (EGFR) and transforming growth factor beta (TGF-beta) moieties. Syndecan-4 (SDC4) is a transmembrane HSPG that has critical roles in focal adhesion formation, cytoskeletal attachment, cell contractility and motility, and cell signaling, especially in relation to pathways activated by heparin-binding growth factors. To date, the role of SDC4 in PDAC has not been clearly elucidated. Yet, data from 1,034 PDAC samples in The Cancer Genome Atlas (TCGA) revealed that 19% of these samples exhibited altered SDC4 expression, a majority of which were either gain-of-function or amplification alterations often co-occurring with enhanced TGF-beta1 expression, thus underscoring the potential importance of SDC4 in PDAC. Given that TGF-beta1 increases SDC4 expression whereas SDC4 modulates TGF-beta1 actions and interacts with EGFR, we sought to test the hypothesis that SDC4 contributes to PDAC’s progression. Accordingly, we established a novel genetically engineered mouse model (GEMM) of PDAC in which pancreas-specific Cre-mediated expression of oncogenic Kras was combined with deletion of a conditional Ink4a/Arf allele in the presence or absence of SDC4: Pdx1-Cre;LSL-KrasG12D;Ink4a/Arflox/lox;Sdc4-/-, termed KIS4-/-C mice. By comparison with control KIS4 animals, KIS4-/-C mice exhibited attenuated pancreatic tumor growth and markedly prolonged survival. There was attenuation of cancer cell proliferation in the tumors, as determined by Ki67 staining, as well as decreased desmoplasia as evidenced by Mason trichrome staining. By contrast, KIS4+/-C animals with heterozygous loss of SDC4 only exhibited a modest survival advantage by comparison with control KIS4 animals. Taken together, these findings suggest that strategies based on suppressing SDC4 expression and/or function may yield new therapeutic approaches in PDAC. (Supported in part by National Cancer Institute grant CA-075059 to M.K.) Citation Format: Samantha McElyea, Murray Korc. The glycosaminoglycan syndecan-4 facilitates pancreatic cancer progression and biologic aggressiveness [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C31.

Sphingosine 1-phosphate but not Fingolimod protects neurons against excitotoxic cell death by inducing neurotrophic gene expression in astrocytes.

Sphingosine 1-phosphate (S1P) is an essential lipid metabolite that signals through a family of five G protein-coupled receptors, S1PR1-S1PR5, to regulate cell physiology. The multiple sclerosis drug Fingolimod (FTY720) is a potent S1P receptor agonist that causes peripheral lymphopenia. Recent research has demonstrated direct neuroprotective properties of FTY720 in several neurodegenerative paradigms; however, neuroprotective properties of the native ligand S1P have not been established. We aimed to establish the significance of neurotrophic factor up-regulation by S1P for neuroprotection, comparing S1P with FTY720. S1P induced brain-derived neurotrophic factor(BDNF), leukemia inhibitory factor (LIF), platelet-derived growth factor B (PDGFB), and heparin-binding EGF-like growth factor (HBEGF)gene expression in primary human and murine astrocytes, but not in neurons, and to a much greater extent than FTY720. Accordingly, S1P but not FTY720 protected cultured neurons against excitotoxic cell death in a primary murine neuron-glia coculture model, and a neutralizing antibody to LIF blocked this S1P-mediated neuroprotection. Antagonists of S1PR1and S1PR2both inhibited S1P-mediated neurotrophic geneinduction in human astrocytes, indicating that simultaneous activation of both receptors is required. S1PR2 signaling was transduced through Gα13 and the small GTPase Rho, and was necessary for the up-regulation and activation of the transcription factors FOS and JUN, which regulate LIF, BDNF, andHBEGF transcription. In summary, we show that S1P protects hippocampal neurons against excitotoxic cell death through up-regulation of neurotrophic gene expression, particularly LIF, in astrocytes. This up-regulation requires both S1PR1 and S1PR2 signaling. FTY720 does not activate S1PR2, explaining its relative inefficacy compared to S1P.

Circulating levels of inflammatory parameters pentraxin-3, cyclophilin and heparin-binding epidermal growth factor-like growth factor in patients with ST-elevation myocardial infarction

Inflammatory biomarkers – pentraxin-3 (PTX3), cyclophilin A (CypA) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) were examined in patients with ST-segment elevation myocardial infarction (STEMI) undergoing revascularization with primary percutaneous coronary intervention (pPCI) and stent implanting. Investigated parameters were compared between patients with and without obstructive coronary artery disease (CAD). In addition, their changes were tested in circulation before and immediately after pPCI. The study group consisted of 81 STEMI patients. Patients were classified in the STEMI-CAD group if they had significant obstructive CAD or in MINOCA group if they had no significant stenosis. In STEMI-CAD patients inflammatory parameters were determined prior to and after pPCI intervention. Immediately after pPCI, in STEMI-CAD patients levels of PTX3 were significantly lower (1.52 vs. 2.17 μg/L, p < .001), while the levels of HB-EGF (14.61 vs. 12.03 pg/L, p < .001) and CyPA (15.95 vs. 8.62 μg/L, p < .001) were significantly higher compared to levels before pPCI. STEMI–CAD patients had lower PTX3 values 2.17 μg/L (1.55–5.10 μg/L) than MINOCA patients 5.06 μg/L (2.77–6.7 μg/L), p = .046. Diagnostic accuracy of PTX3 for discrimination MINOCA from STEMI-CAD patients was low (area under receiver operating characteristic curve = 0.770). Evaluation of PTX3 values may be helpful in the understanding of MINOCA aetiology but they couldn’t distinguish stenosis severity in STEMI patients. Inflammatory biomarkers significantly changed after pPCI but the possibility of clinical use of these biomarkers needs to be evaluated in a larger prospective study.

Pomalidomide Reduces Bleeding and Alters Expression of Angiogenesis-Related Proteins in Patients with Hereditary Hemorrhagic Telangiectasia

Background: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder characterized by abnormal blood vessel formation, recurrent and severe epistaxis, gastrointestinal bleeding, as well as pulmonary, hepatic and cerebral arteriovenous malformation (AVM). The pathogenesis of HHT appears to involve dysregulation of TGF-β signaling in endothelial cells, though other mechanisms may also contribute. Elevated levels of VEGF have been described in some studies of HHT, though detailed molecular profiling of factors that regulate angiogenesis has not been performed, particularly before and after effective therapy. We have recently completed a pilot study of patients with HHT treated with pomalidomide that demonstrated safety and efficacy and suggested that pomalidomide may be a potential treatment option for this disorder (NCT02287558). Here, we describe the outcomes of this study and present correlative studies of potential biomarkers in the plasma of treated patients before, during and after treatment with pomalidomide. Methods: Eligible patients had 1) GI bleeding requiring transfusion of ≥ 4 units PRBC or 4 iron infusions, or 2) epistaxis with epistaxis severity score (ESS) ≥ 4 requiring ≥ 2 units PRBC or 500 mg intravenous iron, each within the preceding four months. The primary efficacy endpoint was a 50% reduction in parenteral iron infusion or blood transfusion. A key secondary endpoint was the effect of pomalidomide on the ESS. Treatment was initiated with 1 mg of pomalidomide daily, increasing by 1 mg/month to a maximum of 5 mg of daily. This dose, or a lower dose on which the patient had ceased bleeding was continued for four months and then tapered. The relative expression of 55 angiogenesis-related proteins was determined semi-quantitatively in the plasma of study subjects using the human angiogenesis proteome profiler array. In addition, the expression of seven angiogenesis-related proteins was determined quantitatively by electrochemiluminescence using a validated immunoassay (Meso Scale Discovery). Heparin-binding EGF-like growth factor (HB-EGF) levels were determined using a conventional sandwich ELISA. Results: Nine patients provided informed consent. One subject was not treated due to an intervening cardiac event. Two subjects developed a drug-related AE (rash) within three weeks of treatment initiation and were removed from the study, thus we report on 6 patients. Two subjects were on study for only 5 months and removed for non-drug related AEs; both had primarily GI bleeding. One of these met the primary endpoint while the other did not; however, this individual demonstrated a significant reduction in the ESS. The remaining four patients completed the study and all met the primary endpoint and demonstrated significant reductions in the ESS. Proteomic profiling of angiogenic biomarkers revealed a statistically significant basal elevation of vascular endothelial growth factor A (VEGF-A), VEGF-C, placental growth factor (PIGF), fibroblast growth factor-basic (FGF-basic) and heparin-binding EGF-like growth factor (HB-EGF) when compared to healthy, age matched controls. Levels of VEGF-D, soluble vascular endothelial growth factor receptor-1 (VEGFR1/Flt1), and tyrosine kinase-2 (Tie-2) exhibited a wide distribution at baseline, but overall were not significantly higher than those in healthy, matched controls. Responses to pomalidomide were associated with significant reductions of several angiogenic mediators including VEGF-A, VEGF-C, MMP8, MMP9, TIMP-4, Angiopoietin 1, and most notably HB-EGF. Conclusion: This pilot study conducted at the Cleveland Clinic HHT center provides demonstrates safety and efficacy of pomalidomide, a third generation thalidomide analogue, in patients with HHT. Differences in levels of several angiogenic mediators in patients HHT suggest a complex underlying pathogenesis with significant differences between individual patients. Angiogenic profiling indicates that pomalidomide treatment of HHT patients promotes the reduction of VEGF-A, VEGF-C, MMP8, MMP9, TIMP-4, Angiopoietin 1, and most notably HB-EGF, which may serve as potential biomarkers of pomalidomide efficacy. This study was supported by Celgene, Summit NJ Disclosures McCrae: Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Pomalidomide in Hereditary Hemorrhagic Telangiectasia

Chronic exposure to submicromolar arsenite promotes the migration of human esophageal Het1A cells induced by heparin-binding EGF-like growth factor.

Chronic arsenic exposure causes cancers in multiple organs in humans. However, the mechanisms underlying arsenic-induced carcinogenesis remain obscure. Here, we examined whether chronic arsenite (As(III)) exposure promotes cell migration induced by heparin-binding EGF-like growth factor (HB-EGF) in human esophageal immortalized Het1A cells. When Het1A cells were exposed to 0.5μM As(III) for 4months, HB-EGF-induced migration was enhanced in As(III)-exposed Het1A cells compared to controls. To elucidate the mechanisms underlying the promotion of HB-EGF-induced migration by chronic exposure to As(III), we compared ERK phosphorylation between As(III)-exposed and control Het1A cells and found that HB-EGF-induced ERK phosphorylation was enhanced in the As(III)-exposed cells. We next measured mRNA levels of 88 genes related to cell cycle regulation. The results showed elevated cyclin D1 mRNA levels in As(III)-exposed Het1A cells. The inhibitors of ERK and cyclin D/Cdk4 markedly suppressed HB-EGF-induced upregulation of cyclin D1 and the migration of Het1A cells, respectively, suggesting that cyclin D1 is located downstream of ERK and is required for HB-EGF-induced migration of Het1A cells. Collectively, these findings indicate that the promotion of HB-EGF-induced migration of Het1A cells chronically exposed to submicromolar As(III) might be caused by increased expression of cyclin D1 mediated by enhanced activation of the ERK pathway.

CRM197 reverses paclitaxel resistance by inhibiting the NAC-1/Gadd45 pathway in paclitaxel-resistant ovarian cancer cells.

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a new promising target for the treatment of ovarian cancer. Our previous study showed that cross‐reacting material 197 (CRM197), a specific HB‐EGF inhibitor, significantly reverses resistance against paclitaxel in paclitaxel‐resistant ovarian cancer cells. However, the mechanism of the effect of CRM197 on the reversion of paclitaxel resistance was unclear. In this study, in vitro and in vivo data suggested that CRM197 treatment sensitized paclitaxel‐resistant ovarian cancer cells to paclitaxel, at least in part, via nucleus accumbens‐1 (NAC‐1) and its downstream pathway, DNA damage‐inducible 45‐γ interacting protein (Gadd45gip1)/growth arrest and DNA damage‐inducible 45 (Gadd45), in A2780/Taxol and SKOV3/Taxol cells. The results also showed that CRM197 activated the proapoptotic JNK/p38MAPK pathway to enhance caspase‐3 activity and apoptosis by downregulation of the NAC‐1/Gadd45gip1/Gadd45 pathway, leading to reversion of paclitaxel resistance in A2780/Taxol and SKOV3/Taxol cells. This study provides the first mechanism through which CRM197 significantly reverses resistance against paclitaxel by modulating the NAC‐1/Gadd45gip1/Gadd45 pathway in paclitaxel‐resistant ovarian cancer cells, and the mechanism of HB‐EGF inhibition as a novel therapeutic strategy for patients with paclitaxel‐resistant ovarian cancer.

Peptides derived from a short stretch of diphtheria toxin bind to heparin-binding epidermal growth factor-like growth factor

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) is the receptor for diphtheria toxin (DT). Mutated or truncated, non-toxic DT has been used earlier for HB-EGF-targeted drug delivery and to modulate HB-EGF signaling. In the present work, we have synthesized a peptide corresponding to a 26 amino acid long stretch of the receptor-binding domain of DT. This region of DT makes multiple contacts with HB-EGF and has residues critical for binding to HB-EGF. We show that this peptide and two of its mutants bind to HB-EGF. We have also created recombinant proteins by fusing Maltose-binding Protein (MBP) with these peptides. These recombinant MBP-tagged peptides bind to HB-EGF with affinities in the range of 10−7 to 10−8 M. We have observed that these MBP-tagged peptides can modulate molecular signaling of HB-EGF. Therefore, this 26 amino acid long stretch of DT can be considered as an independent functional segment for binding to HB-EGF. Peptides corresponding to this region may be used for HB-EGF targeted cellular delivery of molecular cargo or to modulate HB-EGF signaling.

The expression and clinical significance of secretory leukocyte proteinase inhibitor (SLPI) in mammary carcinoma using bioinformatics analysis

BackgroundSecretory leukocyte protease inhibitor (SPLI) was a secreted protein which belongs to a member of whey acidic protein four-disulfide core family. In breast cancer (BC) it may inhibit cell proliferation and promote cancer metastasis. In this study, a comprehensive bioinformatics analysis was performed to identify the expression and prognostic value of SLPI in breast cancer. MethodsSLPI expression in breast cancer was analyzed in Oncomine online database, which was subsequently confirmed by quantitative PCR (qPCR) in 18 BC samples and western blotting in 26 BC samples. Breast cancer gene-expression miner v4.1 was used to access the expression level with clinicopathological parameters in breast cancer patients. The prognostic values of SLPI in breast cancer were evaluated using the PrognoScan database. ResultsOur results indicated that SLPI was downregulated in breast cancer than in normal tissues. SLPI expression was found to be negatively correlated with estrogen receptor (ER) and progesterone receptor (PR) status. SLPI expression level was decreased in negative basal-like status patients compared with positive basal-like status. Meanwhile, triple-negative breast cancer status positive correlated with SLPI. We confirmed a positive correlation between SLPI and interleukin 17 receptor B (IL17RB) express in breast cancer tissues via oncomine co-expression analysis. Ten proteins: Elastase, Granulin, Lipocalin, Defensin beta 103B, Defensin beta 103A, Tubulin, Heparin-binding EGF-like growth factor, Interleukin 6, Epidermal growth factor, Phospholipid scramblase 1 were determinate interactions with SLPI by STRING. ConclusionSLPI could as a biomarker to predict the prognosis values of breast cancer. However, further comprehensive study and mining more evidence are needed to clarify our results.

A nongenomic mechanism for "metalloestrogenic" effects of cadmium in human uterine leiomyoma cells through G protein-coupled estrogen receptor.

Cadmium (Cd) is a ubiquitous environmental metal that is reported to be a "metalloestrogen." Uterine leiomyomas (fibroids) are estrogen-responsive gynecologic neoplasms that can be the target of xenoestrogens. Previous epidemiology studies have suggested Cd may be associated with fibroids. We have shown that Cd can stimulate proliferation of human uterine leiomyoma (ht-UtLM) cells, but not through classical estrogen receptor (ER) binding. Whether nongenomic ER pathways are involved in Cd-induced proliferation is unknown. In the present study, by evaluating G protein-coupled estrogen receptor (GPER), ERα36, and phospho-epidermal growth factor receptor (EGFR) expression in human tissues, we found that GPER, ERα36 and phospho-EGFR wereallhighly expressed in fibroids compared to patient-matched myometrial tissues. In ht-UtLM cells, cell proliferation was increased by low doses of Cd (0.1µM and 10µM), and this effect could be inhibited by GPER-specific antagonist (G15) pretreatment, or silencing (si) GPER, but not by siERα36. Cd-activated MAPK was dependent on GPER/EGFR transactivation, through significantly increased phospho-Src, matrix metalloproteinase-2 (MMP2) and MMP9, and heparin-binding EGF-like growth factor (HB-EGF) expression/activation. Also, phospho-Src could interact directly to phosphorylate EGFR. Overall, Cd-induced proliferation of human fibroid cells was through a nongenomic GPER/p-src/EGFR/MAPK signaling pathway that did not directly involve ERα36. This suggests that Cd may be a risk factor for uterine fibroids through cross talk between hormone and growth factor receptorpathways.

Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor–Dependent Serrated Polyps in Mouse Cecum

Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genesthat regulate matrix fibroblasts and inflammation. In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

HIF-1α is Overexpressed in Odontogenic Keratocyst Suggesting Activation of HIF-1α and NOTCH1 Signaling Pathways.

Background: The odontogenic keratocyst (OKC) is an odontogenic cyst that shows aggressive and intriguing biological behavior. It is suggested that a hypoxic environment occurs in OKC, which led us to investigate the immunoexpression and location of hypoxia-inducible factor 1-alpha (HIF-1α) and other hypoxia-related proteins. Methods: Twenty cases of OKC were evaluated for the expression of Notch homolog 1 (NOTCH1), HIF-1α, disintegrin and metalloproteinase domain-containing protein 12 (ADAM-12), and heparin-binding epidermal growth factor-like growth factor (HBEGF) by immunohistochemistry and compared to eight control cases of calcifying odontogenic cystic (COC), orthokeratinized odontogenic cyst (OOC), and normal oral mucosa (OM) in basal and parabasal layers. Results: In OKC, all the proteins tested were expressed significantly higher in both basal (except for NOTCH1 and HBEGF in OOC) and suprabasal epithelial layers compared to controls. Looking at the epithelial layers within OKC, we observed an increased NOTCH1 and HIF-1α expression in parabasal layers. Conclusions: These results suggest that hypoxia occurs more intensively in OKC compared to COC, OM, and OOC. Hypoxia appeared to be stronger in parabasal layers as observed by higher HIF-1α expression in upper cells. Overexpression of NOTCH1, ADAM-12, and HBEGF in OKC was observed, which suggests that microenvironmental hypoxia could potentially regulate the expression of hypoxia-related proteins, and consequently, its clinical and biological behavior.

Anti-Factor XII Autoantibodies in Patients with Recurrent Pregnancy Loss Recognize the Second Epidermal Growth Factor-Like Domain.

Summary Background Factor XII (FXII) deficiency and autoantibodies that bind to FXII (anti-FXII) have been described in patients with adverse pregnancy outcomes, including recurrent pregnancy loss. It has been reported that FXII functions not only as a coagulation protein but also as a growth factor. Objectives We studied the association between anti-FXII and the epidermal growth factor (EGF) system in patients with recurrent pregnancy loss. Patients/Methods We used synthetic peptides that span the second EGF-like domain in the heavy chain of FXII (EGF2) in inhibition and direct binding studies to determine if anti-FXII antibodies recognize EGF2. Furthermore, we examined whether anti-FXII antibodies, which recognize EGF2, also recognize recombinant EGF and heparin-binding EGF-like growth factor (HB-EGF). Results Among 100 patients with recurrent pregnancy loss, the plasma of 23 patients (23.0%) recognized the synthetic peptide ASQ41, which covers EGF2. Among the 23 anti-ASQ41-positive patients, plasma samples from 13 patients (56.5%) recognized the 22-residue segment C-terminal half of ASQ41. Among the 23 anti-ASQ41-positive patients, the plasma of 17 patients (73.9%) recognized recombinant human EGF. Affinity-purified anti-FXII antibodies, which recognize ASQ41, also recognized recombinant EGF family proteins such as EGF and HB-EGF. Conclusions The autoantibodies in patients with recurrent pregnancy loss recognized the EGF2 domain in FXII and other proteins of the EGF family. Since proteins in the EGF family play an important role in normal pregnancy, autoantibody-associated disruption of the EGF system may cause pregnancy loss.

2196-P: HB-EGF Signaling Is Required for Glucose-Induced Pancreatic ß-Cell Proliferation in Rats

Background: Glucose is a major β-cell mitogen. Despite recent progress, the underlying mechanisms remain unclear. In a rat model of nutrient excess we previously showed that nutrient-induced β-cell proliferation is blocked when either EGF receptor (EGFR) or mTOR signaling is inhibited. Parallel transcriptomic analyses identified the EGFR ligand, HB-EGF as a potential mediator of nutrient-induced β-cell proliferation. Objective: To determine the role of HB-EGF in glucose-induced β-cell proliferation. Methods: HB-EGF mRNA levels were assessed by real-time PCR in isolated rat islets following a 24-h exposure to 2.8 or 16.7 mM glucose. The Carbohydrate-Responsive Element-Binding Protein (ChREBP) transcription factor was down-regulated by siRNA in dispersed rat islets. For β-cell proliferation studies islets were exposed to 16.7 mM glucose or HB-EGF (100 ng/ml) in the presence of 2.8 mM glucose for 72 h. Islets were co-cultured with the EGFR inhibitor AG1478 (300 nM) or the HB-EGF inhibitor CRM197 (10 ug/ml). shRNA was used to knockdown HB-EGF in isolated islets and either cultured ex vivo or transplanted under the kidney capsule of glucose-infused rats. β-cell proliferation was assessed by immunohistochemistry for Ki67 and insulin. Results: Glucose increased HB-EGF mRNA levels and this was prevented by ChREBP knockdown. HB-EGF potently stimulated β-cell proliferation. Inhibition of the EGFR or HB-EGF completely blocked not only the proliferative response to HB-EGF but also the response to 16.7 mM glucose. Knockdown of HB-EGF blocked the β-cell proliferative response to glucose in isolated rat islets as well as in transplanted islets. Conclusion: HB-EGF is a potent β-cell mitogen in rat islets. Glucose increases HB-EGF gene expression via ChREBP. The proliferative response to glucose requires an intact HB-EGF - EGFR pathway. Our findings identify a novel player in the complex mechanisms controlling β-cell proliferation in response to glucose. Disclosure H. Maachi: None. D. Scott: None. J. Ghislain: None. V. Poitout: None. Funding National Institutes of Health; Fonds de recherche du Québec-Santé

MiR-376c-3p targets heparin-binding EGF-like growth factor (HBEGF) to inhibit proliferation and invasion in medullary thyroid carcinoma cells.

IntroductionAggressive medullary thyroid carcinomas (MTC) have a high mortality rate and the treatment for patients diagnosed with advanced MTC is comparatively ineffective. We hence aimed to test the effects of miR-376c-3p on MTC and to explore the relevant mechanism.Material and methodsCell Counting Kit-8 (CCK-8) and soft agar colony formation assay were applied to evaluate the proliferation of transfected MZ-CRC-1 cells. Wound healing and transwell assay were employed to evaluate MTC cell migration and invasion, respectively. Luciferase assay was performed to validate the downstream target of miR-376c-3p in MZ-CRC-1 cells. Quantitative polymerase chain reaction was used to detect mRNA abundance of key genes. Western blot technique was used to analyze protein levels of HBEGF, E-cadherin, ZO-1, N-cadherin and vimentin.ResultsMiR-376c-3p inhibited the viability, migration and invasion of MZ-CRC-1 cells. Moreover, miR-376c-3p mimic downregulated expression of N-cadherin and vimentin but upregulated that of E-cadherin and ZO-1 in MZ-CRC-1 cells. Results for the luciferase reporter assay showed that miR-376c-3p was able to bind the 3′ untranslated region of heparin-binding EGF-like growth factor (HBEGF), of which overexpression nearly nullified the miR-376c-3p mimic-induced inhibitory effects in the MTC cells.ConclusionsMiR-376c-3p showed suppressive effects on MZ-CRC-1 cells via targeting and downregulating HBEGF, suggesting that miR-376c-3p could potentially be targeted for the treatment of MTC.

Novel protein-lipid factors in the pathomechanism of mild preeclampsia

The aim of this review was discussion on the new protein and lipid factors and their role in the pathomechanism of preeclampsia. Sphingolipids discussed in the review: Sph (sphingosine), S1P (sphingosine- 1-phosphate), C20-Cer (ceramides containing arachidic acid), C18-Cer (ceramides containing stearic acid), C16-Cer (ceramides containing palmitic acid), C18: 1-Cer (ceramides containing oleic acid), C22-Cer (ceramide containing behenic acid) and C24: 1-Cer (ceramides containing nervonic acid). Angiogenic factors discussed in the review: Hb-EGF (heparin-binding EGF-like growth factor), HGF (hepatocyte growth factor), LIF (leukaemia inhibitory factor), leptin, PDGF-BB (platelet-derived growth factor BB) and follistatin. Based on literature findings, it seems that protein and lipid factors may play an important role in the pathogenesis of preeclampsia, and there are many proteins and lipids that have not been studied in plasma of women with PE to date. We showed a few new pathways in pathomechanism of preeclampsia.

Astrocyte-to-astrocyte contact and a positive feedback loop of growth factor signaling regulate astrocyte maturation.

Astrocytes are critical for the development and function of the central nervous system. In developing brains, immature astrocytes undergo morphological, molecular, cellular, and functional changes as they mature. Although the mechanisms that regulate the maturation of other major cell types in the central nervous system such as neurons and oligodendrocytes have been extensively studied, little is known about the cellular and molecular mechanisms that control astrocyte maturation. Here, we identified molecular markers of astrocyte maturation and established an in vitro assay for studying the mechanisms of astrocyte maturation. Maturing astrocytes in vitro exhibit similar molecular changes and represent multiple molecular subtypes of astrocytes found in vivo. Using this system, we found that astrocyte‐to‐astrocyte contact strongly promotes astrocyte maturation. In addition, secreted signals from microglia, oligodendrocyte precursor cells, or endothelial cells affect a small subset of astrocyte genes but do not consistently change astrocyte maturation. To identify molecular mechanisms underlying astrocyte maturation, we treated maturing astrocytes with molecules that affect the function of tumor‐associated genes. We found that a positive feedback loop of heparin‐binding epidermal growth factor‐like growth factor (HBEGF) and epidermal growth factor receptor (EGFR) signaling regulates astrocytes maturation. Furthermore, HBEGF, EGFR, and tumor protein 53 (TP53) affect the expression of genes important for cilium development, the circadian clock, and synapse function. These results revealed cellular and molecular mechanisms underlying astrocytes maturation with implications for the understanding of glioblastoma.

Role and mechanism of the regulation of nuclear factor-κB by heparin binding-epidermal growth factor-like growth factor in the induction of paclitaxel resistance of ovarian cancer

Objective: To investigate the role and mechanism of the regulation of nuclear factor-κB (NF-κB) by heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel resistance of ovarian cancer in vitro and in vivo. Methods: (1) The detection of NF-κB expression: parental (A2780) and paclitaxel-resistant (A2780/Taxol) ovarian carcinoma cells were divided into four groups, named A2780 group, A2780+cross-reacting material 197 (CRM197, HB-EGF inhibitor) group, A2780/Taxol group and A2780/Taxol+CRM197 group. Among four groups, the expression level HB-EGF and epidermal growth factor receptor (EGFR) were examined by immunofluorescence double staining on confocal microscopy. Western blot was used to detect the expression level of NF-κB. In vivo, A2780 and A2780/Taxol cells were injected intraperitoneally to nude mouse to determine the expression level of NF-κB of the tumors from these four groups by immunohistochemistry method. (2) The detection on the function of NF-κB: A2780/Taxol cells were divided into four groups, named transfected with empty vector+saline group, NF-κB small interference RNA (siRNA)+saline group, empty vector+CRM197 group and NF-κB siRNA+CRM197 group respectively. Among four groups, the 50% inhibitory concentrations (IC(50)) of A2780/Taxol cells to paclitaxel, the expression level of plasma membrane glycoprotein (P-gp) and the effect of intracellular rhodomine123 (Rh123) accumulation were detected. Results: (1) The detection of NF-κB expression: the expression scores of HB-EGF protein among four groups were 5.6±1.3, 2.1±1.2, 11.7±3.5 and 6.2±1.4; the expression scores of EGFR protein were 5.1±1.6, 2.8±0.6, 10.4±3.1 and 5.6±1.9, respectively. The expression levels of NF-κB protein in the cells of the group named A2780, A2780+CRM197, A2780/Taxol and A2780/Taxol+CRM197 group were 1.89±0.23, 0.74±0.12, 3.45±0.16 and 1.31±0.08, respectively; the expression scores of NF-κB protein in the tissue tumors from four groups were 3.3±1.1, 1.4±0.4, 8.7±2.3 and 3.6±1.2, respectively. The expression level of HB-EGF, EGFR and NF-κB protein between A2780 and A2780/Taxol groups in vivo and in vitro were higher than these in A2780+CRM197 and A2780/Taxol+CRM197 group, while the expression level of HB-EGF, EGFR and NF-κB protein in A2780 group were lower than those in A2780/Taxol groups in vivo and in vitro (P<0.05). (2) The examination of NF-κB function: the IC(50) of A2780/Taxol cells to paclitaxel in groups transfected with empty vector+saline, NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were respectively (39.4±0.8), (7.6±0.6), (6.7±0.5) and (4.2±0.4) μmol/L, while the expression levels of P-gp protein among four groups were respectively 3.11±0.23,1.45±0.16, 1.73±0.21 and 0.68±0.14, the cellular Rh123 accumulation among four groups were respectively 110±15, 246±19, 231±22 and 296±24. The expression levels of IC(50) and P-gp protein in groups transfected with NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were significantly higher than those in group transfected with empty vector+saline group (P<0.01), while the cellular Rh123 accumulation among three groups were significantly lower than that in group transfected with empty vector+saline (P<0.01). Conclusions: The expression of NF-κB may contributes to the paclitaxel resistance to ovarian cancer. HB-EGF may induce the paclitaxel resistance of ovarian cancer by the regulation of EGFR/NF-κB/P-gp pathway.

The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification.

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.

Discovery of HB-EGF binding peptides and their functional characterization in ovarian cancer cell lines

Ovarian cancer is one of the most frequent causes of cancer death among all gynecologic cancers. Though standard therapy often results in temporary clinical remission, most patients suffer from recurrence and metastasis of ovarian cancer, which highlights the need for developing new therapeutic agents targeting specific molecules. Previous studies have demonstrated that the native ligand of epidermal growth factor receptor (EGFR) and ErbB4, heparin-binding EGF-like growth factor (HB-EGF), plays a critical role in the progression of ovarian cancer and is associated with prognosis of ovarian cancer. In the current study, we tried to develop a peptide-based treatment for ovarian cancer by targeting HB-EGF. After the functions of HB-EGF in promoting migration and invasion of SKOV3 and HO-8910 cells were confirmed, phage display was used to discover peptides binding to HB-EGF. Two peptides, no. 7 and no. 29 were found mildly binding to HB-EGF. Then the effects of these peptides on HB-EGF functions were examined and both peptides no. 7 and no. 29 were found indeed inhibiting the functions of HB-EGF in promoting migration and invasion of SKOV3 and HO-8910 cells in vitro. Further mechanism investigation showed that peptides no. 7 and no. 29 inhibited HB-EGF-promoted cell migration and invasion through attenuating activation of the EGFR signaling pathway manifested by decreased p-Erk1/2 and Snail levels. More importantly, peptides no. 7 and no. 29 showed strong activities in inhibiting migration of SKOV3 cells in vivo. These results provide a proof of concept method for developing novel peptide drugs to combat ovarian cancer through interfering with HB-EGF mediated signaling pathways.