Lactate-Induced HBEGF Shedding and EGFR Activation: Paving the Way to a New Anticancer Therapeutic Opportunity.

Cancer cells can release EGF-like peptides, acquiring the capacity of autocrine stimulation via EGFR-mediated signaling. One of these peptides (HBEGF) was found to be released from a membrane-bound precursor protein and is critically implicated in the proliferative potential of cancer cells. We observed that the increased lactate levels characterizing neoplastic tissues can induce the release of uPA, a protease promoting HBEGF shedding. This effect led to EGFR activation and increased ERK1/2 phosphorylation. Since EGFR-mediated signaling potentiates glycolytic metabolism, this phenomenon can induce a self-sustaining deleterious loop, favoring tumor growth. A well characterized HBEGF inhibitor is CRM197, a single-site variant of diphtheria toxin. We observed that, when administered individually, CRM197 did not trigger evident antineoplastic effects. However, its association with a uPA inhibitor caused dampening of EGFR-mediated signaling and apoptosis induction. Overall, our study highlights that the increased glycolytic metabolism and lactate production can foster the activated state of EGFR receptor and suggests that the inhibition of EGFR-mediated signaling can be attempted by means of CRM197 administered with an appropriate protease inhibitor. This attempt could help in overcoming the problem of the acquired resistance to the conventionally used EGFR inhibitors.

HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures.

Hippocampal seizures mimicking mesial temporal lobe epilepsy cause a profound disruption of the adult neurogenic niche in mice. Seizures provoke neural stem cells to switch to a reactive phenotype (reactive neural stem cells, React-NSCs) characterized by multibranched hypertrophic morphology, massive activation to enter mitosis, symmetric division, and final differentiation into reactive astrocytes. As a result, neurogenesis is chronically impaired. Here, using a mouse model of mesial temporal lobe epilepsy, we show that the epidermal growth factor receptor (EGFR) signaling pathway is key for the induction of React-NSCs and that its inhibition exerts a beneficial effect on the neurogenic niche. We show that during the initial days after the induction of seizures by a single intrahippocampal injection of kainic acid, a strong release of zinc and heparin-binding epidermal growth factor, both activators of the EGFR signaling pathway in neural stem cells, is produced. Administration of the EGFR inhibitor gefitinib, a chemotherapeutic in clinical phase IV, prevents the induction of React-NSCs and preserves neurogenesis.

A subpopulation of tissue remodeling monocytes stimulates revascularization of the ischemic limb.

Despite decades of effort aimed at developing clinically effective cell therapies, including mixed population mononuclear cells, to revascularize the ischemic limb, there remains a paucity of patient-based studies that inform the function and fate of candidate cell types. In this study, we showed that circulating proangiogenic/arteriogenic monocytes (PAMs) expressing the FcγIIIA receptor CD16 were elevated in patients with chronic limb-threatening ischemia (CLTI), and these amounts decreased after revascularization. Unlike CD16-negative monocytes, PAMs showed large vessel remodeling properties in vitro when cultured with endothelial cells and smooth muscle cells and promoted salvage of the ischemic limb in vivo in a mouse model of hindlimb ischemia. PAMs showed a propensity to migrate toward and bind to ischemic muscle and to secrete angiogenic/arteriogenic factors, vascular endothelial growth factor A (VEGF-A) and heparin-binding epidermal growth factor. We instigated a first-in-human single-arm cohort study in which autologous PAMs were injected into the ischemic limbs of five patients with CLTI. Greater than 25% of injected cells were retained in the leg for at least 72 hours, of which greater than 80% were viable, with evidence of enhanced large vessel remodeling in the injected muscle area. In summary, we identified up-regulation of a circulatory PAM subpopulation as an endogenous response to limb ischemia in CLTI and tested a potentially clinically relevant therapeutic strategy.

LMWH prevents thromboinflammation in the placenta via HBEGF-AKT signaling.

ABSTRACT: Low molecular weight heparins (LMWH) are used to prevent or treat thromboembolic events during pregnancy. Although studies suggest an overall protective effect of LMWH in preeclampsia (PE), their use in PE remains controversial. LMWH may convey beneficial effects in PE independent of their anticoagulant activity, possibly by inhibiting inflammation. Here, we evaluated whether LMWH inhibit placental thromboinflammation and trophoblast NLRP3 inflammasome activation. Using an established procoagulant extracellular vesicle-induced and platelet-dependent PE-like mouse model, we show that LMWH reduces pregnancy loss and trophoblast inflammasome activation, restores altered trophoblast differentiation, and improves trophoblast proliferation in vivo and in vitro. Moreover, LMWH inhibits platelet-independent trophoblast NLRP3 (NLR family pyrin domain containing 3) inflammasome activation. Mechanistically, LMWH activates via heparin-binding epidermal growth factor (HBEGF) signaling the PI3-kinase-AKT pathway in trophoblasts, thus preventing inflammasome activation. In human PE placental explants, inflammasome activation and PI3-kinase-AKT signaling events were reduced with LMWH treatment compared with those without LMWH treatment. Thus, LMWH inhibits sterile inflammation via the HBEGF signaling pathway in trophoblasts and ameliorates PE-associated complications. These findings suggest that drugs targeting the inflammasome may be evaluated in PE and identify a signaling mechanism through which LMWH ameliorates PE, thus providing a rationale for the use of LMWH in PE.

Novel IL-4/HB-EGF-dependent crosstalk between eosinophils and macrophages controls liver regeneration after ischaemia and reperfusion injury.

OBJECTIVE: Previous studies indicate that eosinophils are recruited into the allograft following orthotopic liver transplantation and protect from ischaemia reperfusion (IR) injury. In the current studies, we aim to explore whether their protective function could outlast during liver repair. DESIGN: Eosinophil-deficient mice and adoptive transfer of bone marrow-derived eosinophils (bmEos) were employed to investigate the effects of eosinophils on tissue repair and regeneration after hepatic IR injury. Aside from exogenous cytokine or neutralising antibody treatments, mechanistic studies made use of a panel of mouse models of eosinophil-specific IL-4/IL-13-deletion, cell-specific IL-4rα-deletion in liver macrophages and hepatocytes and macrophage-specific deletion of heparin-binding epidermal growth factor-like growth factor (hb-egf). RESULT: We observed that eosinophils persisted over a week following hepatic IR injury. Their peak accumulation coincided with that of hepatocyte proliferation. Functional studies showed that eosinophil deficiency was associated with a dramatic delay in liver repair, which was normalised by the adoptive transfer of bmEos. Mechanistic studies demonstrated that eosinophil-derived IL-4, but not IL-13, was critically involved in the reparative function of these cells. The data further revealed a selective role of macrophage-dependent IL-4 signalling in liver regeneration. Eosinophil-derived IL-4 stimulated macrophages to produce HB-EGF. Moreover, macrophage-specific hb-egf deletion impaired hepatocyte regeneration after IR injury. CONCLUSION: Together, these studies uncovered an indispensable role of eosinophils in liver repair after acute injury and identified a novel crosstalk between eosinophils and macrophages through the IL-4/HB-EGF axis.

Can the HB-EGF/EGFR pathway restore injured neurons?

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a transmembrane protein that, when cleaved by metalloproteases through a process called ectodomain shedding, binds to the EGF receptor (EGFR), activating downstream signaling. The HB-EGF/EGFR pathway is crucial in development and is involved in numerous pathophysiological processes. In this issue of The FEBS Journal, Sireci et al. reveal a previously unexplored function of the HB-EGF/EGFR pathway in promoting neuronal progenitor proliferation and sensory neuron regeneration in the zebrafish olfactory epithelium in response to injury.

The Multiple Functions of HB-EGF in Female Reproduction and Related Cancer: Molecular Mechanisms and Targeting Strategies.

Heparin-binding growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) ligand family which has a crucial role in women's health. However, there is a lack of comprehensive review to summarize the significance of HB-EGF. Therefore, this work first described the expression patterns of HB-EGF in the endometrium and ovary of different species and gestational time. Then, the focus was on exploring how it promotes the successful implantation and regulates the process of decidualization and the function of ovarian granulosa cells as an intermediate molecule. Otherwise, we also focused on the clinical and prognostic significance of HB-EGF in female-related cancers (including ovarian cancer, cervical cancer, and endometrial cancer) and breast cancer. Lastly, the article also summarizes the current drugs targeting HB-EGF in the treatment of ovarian cancer and breast cancer. Overall, these studies found that the expression of HB-EGF in the endometrium is spatiotemporal and species-specific. And it mediates the dialogue between the blastocyst and endometrium, promoting synchronous development of the blastocyst and endometrium as an intermediate molecule. HB-EGF may serve as a potentially valuable prognostic clinical indicator in tumors. And the specific inhibitor of HB-EGF (CRM197) has a certain anti-tumor ability, which can exert synergistic anti-tumor effects with conventional chemotherapy drugs. However, it also suggests that more research is needed in the future to elucidate its specific mechanisms and to accommodate clinical studies with a larger sample size to clarify its clinical value.

Role of an interdependent Wnt, GSK3-ß/ß-catenin and HB-EGF/EGFR mechanism in arsenic-induced hippocampal neurotoxicity in adult mice.

We previously reported the neurotoxic effects of arsenic in the hippocampus. Here, we explored the involvement of Wnt pathway, which contributes to neuronal functions. Administering environmentally relevant arsenic concentrations to postnatal day-60 (PND60) mice demonstrated a dose-dependent increase in hippocampal Wnt3a and its components, Frizzled, phospho-LRP6, Dishevelled and Axin1 at PND90 and PND120. However, p-GSK3-ß(Ser9) and ß-catenin levels although elevated at PND90, decreased at PND120. Additionally, treatment with Wnt-inhibitor, rDkk1, reduced p-GSK3-ß(Ser9) and ß-catenin at PND90, but failed to affect their levels at PND120, indicating a time-dependent link with Wnt. To explore other underlying factors, we assessed epidermal growth factor receptor (EGFR) pathway, which interacts with GSK3-ß and appears relevant to neuronal functions. We primarily found that arsenic reduced hippocampal phosphorylated-EGFR and its ligand, Heparin-binding EGF-like growth factor (HB-EGF), at both PND90 and PND120. Moreover, treatment with HB-EGF rescued p-GSK3-ß(Ser9) and ß-catenin levels at PND120, suggesting their HB-EGF/EGFR-dependent regulation at this time point. Additionally, rDkk1, LiCl (GSK3-ß-activity inhibitor), or ß-catenin protein treatments induced a time-dependent recovery in HB-EGF, indicating potential inter-dependent mechanism between hippocampal Wnt/ß-catenin and HB-EGF/EGFR following arsenic exposure. Fluorescence immunolabeling then validated these findings in hippocampal neurons. Further exploration of hippocampal neuronal survival and apoptosis demonstrated that treatment with rDkk1, LiCl, ß-catenin and HB-EGF improved Nissl staining and NeuN levels, and reduced cleaved-caspase-3 levels in arsenic-treated mice. Supportively, we detected improved Y-Maze and Passive Avoidance performances for learning-memory functions in these mice. Overall, our study provides novel insights into Wnt/ß-catenin and HB-EGF/EGFR pathway interaction in arsenic-induced hippocampal neurotoxicity.

HB-EGF-loaded nanovesicles enhance trophectodermal spheroid attachment and invasion.

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential. We have previously established generation of cell-derived nanovesicles (NVs) from human trophectodermal cells (hTSCs) and their capacity to reprogram endometrial cells to enhance adhesion and blastocyst outgrowth. Here, we employed a rapid NV loading strategy to encapsulate potent implantation molecules such as HB-EGF (NVHBEGF). We show these loaded NVs elicit EGFR-mediated effects in recipient endometrial cells, activating kinase phosphorylation sites that modulate their activity (AKT S124/129, MAPK1 T185/Y187), and downstream signalling pathways and processes (AKT signal transduction, GTPase activity). Importantly, they enhanced target cell attachment and invasion. The phosphoproteomics and proteomics approach highlight NVHBEGF-mediated short-term signalling patterns and long-term reprogramming capabilities on endometrial cells which functionally enhance trophectodermal-endometrial interactions. This proof-of-concept study demonstrates feasibility in enhancing the functional potency of NVs in the context of embryo implantation.

MicroRNA-194-5p/Heparin-binding EGF-like growth factor signaling mediates dexamethasone-induced activation of pseudorabies virus in rat pheochromocytoma cells.

Pseudorabies virus (PRV) is a neurotropic virus, which infects a wide range of mammals. The activity of PRV is gradually suppressed in hosts that have tolerated the primary infection. Increased glucocorticoid levels resulting from stressful stimuli overcome repression of PRV activity. However, the host cell mechanism involved in the activation processes under stressful conditions remains unclear. In this study, infection of rat PC-12 pheochromocytoma cells with neuronal properties using PRV at a multiplicity of infection (MOI) = 1 for 24 h made the activity of PRV be the relatively repressed state, and then incubation with 0.5 µM of the corticosteroid dexamethasone (DEX) for 4 h overcomes the relative repression of PRV activity. RNA-seq deep sequencing and bioinformatics analyses revealed different microRNA and mRNA profiles of PC-12 cells with/without PRV and/or DEX treatment. qRT-PCR and western blot analyses confirmed the negative regulatory relationship of miRNA-194-5p and its target heparin-binding EGF-like growth factor (Hbegf); a dual-luciferase reporter assay revealed that Hbegf is directly targeted by miRNA-194-5p. Further, miRNA-194-5p mock transfection contributed to PRV activation, Hbegf was downregulated in DEX-treated PRV infection cells, and Hbegf overexpression contributed to returning activated PRV to the repression state. Moreover, miRNA-194-5p overexpression resulted in reduced levels of HBEGF, c-JUN, and p-EGFR, whereas Hbegf overexpression suppressed the reduction caused by miRNA-194-5p overexpression. Overall, this study is the first to report that changes in the miR-194-5p-HBEGF/EGFR pathway in neurons are involved in DEX-induced activation of PRV, laying a foundation for the clinical prevention of stress-induced PRV activation.

Spinal cord repair is modulated by the neurogenic factor Hb-egf under direction of a regeneration-associated enhancer.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.

An unanticipated discourse of HB-EGF with VANGL2 signaling during embryo implantation.

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo-uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

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.

The regulation and function of acetylated high-mobility group box 1 during implantation and decidualization.

Introduction: High-mobility group box 1 (HMGB1) is a non-histone nuclear protein and can be extracellularly secreted to induce sterile inflammation. Although uterine deletion of HMGB1 causes implantation and decidualization defects, how secreted HMGB1 is involved in mouse early pregnancy is still unknown. Methods: Mouse models, mouse primary endometrial cells and human endometrial cell lines were used in this study. Both immunofluorescence and Western blot were performed to show the localization and relative level of HMGB1 and acetylated HMGB1, respectively. Relative mRNA levels were analyzed by real time RT-PCR. Results: The secreted HMGB1 was detected in uterine lumen fluid in mouse periimplantation uterus. There is an obvious difference for secreted HMGB1 levels in uterine fluid between day 4 of pregnancy and day 4 of pseudopregnancy, suggesting the involvement of blastocysts during HMGB1 secretion. Trypsin is clearly detected in mouse blastocyst cavity and in the supernatant of cultured blastocysts. Trypsin significantly stimulates HB-EGF production through activating PAR2 and ADAM17. Uterine injection of PAR2 inhibitor into day 4 pregnant mice significantly reduces the number of implantation sites. HB-EGF released from luminal epithelium can induce mouse in vitro decidualization. The conditioned medium collected from trypsin-treated luminal epithelium is able to induce in vitro decidualization, which is suppressed by EGFR inhibitor. Intrauterine injection of glycyrrhizin (HMGB1 inhibitor) can significantly inhibit mouse embryo implantation. We also showed that exogenous HMGB1 released from human epithelial cells are able to induce human in vitro decidualization. Conclusion: Trypsin can induce decidualization of stromal cells via PAR2-HMGB1-ADAM17-HB-EGF from luminal epithelium.

Hypothyroidism induces motor deficit via altered cerebellar HB-EGF/EGFR and autophagy.

Thyroid hormones (TH) are vital for brain functions, while TH deficiency, i.e. hypothyroidism, induces neurological impairment in children and adults. Cerebellar neuronal apoptosis and motor deficits are crucial events in hypothyroidism; however, the underlying mechanism is less-known. Using a methimazole-treated hypothyroidism rat model, we investigated cerebellar autophagy, growth factor, and apoptotic mechanisms that participate in motor functions. We first identified that methimazole up-regulated cerebellar autophagy, marked by enhanced LC3B-II, Beclin-1, ATG7, ATG5-12, p-AMPKα/AMPKα, and p62 degradation as well as reduced p-AKT/AKT, p-mTOR/mTOR, and p-ULK1/ULK1 in developing and young adult rats. We probed upstream effectors of this abnormal autophagy and detected a methimazole-induced reduction in cerebellar phospho-epidermal growth factor receptor (p-EGFR)/EGFR and heparin-binding EGF-like growth factor (HB-EGF). Here, while a thyroxine-induced TH replenishment alleviated autophagy process and restored HB-EGF/EGFR, HB-EGF treatment regulated AKT-mTOR and autophagy signaling in the cerebellum. Moreover, neurons of the rat cerebellum demonstrated this reduced HB-EGF-dependent increased autophagy in hypothyroidism. We further checked whether the above events were related to cerebellar neuronal apoptosis and motor functions. We detected that comparable to thyroxine, treatment with HB-EGF or autophagy inhibitor, 3-MA, reduced methimazole-induced decrease in Nissl staining and increase in c-Caspase-3 and TUNEL-+ve apoptotic count of cerebellar neurons. Additionally, 3-MA, HB-EGF, and thyroxine attenuated the methimazole-induced diminution in riding time on rota-rod and grip strength for the motor performance of rats. Overall, our study enlightens HB-EGF/EGFR-dependent autophagy mechanism as a key to cerebellar neuronal loss and functional impairments in developmental hypothyroidism, which may be inhibited by HB-EGF and 3-MA treatments, like thyroxine.

Egr3 as an important regulator of uterine decidualization through targeting Hand2.

Early growth response 3 (Egr3) is required for embryogenesis, but little understanding is usable about its function in embryo implantation and decidualization. The present study exhibited an obvious localization of Egr3 in luminal epithelium and subluminal stroma at implantation sites. Administration of estrogen brought about a distinct gather of Egr3 mRNA in uterine luminal and glandular epithelia. Meanwhile, Egr3 was visualized in the decidua where it might facilitate the proliferation of stromal cells via Ccnd3 and accelerate stromal differentiation, testifying the significance of Egr3 in decidualization. In ovariectomized mice uteri or stromal cells, progesterone advanced the expression of Egr3 whose obstruction counteracted the inducement of stromal differentiation by progesterone. Consistently, Egr3 mediated the influence of cAMP and heparin-binding EGF-like growth factor (HB-EGF) on the differentiation program. Additionally, cAMP-protein kinase A (PKA) signaling mediated the adjustment of progesterone on Egr3. Impediment of HB-EGF antagonized the ascendance of Egr3 conferred by cAMP. In stromal cells, Egr3 activated the transcription of Hand2 whose promoter region exhibited the binding enrichment of Egr3. Activation of Hand2 relieved the weakness of stromal differentiation by Egr3 hinderance, whereas knockdown of Hand2 neutralized the guidance of Egr3 overexpression on the differentiation program. Collectively, Egr3 was identified as an important regulator of uterine decidualization through targeting Hand2 in response to progesterone/cAMP/HB-EGF pathway.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

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.

Peribronchial Inflammation Resulting from Regulatory T Cell Deficiency Damages the Respiratory Epithelium and Disturbs Barrier Function.

Regulatory T cells (Tregs) that express the transcription factor Foxp3 have a critical role in limiting inflammatory processes and tissue damage. Whether Tregs are functional in maintaining epithelial barriers and in control of tight junction expression has not yet been explored. In this study, we investigated the effect of Treg deficiency on the airway epithelial barrier in an experimental murine model in which diphtheria toxin was repeatedly injected in Foxp3-diphtheria toxin receptor (DTR) mice to deplete Tregs. This resulted in spontaneous peribronchial inflammation and led to a systemic and local increase of IL-4, IL-5, CCL3, IFN-γ, and IL-10 and a local (lung) increase of IL-6 and IL-33 and decreased amphiregulin levels. Moreover, Treg depletion increased airway permeability and decreased epithelial tight junction (protein and mRNA) expression. CTLA4-Ig treatment of Treg-depleted mice almost completely prevented barrier dysfunction together with suppression of lung inflammation and cytokine secretion. Treatment with anti-IL-4 partly reversed the effects of Treg depletion on tight junction expression, whereas neutralization of IL-6 of IFN-γ had either no effect or only a limited effect. We conclude that Tregs are essential to protect the epithelial barrier at the level of tight junctions by restricting spontaneous T cell activation and uncontrolled secretion of cytokines, in particular IL-4, in the bronchi.

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.