Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

Schwann cell-derived amphiregulin enhances nerve regeneration via supporting the proliferation and migration of Schwann cells and the elongation of axons.

Peripheral nerves have limited regeneration ability following nerve injury. Applying growth factors with neurotrophic roles is beneficial for accelerating peripheral nerve regeneration. Here we show that after rat sciatic nerve injury, growth factor amphiregulin (AREG) is upregulated in Schwann cells of sciatic nerves. Elevated AREG stimulates the proliferation and migration of Schwann cells by activating ERK1/2 cascade. Schwann cell-secreted AREG further facilitates the outgrowth of neurites and the elongation of injured axons. Administration of AREG to injured sciatic nerves stimulates the proliferation of Schwann cells to replace lost cell population, encourages the migration of Schwann cells to form cell cords, and facilitates the regrowth of axons. Overall, our results identify AREG as an important neurotrophic factor and thus provide a promising therapeutic avenue towards peripheral nerve injury.

Quercetin inhibits the amphiregulin/EGFR signaling-mediated renal tubular epithelial-mesenchymal transition and renal fibrosis in obstructive nephropathy.

Quercetin is a widely distributed, bioactive flavonoid compound, which displays potential to inhibit fibrosis in several diseases. The purpose of our study was to determine the effect of quercetin treatment on renal fibrosis and investigate the mechanism. Human proximal tubular epithelial cells (HK-2) stimulated by transforming growth factor-ß1 (TGF-ß1) and a rat model of unilateral ureter obstruction (UUO) that contributes to fibrosis were used to investigate the role and molecular mechanism of quercetin. PD153035 (N-[3-Bromophenyl]-6,7-dimethoxyquinazolin-4-amine) was used to inactivate EGFR (epidermal growth factor receptor). The level of fibrosis, proliferation, apoptosis, and oxidative stress in HK-2 were measured. All data are presented as means ± standard deviation (SD). p-value < .05 was considered statistically significant. In UUO rats, quercetin reduced the area of fibrosis as well as inflammation, oxidative stress, and cell apoptosis. In cultured HK-2 cells, quercetin significantly ameliorated the EMT induced by TGF-ß1, which was accompanied by increased amphiregulin (AREG) expression. Moreover, quercetin inhibited AREG binding to the EGFR receptor, thereby further affecting other downstream pathways. Quercetin may alleviate fibrosis in vitro and in vivo by inhibiting the activation of AREG/EGFR signaling indicating a potential therapeutic effect of quercetin in renal fibrosis.

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.

The in-vitro effect of gonadotropins' type and combination on Granulosa cells gene expressions.

OBJECTIVE: Nowadays, different modes and timing of GnRH-agonist combined with hCG trigger, for final follicular maturation, have been described. While LH + FSH are the naturally occurring final follicular maturation trigger, hCG is commonly use during stimulated cycle, and recently the introduction of the Dual/Double trigger combines LH + FSH + hCG. In the present study we aim to investigate the messenger RNA (mRNA) expression of reproduction-related genes in human granulosa cells (GCs) exposed to the aforementioned different types and combinations of gonadotropins. MATERIAL AND METHODS: Mural GCs were obtained from follicular fluid aspirated during IVF protocol. GCs were seeded in culture for 4 days with daily medium exchange followed by administration of either hCG (1 U/ml); FSH (1 U/ml) and LH (8 U/ml); or hCG (1 U/ml) and FSH (1 U/ml) and LH (8 U/ml) for 16 h. mRNA was purified from harvested GCs and gene expression was quantitative by qPCR. MAIN OUTCOME MEASURES: The expression of genes related to steroidogenesis (StAR/ CYP19) and oocyte maturation (COX2/Amphiregulin) in cultured GCs. RESULTS: The Dual/Double trigger (LH + FSH + hCG) showed higher activation of steroidogenesis (StAR/CYP19) and maturation (COX2/Amphiregulin) as compared to the naturally occurring trigger (LH + FSH) and the hCG triggers. Moreover, while the naturally occurring trigger (LH + FSH) activated maturation significantly and more intensely than the hCG trigger, no in between group differences were observed with regards to steroidogenic related genes. CONCLUSIONS: Our findings are in agreement with clinical experience, demonstrating the superiority of the double/dual (LH + FSH + hCG) trigger over the naturally occurring and the hCG triggers.

Amphiregulin Supplementation During Pig Oocyte In Vitro Maturation Enhances Subsequent Development of Cloned Embryos by Promoting Cumulus Cell Proliferation.

The oocyte in vitro maturation (IVM) technique is important in animal husbandry, biomedicine, and human-assisted reproduction. However, the developmental potential of in vitro matured oocytes is usually lower than that of in vivo matured (IVVM) oocytes. Amphiregulin (AREG) is an EGF-like growth factor that plays critical roles in the maturation and development of mammalian oocytes. This study investigated the effects of AREG supplementation during pig oocyte IVM on the subsequent development of cloned embryos. The addition of AREG to pig oocyte IVM medium improved the developmental competence of treated oocyte-derived cloned embryos by enhancing the expansion and proliferation of cumulus cells (CCs) during IVM. The positive effect of AREG on enhancing the quality of IVVM pig oocytes might be due to the activation of proliferation-related pathways in CCs by acting on the AREG receptor. The present study provides an AREG treatment-based method to improve the developmental competence of cloned pig embryos.

Amphiregulin regulates odontogenic differentiation of dental pulp stem cells by activation of mitogen-activated protein kinase and the phosphatidylinositol 3-kinase signaling pathways.

BACKGROUND: Human dental pulp stem cells (hDPSCs) have received widespread attention in the fields of tissue engineering and regenerative medicine. Although amphiregulin (AREG) has been shown to play a vital function in the biological processes of various cell types, its effects on DPSCs remain largely unknown. The aim of this study was to explore the specific role of AREG as a biologically active factor in the regeneration of dental pulp tissue. METHODS: The growth of hDPSCs, together with their proliferation and apoptosis, in response to AREG was examined by CCK-8 assay and flow cytometry. We explored the effects of AREG on osteo/odontogenic differentiation in vitro and investigated the regeneration and mineralization of hDPSCs in response to AREG in vivo. The effects of AREG gain- and loss-of-function on DPSC differentiation were investigated following transfection using overexpression plasmids and shRNA, respectively. The involvement of the mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the mineralization process and the expression of odontoblastic marker proteins after AREG induction were investigated by using Alizarin Red S staining and Western blotting, respectively. RESULTS: AREG (0.01-0.1 µg/mL) treatment of hDPSCs from 1 to 7 days increased hDPSCs growth and affected apoptosis minimally compared with negative controls. AREG exposure significantly promoted hDPSC differentiation, shown by increased mineralized nodule formation and the expression of odontoblastic marker protein expression. In vivo micro-CT imaging and quantitative analysis showed significantly greater formation of highly mineralized tissue in the 0.1 µg/mL AREG exposure group in DPSC/NF-gelatin-scaffold composites. AREG also promoted extracellular matrix production, with collagen fiber, mineralized matrix, and calcium salt deposition on the composites, as shown by H&E, Masson, and Von Kossa staining. Furthermore, AREG overexpression boosted hDPSC differentiation while AREG silencing inhibited it. During the differentiation of hDPSCs, AREG treatment led to phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and PI3K/Akt. Notably, a specific inhibitor of ERK, JNK, and PI3K/Akt signaling markedly reduced AREG-induced differentiation, as well as levels of phosphorylated ERK and JNK in hDPSCs. CONCLUSIONS: The data indicated that AREG promoted odontoblastic differentiation and facilitated regeneration and mineralization processes in hDPSCs.

Interrelationships between amphiregulin, kisspeptin, FSH and FSH receptor in promotion of human ovarian cell functions.

The aim of this study was to investigate: (1) the ability of granulosa cells to produce amphiregulin (AREG), kisspeptin (KISS) and FSH receptor (FSHR); (2) the role of AREG and KISS in the control of ovarian functions; (3) the effect of FSH and KISS on AREG; and (4) the ability of KISS to affect FSHR and to modify FSH action on AREG output by human ovarian granulosa cells. We examined: (1) time-dependent accumulation of AREG; (2) effects of AREG (0, 1, 10, 100ng/mL) and KISS (0, 1, 10, 100ng/mL) on granulosa cell functions; and (3) the effects of KISS (0, 1, 10, 100ng/mL), FSH (0, 1, 10, 100ng/mL), and their combinations on AREG release. Viability, markers of proliferation [accumulation ofproliferating cell nuclear antigen (PCNA) cyclin B1 and sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy6-nitro)benzene sulfonic acid hydrate (XTT formazan)] and apoptosis (accumulation of bax, caspase 3 and terminal deoxynucleotidyl transferase dUTP nick-end labelling), accumulation of KISS, FSHR and steroid hormones, and AREG release were analysed by Trypan blue exclusion test, quantitative immunocytochemistry, XTT, terminal deoxynucleotidyl transferase dUTP nick-end labelling assays and enzyme-linked immunosorbent assay. AREG promoted cell viability, proliferation and steroid hormone output, and inhibited apoptosis. KISS (1 and 10ng/mL) stimulated viability, proliferation, steroid hormone release and occurrence of FSHR and suppressed apoptosis and AREG output; KISS (100ng/mL) had the opposite effect. FSH stimulated AREG release, whilst addition of KISS reversed this FSH effect. FSH mimicked and promoted the inhibitory effect of KISS on AREG release. These results suggest an intra-ovarian production and a functional interrelationship between AREG, KISS, FSH and FSHR in direct regulation of basic ovarian cell functions.

Amphiregulin promotes hair regeneration of skin-derived precursors via the PI3K and MAPK pathways.

OBJECTIVES: There are significant clinical challenges associated with alopecia treatment, including poor efficiency of related drugs and insufficient hair follicles (HFs) for transplantation. Skin-derived precursors (SKPs) exhibit great potential as stem cell-based therapies for hair regeneration; however, the proliferation and hair-inducing capacity of SKPs gradually decrease during culturing. MATERIALS AND METHODS: We describe a 3D co-culture system accompanied by kyoto encyclopaedia of genes and genomes and gene ontology enrichment analyses to determine the key factors and pathways that enhance SKP stemness and verified using alkaline phosphatase assays, Ki-67 staining, HF reconstitution, Western blot and immunofluorescence staining. The upregulated genes were confirmed utilizing corresponding recombinant protein or small-interfering RNA silencing in vitro, as well as the evaluation of telogen-to-anagen transition and HF reconstitution in vivo. RESULTS: The 3D co-culture system revealed that epidermal stem cells and adipose-derived stem cells enhanced SKP proliferation and HF regeneration capacity by amphiregulin (AREG), with the promoted stemness allowing SKPs to gain an earlier telogen-to-anagen transition and high-efficiency HF reconstitution. By contrast, inhibitors of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways downstream of AREG signalling resulted in diametrically opposite activities. CONCLUSIONS: By exploiting a 3D co-culture model, we determined that AREG promoted SKP stemness by enhancing both proliferation and hair-inducing capacity through the PI3K and MAPK pathways. These findings suggest AREG therapy as a potentially promising approach for treating alopecia.

Dysregulation of Amphiregulin stimulates the pathogenesis of cystic lymphangioma.

Along with blood vessels, lymphatic vessels play an important role in the circulation of body fluid and recruitment of immune cells. Postnatal lymphangiogenesis commonly occurs from preexisting lymphatic vessels by sprouting, which is induced by lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C). However, the key signals and cell types that stimulate pathological lymphangiogenesis, such as human cystic lymphangioma, are less well known. Here, we found that mouse dermal fibroblasts that infiltrate to sponges subcutaneously implanted express VEGF-D and sushi, Von Willebrand factor type A, EGF, and pentraxin domain containing 1 (SVEP1) in response to PDGFRß signal. In vitro, Pdgfrb knockout (ß-KO) fibroblasts had reduced expression of VEGF-D and SVEP1 and overproduced Amphiregulin. Dysregulation of these three factors was involved in the cyst-like and uneven distribution of lymphatic vessels observed in the ß-KO mice. Similarly, in human cystic lymphangioma, which is one of the intractable diseases and mostly occurs in childhood, fibroblasts surrounding cystic lymphatics highly expressed Amphiregulin. Moreover, fibroblast-derived Amphiregulin could induce the expression of Amphiregulin in lymphatic endothelial cells. The dual source of Amphiregulin activated EGFR expressed on the lymphatic endothelial cells. This exacerbation cascade induced proliferation of lymphatic endothelial cells to form cystic lymphangioma. Ultimately, excessive Amphiregulin produced by fibroblasts surrounding lymphatics and by lymphatic endothelial cells per se results in pathogenesis of cystic lymphangioma and will be a fascinating therapeutic target of cystic lymphangioma.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

Serum glucose excretion after Roux-en-Y gastric bypass: a potential target for diabetes treatment.

OBJECTIVE: The mechanisms underlying type 2 diabetes resolution after Roux-en-Y gastric bypass (RYGB) are unclear. We suspected that glucose excretion may occur in the small bowel based on observations in humans. The aim of this study was to evaluate the mechanisms underlying serum glucose excretion in the small intestine and its contribution to glucose homeostasis after bariatric surgery. DESIGN: 2-Deoxy-2-[18F]-fluoro-D-glucose (FDG) was measured in RYGB-operated or sham-operated obese diabetic rats. Altered glucose metabolism was targeted and RNA sequencing was performed in areas of high or low FDG uptake in the ileum or common limb. Intestinal glucose metabolism and excretion were confirmed using 14C-glucose and FDG. Increased glucose metabolism was evaluated in IEC-18 cells and mouse intestinal organoids. Obese or ob/ob mice were treated with amphiregulin (AREG) to correlate intestinal glycolysis changes with changes in serum glucose homeostasis. RESULTS: The AREG/EGFR/mTOR/AKT/GLUT1 signal transduction pathway was activated in areas of increased glycolysis and intestinal glucose excretion in RYGB-operated rats. Intraluminal GLUT1 inhibitor administration offset improved glucose homeostasis in RYGB-operated rats. AREG-induced signal transduction pathway was confirmed using IEC-18 cells and mouse organoids, resulting in a greater capacity for glucose uptake via GLUT1 overexpression and sequestration in apical and basolateral membranes. Systemic and local AREG administration increased GLUT1 expression and small intestinal membrane translocation and prevented hyperglycaemic exacerbation. CONCLUSION: Bariatric surgery or AREG administration induces apical and basolateral membrane GLUT1 expression in the small intestinal enterocytes, resulting in increased serum glucose excretion in the gut lumen. Our findings suggest a novel, potentially targetable glucose homeostatic mechanism in the small intestine.

Amphiregulin retains ERα expression in acquired aromatase inhibitor resistant breast cancer cells.

Acquired resistance to aromatase inhibitors (AIs) is a significant clinical issue in endocrine therapy for estrogen receptor (ER) positive breast cancer which accounts for the majority of breast cancer. Despite estrogen production being suppressed, ERα signaling remains active and plays a key role in most AI-resistant breast tumors. Here, we found that amphiregulin (AREG), an ERα transcriptional target and EGF receptor (EGFR) ligand, is crucial for maintaining ERα expression and signaling in acquired AI-resistant breast cancer cells. AREG was deregulated and critical for cell viability in ER+ AI-resistant breast cancer cells, and ectopic expression of AREG in hormone responsive breast cancer cells promoted endocrine resistance. RNA-sequencing and reverse phase protein array analyses revealed that AREG maintains ERα expression and signaling by activation of PI3K/Akt/mTOR signaling and upregulation of forkhead box M1 (FOXM1) and serum- and glucocorticoid-inducible kinase 3 (SGK3) expression. Our study uncovers a previously unappreciated role of AREG in maintaining ERα expression and signaling, and establishes the AREG-ERα crosstalk as a driver of acquired AI resistance in breast cancer.

Prenatal smoke exposure dysregulates lung epithelial cell differentiation in mouse offspring: role for AREG-induced EGFR signaling.

Prenatal smoke exposure is a risk factor for impaired lung development in children. Recent studies have indicated that amphiregulin (AREG), which is a ligand of the epidermal growth factor receptor (EGFR), has a regulatory role in airway epithelial cell differentiation. In this study, we investigated the effect of prenatal smoke exposure on lung epithelial cell differentiation and linked this with AREG-EGFR signaling in 1-day-old mouse offspring. Bronchial and alveolar epithelial cell differentiations were assessed by immunohistochemistry. Areg, epidermal growth factor (Egf), and mRNA expressions of specific markers for bronchial and alveolar epithelial cells were assessed by RT-qPCR. The results in neonatal lungs were validated in an AREG-treated three-dimensional mouse lung organoid model. We found that prenatal smoke exposure reduced the number of ciliated cells and the expression of the cilia-related transcription factor Foxj1, whereas it resulted in higher expression of mucus-related transcription factors Spdef and Foxm1 in the lung. Moreover, prenatally smoke-exposed offspring had higher numbers of alveolar epithelial type II cells (AECII) and lower expression of the AECI-related Pdpn and Gramd2 markers. This was accompanied by higher expression of Areg and lower expression of Egf in prenatally smoke-exposed offspring. In bronchial organoids, AREG treatment resulted in fewer ciliated cells and more basal cells when compared with non-treated bronchiolar organoids. In alveolar organoids, AREG treatment led to more AECII cells than non-treated AECII cells. Taken together, the observed impaired bronchial and alveolar cell development in prenatally smoke-exposed neonatal offspring may be induced by increased AREG-EGFR signaling.

Amphiregulin alleviated concanavalin A-induced acute liver injury via IL-22.

OBJECTIVES: Amphiregulin (Areg), a glycoprotein from the epidermal growth factor receptor (EGFR) ligand family, has a well-documented protective role against tissue injury; however, its effects on immune-mediated liver injury are still unclear. Here, we used a concanavalin A (ConA)-induced acute liver hepatitis model to explore the effects of Areg on immune-mediated acute liver injury. MATERIALS AND METHODS: Some C57BL/6 mice were administered ConA at a dose of 20 mg/kg (model mice), and some received 5 µg of Areg (treated mice). Then, their survival rates over 36 h were analyzed. After 5 h of treatment, liver function, hepatic histology, and apoptosis in liver tissue were investigated, and cytokine expression and neutrophil infiltration and activity in the liver were detected. Moreover, the protective effects of Areg were also evaluated without IL-22 in vivo. RESULTS: Our results showed that Areg administration increased acute liver failure (ALF) mouse survival, restored liver function, and alleviated liver damage. Interestingly, Areg administration increased IL-22 production in hepatic T cells and upregulated IL-22 concentrations in the serum and liver, whereas IL-22 neutralization completely abolished the therapeutic effect of Areg. Meanwhile, Areg administration was concomitant with increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, which are important in the hepatoprotective mechanism of IL-22. CONCLUSIONS: Areg showed direct protective effects against ConA-induced acute liver injury, which suggests the potential therapeutic application of Areg in immune-mediated ALF.

Granulosa secreted factors improve the developmental competence of cumulus oocyte complexes from small antral follicles in sheep.

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.

Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust.

Exposure to agricultural bioaerosols can lead to chronic inflammatory lung diseases. Amphiregulin (AREG) can promote the lung repair process but can also lead to fibrotic remodeling. The objective of this study was to determine the role of AREG in altering recovery from environmental dust exposure in a murine in vivo model and in vitro using cultured human and murine lung fibroblasts. C57BL/6 mice were intranasally exposed to swine confinement facility dust extract (DE) or saline daily for 1 wk or allowed to recover for 3-7 days while being treated with an AREG-neutralizing antibody or recombinant AREG. Treatment with the anti-AREG antibody prevented resolution of DE exposure-induced airway influx of total cells, neutrophils, and macrophages and increased levels of TNF-α, IL-6, and CXCL1. Neutrophils and activated macrophages (CD11c+CD11bhi) persisted after recovery in lung tissues of anti-AREG-treated mice. In murine and human lung fibroblasts, DE induced the release of AREG and inflammatory cytokines. Fibroblast recellularization of primary human lung mesenchymal matrix scaffolds and wound closure was inhibited by DE and enhanced with recombinant AREG alone. AREG treatment rescued the DE-induced inhibitory fibroblast effects. AREG intranasal treatment for 3 days during recovery phase reduced repetitive DE-induced airway inflammatory cell influx and cytokine release. Collectively, these studies demonstrate that inhibition of AREG reduced, whereas AREG supplementation promoted, the airway inflammatory recovery response following environmental bioaerosol exposure, and AREG enhanced fibroblast function, suggesting that AREG could be targeted in agricultural workers repetitively exposed to organic dust environments to potentially prevent and/or reduce disease.

Amphiregulin promotes trophoblast invasion and increases MMP9/TIMP1 ratio through ERK1/2 and Akt signal pathways.

AIMS: The aim of our study is to illustrate the role of amphiregulin in trophoblast invasiveness and underlying signal cascades. MAIN METHODS: An immortalized human early extravillous cell line, HTR-8/SVneo, was used for this investigation. Matrigel-transwell invasion assay was used for testing the effects of amphiregulin on cell invasiveness. MMP9 and MMP2 mRNA expression level and activity were measured using Rt-qPCR and zymographic analysis. Cell signals involved in the invasion process were verified using western blot and specific inhibitors. KEY FINDINGS: Our results showed that amphiregulin could promote HTR-8/SVneo cell invasiveness without interfering cell proliferation, and significantly upregulate the expression of MMP9 and TIMP-1 mRNAs as well as the ratio of MMP9/TIMP-1. Using specific inhibitors for MEK and PI3K signaling further indicated that, both ERK1/2 and Akt signal pathways were required for amphiregulin-induced cell invasiveness. The co-ordination between ERK1/2 and Akt signaling pathway was needed for the upregulation of MMP9 mRNA, while ERK1/2 was more essential for the upregulation of TIMP-1 mRNA. Meanwhile, we first put forward that the deficiency of amphiregulin expression in trophoblast might be compensated by the upregulation of epidermal growth factor receptor (EGFR) and heparin-binding EGF (HB-EGF) mRNA. SIGNIFICANCE: ERK1/2 and Akt signaling pathways mediate amphiregulin-induced upregulation of MMP9 mRNA and the MMP9/TIMP-1 ratio, which subsequently contribute to amphiregulin-promotion of HTR-8/SVneo cell invasion.

Lapatinib Inhibits Amphiregulin-induced BeWo Choriocarcinoma Cell Proliferation by Reducing ERK1/2 and AKT Signaling Pathways.

BACKGROUND: Human choriocarcinoma is the most aggressive type of gestational trophoblastic neoplasia. The expression of epidermal growth factor receptor (EGFR) in choriocarcinomas is significantly higher than those of trophoblastic cells in healthy placentas. Lapatinib is a potent EGFR and human epidermal growth factor receptor 2 (HER2) inhibitor that inhibits cell proliferation and induces apoptosis in various human cancer cells. Amphiregulin (AREG) is the most abundant EGFR ligand in amniotic fluid during human pregnancy. AIM: To explore the role of AREG in human choriocarcinoma cell proliferation. MATERIALS AND METHODS: The effect of lapatinib and AREG on cell proliferation was examined by the MTT assay. Western blots were used to investigate EGFR and HER2 expression, and the activation of caspase-3, extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase /protein kinase B (PI3K/AKT) signaling pathways. RESULTS: Treatment with lapatinib reduced BeWo cell proliferation by inducing apoptosis. Moreover, AREG treatment stimulated BeWo cell proliferation by activating ERK1/2 and PI3K/AKT signaling pathways, which was blocked by lapatinib. CONCLUSION: Targeting EGFR/HER2 might be a useful therapeutic strategy for human choriocarcinoma.

Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and the Bcl-2 Protein Family.

Neonates are extremely susceptible to bacterial infections, and evidences suggest that phagocytosis-induced cell death (PICD) is less frequently triggered in neonatal monocytes than in monocytes from adult donors. An insufficient termination of the inflammatory response, leading to a prolonged survival of neonatal monocytes with ongoing proinflammatory cytokine release, could be associated with the progression of various inflammatory diseases in neonates. Our previous data indicate that amphiregulin (AREG) is increasingly expressed on the cell surface of neonatal monocytes, resulting in remarkably higher soluble AREG levels after proteolytic shedding. In this study, we found that E. coli-infected neonatal monocytes show an increased phosphorylation of ERK, increased expression of Bcl-2 and Bcl-XL, and reduced levels of cleaved caspase-3 and caspase-9 compared to adult monocytes. In both cell types, additional stimulation with soluble AREG further increased ERK activation and expression of Bcl-2 and Bcl-XL and reduced levels of cleaved caspase-3 and caspase-9 in an EGFR-dependent manner. These data suggest that reduced PICD of neonatal monocytes could be due to reduced intrinsic apoptosis and that AREG can promote protection against PICD. This reduction of the intrinsic apoptosis pathway in neonatal monocytes could be relevant for severely prolonged inflammatory responses of neonates.