Myeloid- and Epithelial-derived Heparin-Binding Epidermal Growth Factor-like Growth Factor Promotes Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a poorly understood, progressive lethal lung disease with no known cure. In addition to alveolar epithelial cell (AEC) injury and excessive deposition of extracellular matrix proteins, chronic inflammation is a hallmark of IPF. Literature suggests that the persistent inflammation seen in IPF primarily consists of monocytes and macrophages. Recent work demonstrates that monocyte-derived alveolar macrophages (moAMs) drive lung fibrosis, but further characterization of critical moAM cell attributes is necessary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an important epidermal growth factor receptor ligand that has essential roles in angiogenesis, wound healing, keratinocyte migration, and epithelial-mesenchymal transition. Our past work has shown HB-EGF is a primary marker of profibrotic M2 macrophages, and this study seeks to characterize myeloid-derived HB-EGF and its primary mechanism of action in bleomycin-induced lung fibrosis using Hbegff/f;Lyz2Cre+ mice. Here, we show that patients with IPF and mice with pulmonary fibrosis have increased expression of HB-EGF and that lung macrophages and transitional AECs of mice with pulmonary fibrosis and humans all express HB-EGF. We also show that Hbegff/f;Lyz2Cre+ mice are protected from bleomycin-induced fibrosis and that this protection is likely multifactorial, caused by decreased CCL2-dependent monocyte migration, decreased fibroblast migration, and decreased contribution of HB-EGF from AEC sources when HB-EGF is removed under the Lyz2Cre promoter.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments. RESULTS: Western blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway. CONCLUSION: This study increases the understanding of the physiological role of HB-EGF in human luteal functions. Video Abstract.

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 0.5 µM As(III) for 4 months, 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.

Stat5 phosphorylation is responsible for the excessive potency of HB-EGF.

Heparin-binding EGF-like growth factor (HB-EGF) is a potent growth factor involved in wound healing and tumorigenesis. Despite the sequence similarity between HB-EGF and EGF, HB-EGF induces cellular proliferation and migration more potently than EGF. However, the differential regulation by HB-EGF and EGF has not been thoroughly elucidated. In this study, we compared signaling pathways activated by HB-EGF and EGF to understand the details of the molecular mechanism of the high potency induced by HB-EGF. HB-EGF specifically induced the phosphorylation of EGFR-Y1045 and activated Stat5, which is responsible for promoting cell proliferation, and migration. The competition of phosphorylated EGFR-Y1045 inhibited Stat5 activation and consequently lowered the effect of HB-EGF on cell proliferation, suggesting that the phosphorylation of EGFR-Y1045 is essential for the activation of Stat5. The phosphorylation of EGFR-Y1045 and Stat5 induced by HB-EGF was prevented by sequestering the heparin-binding domain, suggesting that the heparin-binding domain is critical for HB-EGF-mediated signaling and cellular responses. In conclusion, the heparin-binding domain of HB-EGF was responsible for EGFR-mediated Stat5 activation, resulting in a more potent cellular proliferation, and migration than that mediated by EGF. This molecular mechanism is useful for understanding ligand-specific EGFR signaling and developing biomedicines for wound healing or cancer therapy.

Heparin-binding EGF-like growth factor attenuates lung inflammation and injury in a murine model of pulmonary emphysema.

Pulmonary inflammation and progressive lung destruction are the major causes of chronic obstructive pulmonary disease (COPD), resulting in emphysema and irreversible pulmonary dysfunction. Heparin-binding EGF-like growth factor (HB-EGF), is known to play a protective role in the process of various inflammatory diseases. However, its effect on COPD is poorly understood. This study was designed to determine the effect of HB-EGF on lung inflammation and injury in a murine model of pulmonary emphysema. HB-EGF promoted percent survival and body weight, attenuated lung injury, inflammatory cells, and cytokines infiltration, and prevented lung function decline. Additionally, treatment of rHB-EGF suppressed the nuclear translocation of nuclear factor κB (NF-κB)/p65, decreased TUNEL-positive cells and the expression of caspase 3, and increased the expression of PCNA, HB-EGF, and EGF receptor (EGFR). We conclude that HB-EGF attenuates lung inflammation and injury, probably through the activation of EGFR, followed by suppression of NF-ΚB signalling, promotion of cell proliferation, and inhibition of apoptosis.

Egr2 mediates the differentiation of mouse uterine stromal cells responsiveness to HB-EGF during decidualization.

Although Egr2 is involved in regulating the folliculogenesis and ovulation, there is almost no data describing its physiological function in embryo implantation and decidualization. Here, we showed that Egr2 mRNA was distinctly accumulated in subluminal stromal cells around implanting blastocyst on day 5 of pregnancy as well as in estrogen-activated implantation uterus. Estrogen induced the expression of Egr2 in uterine epithelia. Elevated expression of Egr2 mRNA was also observed in the decidual cells. Silencing of Egr2 by specific siRNA weakened the proliferation of uterine stromal cells and reduced the expression of Ccnd1, Ccnd3, Cdk4, and Cdk6. Furthermore, Egr2 advanced the expression of Prl8a2, Prl3c1, and Pgr, the well-established differentiation markers for decidualization. Administration of exogenous recombinant heparin-binding EGF-like growth factor (rHB-EGF) to uterine stromal cells resulted in an increase in the level of Egr2 mRNA. Moreover, siRNA-mediated attenuation of Egr2 impeded the stimulation of HB-EGF on stromal cell differentiation. Knockdown of Egr2 led to a reduction in the expression of Cox-2, mPGES-1, Vegf, Trp53, and Mmp2. Further analysis found that Egr2 may serve as an intermediate to mediate the regulation of HB-EGF on Cox-2, mPGES-1, Vegf, Trp53, Mmp2, and Ccnd3. Collectively, Egr2 may play an important role during embryo implantation and decidualization.

HB-EGF-Promoted Airway Smooth Muscle Cells and Their Progenitor Migration Contribute to Airway Smooth Muscle Remodeling in Asthmatic Mouse.

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.

Epithelial separation theory for post-tonsillectomy secondary hemorrhage: evidence in a mouse model and potential heparin-binding epidermal growth factor-like growth factor therapy.

OBJECTIVE: To provide histological evidence to investigate a theory for post-tonsillectomy secondary hemorrhage (PTH) in a mouse model and to evaluate the potential for heparin-binding epidermal growth factor-like growth factor (HB-EGF) treatment on wound healing in this model. METHODS: A prospective randomized single-blinded cohort study. A uniform tongue wound was created in 84 mice (day 0). Mice were randomized to HB-EGF (treatment, n = 42) or saline (control, n = 42). In treatment mice, HB-EGF 5 µg/ml was administered intramuscularly into the wound daily (days 0-14). In control mice, normal saline was administered daily. Three mice from each group were sacrificed daily through day 14 and the wounds evaluated histologically by blinded reviewers. RESULTS: Key stages of wound healing, including keratinocyte proliferation and migration, wound contraction, epithelial separation, and neoangiogenesis, are defined with implications for post-tonsillectomy wound healing. Epithelial separation (59 vs. 100%, p = 0.003) and wound reopening (8 vs. 48%, p < 0.001) were reduced with HB-EGF. Epithelial thickness (220 vs. 30 µm, p = 0.04) was greater with HB-EGF. Wound closure (days 4-5 vs. day 6, p = 0.01) occurred earlier with HB-EGF. CONCLUSIONS: In healing of oral keratinocytes on muscle epithelial separation secondary to muscle, contraction occurs concurrently with neoangiogenesis in the base of the wound, increasing the risk of hemorrhage. This potentially explains why post-tonsillectomy secondary hemorrhage occurs and its timing. HB-EGF-treated wounds showed greater epithelial thickness, less frequent epithelial separation and wound reopening, and earlier wound closure prior to neovascularization, suggesting that HB-EGF may be a potential preventative therapy for PTH. LEVEL OF EVIDENCE: NA-animal studies or basic research.

[Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer].

Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) µmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) µmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR protein (0.71±0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84±0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group (1.78±0.27) , MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79±0.13, P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5±3.1,100.4±7.4, and the pNA of the A2780/Taxol cells was (11.4±1.2) , (52.8±0.9) , (71.2±3.6) , (82.7±3.8) µmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4±1.4) were lower than that in A2780/Taxol tumors (10.2±3.1, P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8±1.1) were lower than that in A2780/Taxol tumors (8.8±2.7, P<0.05). Conclusion: CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas.

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.

M1 to M2 macrophage polarization in heparin-binding epidermal growth factor-like growth factor therapy for necrotizing enterocolitis.

BACKGROUND: Macrophages can be polarized into proinflammatory (M1) and anti-inflammatory (M2) subtypes. However, whether macrophage polarization plays a role in necrotizing enterocolitis (NEC) remains unknown. MATERIALS AND METHODS: Macrophages were derived from the THP-1 human monocyte cell line. Apoptosis of human fetal small intestinal epithelial FHs-74 cells was determined by Annexin V/propidium iodide flow cytometry and by Western blotting to detect cleaved caspase-3. The effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on macrophage polarization was determined by flow cytometry with M1/M2 markers and real time polymerase chain reaction. In vivo, experimental NEC was induced in mouse pups by repeated exposure to hypoxia, hypothermia, and hypertonic feedings. Intestinal histologic sections were subjected to immunohistochemical staining for the detection of M1 and M2 macrophages. RESULTS: In vitro, FHs-74 cell apoptosis was increased after coculture with macrophages and lipopolysaccharide (LPS). This apoptosis was increased by exposure to M1-conditioned medium and suppressed by exposure to M2-conditioned medium. HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via signal transducers and activators of transcription 3 activation. Addition of HB-EGF to LPS-stimulated macrophages suppressed the proapoptotic effects of the macrophages on FHs-74 cells. In vivo, we found enhanced intestinal macrophage infiltration in pups subjected to NEC, most of which were M1 macrophages. HB-EGF treatment of pups subjected to experimental NEC significantly reduced M1 and increased M2 polarization and protected the intestines from NEC. CONCLUSIONS: M1 macrophages promote NEC by increasing intestinal epithelial apoptosis. HB-EGF protects the intestines from NEC by preventing M1 and promoting M2 polarization.

SAH-induced MMP activation and K V current suppression is mediated via both ROS-dependent and ROS-independent mechanisms.

Voltage-gated potassium (K V) channels regulate cerebral artery tone and have been implicated in subarachnoid hemorrhage (SAH)-induced pathologies. Here, we examined whether matrix metalloprotease (MMP) activation contributes to SAH-induced K V current suppression and cerebral artery constriction via activation of epidermal growth factor receptors (EGFRs). Using patch clamp electrophysiology, we observed that K V currents were selectively decreased in cerebral artery myocytes isolated from SAH model rabbits. Consistent with involvement of enhanced MMP and EGFR activity in SAH-induced K V current suppression, we found that: (1) oxyhemoglobin (OxyHb) and/or the exogenous EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), failed to induce further K V current suppression after SAH and (2) gelatin zymography detected significantly higher MMP-2 activity after SAH. The removal of reactive oxygen species (ROS) by combined treatment with superoxide dismutase (SOD) and catalase partially inhibited OxyHb-induced K V current suppression. However, these agents had little effect on OxyHb-induced MMP-2 activation. Interestingly, in the presence of a broad-spectrum MMP inhibitor (GM6001), OxyHb failed to cause K V current suppression. These data suggest that OxyHb suppresses K V currents through both ROS-dependent and ROS-independent pathways involving MMP activation. The ROS-independent pathway involves activation of MMP-2, whereas the ROS-dependent pathway involves activation of a second unidentified MMP or ADAM (a disintegrin and metalloprotease domain).

MiR-760 targets HBEGF to control cartilage extracellular matrix degradation in osteoarthritis.

The present study was developed to explore whether microRNA (miR)-760 targets heparin-binding EGF-like growth factor (HBEGF) to control cartilage extracellular matrix degradation in osteoarthritis. Both miR-760 and HBEGF expression levels were analysed in human degenerative cartilage tissues and in interleukin (IL)-1ß/tumour necrosis factor (TNF)-α-treated chondrocytes in vitro. A series of knockdown and overexpression assays were then used to gauge the functional importance of miR-760 and HBEGF in OA, with qPCR and western immunoblotting analyses. Bioinformatics assays were used to identify putative miR-760 target genes, with these predictions then being validated through RNA pulldown and luciferase reporter assays. A murine anterior cruciate ligament transection model of OA was then established to prove the in vivo relevance of these findings. These experiments revealed that human degenerative cartilage tissues exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF levels. IL-1ß/TNF-α-treated chondrocytes also exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF expression. When chondrocytes were transfected with either miR-760 inhibitor or HBEGF overexpression constructs, this was sufficient to interfere with degradation of the extracellular matrix (ECM). Moreover, miR-760 was confirmed to control chondrocyte matrix homeostasis by targeting HBEGF, and the overexpression of HBEGF partially reversed the effects of miR-760 mimic treatment on the degradation of the cartilage ECM. When OA model mice were administered an intra-articular knee injection of an adenoviral vector encoding a miR-760 mimic construct, cartilage ECM degradation was aggravated. Conversely, the overexpression of HBEGF in OA model mice partially reversed the effects of miR-760 overexpression, restoring appropriate ECM homeostasis. In summary, these data indicated that the miR-760/HBEGF axis plays a central role in orchestrating the pathogenesis of OA, making it a candidate target for therapeutic efforts in OA.

HB-EGF induces mitochondrial dysfunction via estrogen hypersecretion in granulosa cells dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway.

Polycystic ovarian syndrome (PCOS) is one of the most prevalent endocrinopathies and the leading cause of anovulatory infertility, but its pathogenesis remains elusive. Although HB-EGF is involved in ovarian cancer progression, there is still no clarity about its relevance with PCOS. The present study exhibited that abundant HB-EGF was noted in follicular fluid from PCOS women, where it might induce the granulosa cells (GCs) production of more estrogen via the elevation of CYP19A1 expression after binding to EGFR. Furthermore, HB-EGF transduced intracellular downstream cAMP-PKA signaling to promote the phosphorylation of JNK and ERK whose blockage impeded the induction of HB-EGF on estrogen secretion. Meanwhile, HB-EGF enhanced the accumulation of intracellular Ca2+ whose chelation by BAPTA-AM abrogated the stimulation of HB-EGF on FOXO1 along with an obvious diminishment for estrogen production. cAMP-PKA-JNK/ERK-Ca2+ pathway played an important role in the crosstalk between HB-EGF and FOXO1. Treatment of GCs with HB-EGF resulted in mitochondrial dysfunction as evinced by the reduction of ATP content, mtDNA copy number and mitochondrial membrane potential. Additionally, HB-EGF facilitated the opening of mitochondrial permeability transition pore via targeting BAX and raised the release of cytochrome C from mitochondria into the cytosol to trigger the apoptosis of GCs, but this effectiveness was counteracted by estrogen receptor antagonist. Collectively, HB-EGF might induce mitochondrial dysfunction and GCs apoptosis through advancing estrogen hypersecretion dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway and act as a promising therapeutic target for PCOS.

High-throughput formation and image-based analysis of basal-in mammary organoids in 384-well plates.

This manuscript describes a new method for forming basal-in MCF10A organoids using commercial 384-well ultra-low attachment (ULA) microplates and the development of associated live-cell imaging and automated analysis protocols. The use of a commercial 384-well ULA platform makes this method more broadly accessible than previously reported hanging drop systems and enables in-incubator automated imaging. Therefore, time points can be captured on a more frequent basis to improve tracking of early organoid formation and growth. However, one major challenge of live-cell imaging in multi-well plates is the rapid accumulation of large numbers of images. In this paper, an automated MATLAB script to handle the increased image load is developed. This analysis protocol utilizes morphological image processing to identify cellular structures within each image and quantify their circularity and size. Using this script, time-lapse images of aggregating and non-aggregating culture conditions are analyzed to profile early changes in size and circularity. Moreover, this high-throughput platform is applied to widely screen concentration combinations of Matrigel and epidermal growth factor (EGF) or heparin-binding EGF-like growth factor (HB-EGF) for their impact on organoid formation. These results can serve as a practical resource, guiding future research with basal-in MCF10A organoids.

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans.

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

Myeloid Cell-Derived HB-EGF Drives Tissue Recovery After Pancreatitis.

BACKGROUND & AIMS: Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis. METHODS: To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbegff/f;LysM-Cre mice were used to ablate myeloid cell-derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfrf/f;Ptf1aFlpO/+;FSF-Rosa26CAG-CreERT2 mice followed by tamoxifen treatment. RESULTS: Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage. CONCLUSIONS: By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) protected intestinal ischemia-reperfusion injury through JNK and p38/MAPK-dependent pathway for anti-apoptosis.

BACKGROUND: Heparin-Binding Epidermal Growth Factor-Like Growth Factor (HB-EGF) is a potent cytoprotective factor in various body systems, including gastrointestinal tract. In this study, we intended to examine whether HB-EGF exerts its protective effects through MAPK dependent anti-apoptosis after intestinal I/R injury. METHODS: We randomly divided 30 laboratory 30 rats into 5 groups: (A) normal control group, (B) ischemia group with normal saline, (C) I/R group with normal saline, (D) ischemia group with HB-EGF (400 ug/kg), and (E) I/R group with HB-EGF (400 ug/kg). With Western blotting study, we determined JNK and p38/MAPK pathway and caspase-3 activity protein levels using Western analyses. RESULTS: The JNK phosphorylation protein levels increased after intestinal ischemia or intestinal reperfusion phase, and HB-EGF pre-treatment was significantly decreased in JNK phosphorylation protein levels (p < 0.01). We found that p38 protein levels was increased after intestinal reperfusion phase, and that HB-EGF pre-treatment significantly decreased p38 protein levels (p < 0.01). The expression protein level of caspase 3 was increased after intestinal ischemia or intestinal reperfusion phase. HB-EGF pre-treatment significantly decreased Caspase 3 proteins. (p < 0.01). CONCLUSION: Our study revealed that pre-treatment of HB-EGF decreased the amount of activity of JNK and p38/MAPK pathway and caspase-3 protein after intestinal I/R injury. These results may further support that the cytoprotective of HB-EGF after I/R injury could be through anti-apoptotic effect of activity of JNK and p38/MAPK pathway.

Engineering molecularly mobile polyrotaxane surfaces with heparin-binding EGF-like growth factors for improving hepatocyte functions.

Hepatocytes in vitro may be useful for treating various types of liver diseases, but these cells immediately lose their functions. Here, we designed sulfonated-polyrotaxane (PRX) surfaces with immobilized heparin binding-epidermal growth factor-like growth factors (HB-EGFs) for improving hepatic functions. Sulfonated-PRX triblock copolymers, composed of sulfopropyl ether-modified α-cyclodextrins (α-CDs) threaded onto a poly(ethylene glycol) (PEG) chain as a PRX segment and poly(benzyl methacrylate) at both terminals of the PEG as anchoring segments, were coated onto polystyrene surfaces by a drop cast method. The sulfonated-PRX surfaces with a small number of threading α-CDs induced cytoplasmic localization of yes-associated proteins in HepG2 cells. Moreover, immobilization of HB-EGFs onto the sulfonated-PRX surfaces with a small number of threading α-CDs promoted hepatic functions, including albumin secretion and gene expression. These results suggest that the combination of modulating the mobility of PRXs and immobilizing growth factors is effective for improving hepatic functions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1080-1085, 2019.