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.

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.

miR-4443/MMP2 suppresses the migration and invasion of trophoblasts through the HB-EGF/EGFR pathway in preeclampsia.

Preeclampsia (PE) is a pregnancy-associated disease that may cause maternal and fetal morbidity and mortality. The dysregulation of microRNAs (miRNAs) and their potential functions has been an important direction for elucidating the mechanism of preeclampsia in recent years. The present study investigated whether miR-4443 was significantly increased in the placentas of severe preeclamptic patients, and the upregulation of miR-4443 inhibited the migration and invasion of HTR-8/SVneo cells according to transwell assays. Matrix metallopeptidase 2 (MMP2), which is involved in the degradation of extracellular matrix (ECM) components and harbors a miR-4443-binding site within its 3'-UTR as confirmed by a luciferase reporter assay, was identified to be directly inhibited by miR-4443. Moreover, siRNA targeting MMP2 imitated the effects of overexpressed miR-4443 on HTR-8/SVneo cell invasion and migration, whereas rescue experiments showed that MMP2 reversed this inhibitory function of miR-4443. Heparin-binding EGF-like growth factor (HB-EGF), as the downstream gene of MMP2, plays an important role in trophoblast invasion, and we confirmed that the expression of HB-EGF/EGFR pathway-related biomolecules was consistent with MMP2 influenced by upregulating and downregulating miR-4443 and that activated EGFR further transmitted intracellular downstream signaling via the MAPK pathway according to western blot assay. In conclusion, we demonstrated that miR-4443 suppresses the migration and invasion of trophoblasts, and its inhibitory effects are at least partially mediated by the suppression of MMP2. This inhibition might further affect the progression of preeclampsia through the HB-EGF/EGFR pathway, thus providing a new clue on the role of miR-4443 in the pathogenesis of preeclampsia.

Clostridium butyricum, a butyrate-producing potential probiotic, alleviates experimental colitis through epidermal growth factor receptor activation.

Inflammatory bowel disease (IBD) is an idiopathic inflammatory bowel disease. Modulation of gut microbiota with dietary and nutritional targets is a feasible strategy for the prevention and treatment of IBD. In this study, we focused on Clostridium butyricum Prazmowski (CB), a butyrate-producing potential probiotic. We found that CB feeding decreased the disease activity index, colon inflammation/injury score and cell apoptosis in an experimental colitis mouse model, as well as elevated the level of SCFAs in cecal feces. CB could also balance the inflammatory cytokines, protect tight junctions, and increase the number of goblet cells and MUC2 production in mice, accompanied by EGFR signaling activation triggered by heparin-binding epidermal growth factor (HB-EGF) and amphiregulin (AREG). From the perspective of mechanism, the CB supernatant (CBS) stimulated EGFR activation in colon epithelial cell lines in concentration-dependent and time-dependent manners. CBS reduced the damage of tight junctions induced by H2O2, and inhibition of EGFR could suppress the protective effect of CBS. In conclusion, CB could protect the gut barrier and alleviate experimental colitis through the transactivation of EGFR signaling in intestinal epithelial cells induced by ligands (HB-EGF and AREG). This study identified the potential efficacy of CB as a preventive strategy for IBD and showed the broad prospect of CB as a food supplement.

Saccharomyces boulardii, a yeast probiotic, inhibits gut motility through upregulating intestinal serotonin transporter and modulating gut microbiota.

Saccharomyces boulardii (Sb) is a widely used fungal probiotic in treating various digestive diseases, including irritable bowel syndrome (IBS). However, the specific mechanisms of Sb relieving IBS remain unclear. The abnormal serotonin transporter (SERT) / 5-hydroxytryptamine (5-HT) system could cause disordered gastrointestinal sensation and motility, which closely related to IBS pathogenesis. The aim of this study was to explore the effects and mechanisms of Sb on regulating gut motility. Sb supernatant (SbS) was administered to intestinal epithelial cells and mice. SbS upregulated SERT expression via enhancing heparin-binding epidermal growth factor (HB-EGF) release to activate epidermal growth factor receptor (EGFR). EGFR kinase inhibitor treatment or HB-EGF siRNA transfection in cells blocked SbS upregulating SERT. Consistently, SbS-treated mice presented inhibited gut motility, and EGFR activation and SERT upregulation were found. Moreover, 16 S rDNA sequence presented an evident decrease in Firmicutes / Bacteroidetes ratio in SbS group. In genus level, SbS reduced Escherichia_Shigella, Alistipes, Clostridium XlVa, and Saccharibacteria_genera_incertae_sedis, meanwhile, increased Parasutterella. The abundance of Saccharibacteria_genera_incertae_sedis positively correlated with defecation parameters and intestinal 5-HT content. Fecal microbiota transplantation showed that SbS could modulate gut microbiota to influence gut motility. Interestingly, elimination of gut microbiota with antibiotic cocktail did not entirely block SbS regulating gut motility. Furthermore, SbS administration to IBS-D mice significantly upregulated SERT and inhibited gut motility. In conclusion, SbS could upregulate SERT by EGFR activation, and modulate gut microbiota to inhibit gut motility. This finding would provide more evidence for the application of this yeast probiotic in IBS and other diarrheal disorders.

Forkhead Box q1 promotes invasion and metastasis in colorectal cancer by activating the epidermal growth factor receptor pathway.

BACKGROUND: Colorectal cancer (CRC) is an extremely malignant tumor with a high mortality rate. Little is known about the mechanism by which forkhead Box q1 (FOXQ1) causes CRC invasion and metastasis through the epidermal growth factor receptor (EGFR) pathway. AIM: To illuminate the mechanism by which FOXQ1 promotes the invasion and metastasis of CRC by activating the heparin binding epidermal growth factor (HB-EGF)/EGFR pathway. METHODS: We investigated the differential expression and prognosis of FOXQ1 and HB-EGF in CRC using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of FOXQ1 and HB-EGF in cell lines and tissues, and we constructed a stable low-expressing FOXQ1 cell line and verified it with the above method. The expression changes of membrane-bound HB-EGF (proHB-EGF) and soluble HB-EGF (sHB-EGF) in the low-expressing FOXQ1 cell line were detected by flow cytometry and ELISA. Western blotting was used to detect changes in the expression levels of HB-EGF and EGFR pathway-related downstream genes when exogenous recombinant human HB-EGF was added to FOXQ1 knockdown cells. Proliferation experiments, transwell migration experiments, and scratch experiments were carried out to determine the mechanism by which FOXQ1 activates the EGFR signaling pathway through HB-EGF, and then to evaluate the clinical relevance of FOXQ1 and HB-EGF. RESULTS: GEPIA showed that the expression of FOXQ1 in CRC tissues was relatively high and was related to a lower overall survival rate. PCR array results showed that FOXQ1 is related to the HB-EGF and EGFR pathways. Knockdown of FOXQ1 suppressed the expression of HB-EGF, and led to a decrease in EGFR and its downstream genes AKT, RAF, KRAS expression levels. After knockdown of FOXQ1 in CRC cell lines, cell proliferation, migration and invasion were attenuated. Adding HB-EGF restored the migration and invasion ability of CRC, but not the cell proliferation ability. Kaplan-Meier survival analysis results showed that the combination of FOXQ1 and HB-EGF may serve to predict CRC survival. CONCLUSION: Based on these collective data, we propose that FOXQ1 promotes the invasion and metastasis of CRC via the HB-EGF/EGFR pathway.

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.

New transgenic mouse models enabling pan-hematopoietic or selective hematopoietic stem cell depletion in vivo.

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in "Vav-DTR" mice. Crossing these mice to Flk2-Cre mice results in "HSC-DTR" mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis.

Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.

HB-EGF-induced IL-8 secretion from airway epithelium leads to lung fibroblast proliferation and migration.

BACKGROUND: We have reported that heparin-binding epidermal growth factor (HB-EGF) is increased in patients with chronic obstructive pulmonary disease (COPD) and associated with collagen deposition, but the mechanisms remain unclear. In the present study, we aimed to investigated the inflammatory cytokines secreted by bronchial epithelial cells following exposure to HB-EGF that promoted proliferation and migration of human lung fibroblast. METHODS: HB-EGF-induced inflammatory cytokines were assayed in two airway epithelial cells (primary human bronchial epithelial cells [HBECs] and BEAS-2B cells). Moreover, the culture supernatants derived from HB-EGF-treated HBECs and BEAS-2B cells were added to human primary lung fibroblasts. The effect of culture supernatants on proliferation and migration of fibroblasts was assessed. RESULTS: IL-8 expression was significantly increased in bronchial epithelial cells treated with HB-EGF, which was at least partially dependent on NF-kB pathways activation. HB-EGF-induced IL-8 was found to further promote lung fibroblasts proliferation and migration, and the effects were attenuated after neutralizing IL-8. CONCLUSIONS: These findings suggest that HB-EGF may be involved in the pathology of airway fibrosis by induction of IL-8 from airway epithelium, subsequently causing lung fibroblasts proliferation and migration. Thus, inhibition of HBEGF and/or IL-8 production could prevent the development of airway fibrosis by modulating fibroblast activation.