Amphiregulin Downregulates E-cadherin Expression by Activating YAP/Egr-1/Slug Signaling in SKOV3 Human Ovarian Cancer Cells.

Amphiregulin (AREG) stimulates human epithelial ovarian cancer (EOC) cell invasion by downregulating E-cadherin expression. YAP is a transcriptional cofactor that has been shown to regulate tumorigenesis. This study aimed to examine whether AREG activates YAP in EOC cells and explore the roles of YAP in AREG-induced downregulation of E-cadherin and cell invasion. Analysis of the Cancer Genome Atlas (TCGA) showed that upregulation of AREG and EGFR were associated with poor survival in human EOC. Treatment of SKOV3 human EOC cells with AREG induced the activation of YAP. In addition, AREG downregulated E-cadherin, upregulated Egr-1 and Slug, and stimulated cell invasion. Using gain- and loss-of-function approaches, we showed that YAP was required for the AREG-upregulated Egr-1 and Slug expression. Furthermore, YAP was also involved in AREG-induced downregulation of E-cadherin and cell invasion. This study provides evidence that AREG stimulates human EOC cell invasion by downregulating E-cadherin expression through the YAP/Egr-1/Slug signaling.

Sustained amphiregulin expression in intermediate alveolar stem cells drives progressive fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal fibrotic disease. Recent studies have highlighted the persistence of an intermediate state of alveolar stem cells in IPF lungs. In this study, we discovered a close correlation between the distribution pattern of intermediate alveolar stem cells and the progression of fibrotic changes. We showed that amphiregulin (AREG) expression is significantly elevated in intermediate alveolar stem cells of mouse fibrotic lungs and IPF patients. High levels of serum AREG correlate significantly with profound deteriorations in lung function in IPF patients. We demonstrated that AREG in alveolar stem cells is both required and sufficient for activating EGFR in fibroblasts, thereby driving lung fibrosis. Moreover, pharmacological inhibition of AREG using a neutralizing antibody effectively blocked the initiation and progression of lung fibrosis in mice. Our study underscores the therapeutic potential of anti-AREG antibodies in attenuating IPF progression, offering a promising strategy for treating fibrotic diseases.

Application of amphiregulin in IVM culture of immature human oocytes and pre-insemination culture for COCs in IVF cycles.

Background: Amphiregulin (AR) is a growth factor that resembles the epidermal growth factor (EGF) and serves various functions in different cells. However, no systematic studies or reports on the role of AR in human oocytes have currently been performed or reported. This study aimed to explore the role of AR in human immature oocytes during in vitro maturation (IVM) and in vitro fertilization (IVF) in achieving better embryonic development and to provide a basis for the development of a pre-insemination culture medium specific for cumulus oocyte complexes (COCs). Methods: First, we examined the concentration of AR in the follicular fluid (FF) of patients who underwent routine IVF and explored the correlation between AR levels and oocyte maturation and subsequent embryonic development. Second, AR was added to the IVM medium to culture immature oocytes and investigate whether AR could improve the effects of IVM. Finally, we pioneered the use of a fertilization medium supplemented with AR for the pre-insemination culture of COCs to explore whether the involvement of AR can promote the maturation and fertilization of IVF oocytes, as well as subsequent embryonic development. Results: A total of 609 FF samples were examined, and a positive correlation between AR levels and blastocyst formation was observed. In our IVM study, the development potential and IVM rate of immature oocytes, as well as the fertilization rate of IVM oocytes in the AR-added groups, were ameliorated significantly compared to the control group (All P < 0.05). Only the IVM-50 group had a significantly higher blastocyst formation rate than the control group (P < 0.05). In the final IVF study, the maturation, fertilization, high-quality embryo, blastocyst formation, and high-quality blastocyst rates of the AR-added group were significantly higher than those of the control group (All P < 0.05). Conclusion: AR levels in the FF positively correlated with blastocyst formation, and AR involvement in pre-insemination cultures of COCs can effectively improve laboratory outcomes in IVF. Furthermore, AR can directly promote the in vitro maturation and developmental potential of human immature oocytes at an optimal concentration of 50 ng/ml.

Histamine promotes mouse decidualization through stimulating epithelial amphiregulin release.

Accumulating evidence shows that inflammation is essential for embryo implantation and decidualization. Histamine, a proinflammatory factor that is present in almost all mammalian tissues, is synthesized through decarboxylating histidine by histidine decarboxylase (HDC). Although histamine is known to be essential for decidualization, the underlying mechanism remains undefined. In the present study, histamine had no obvious direct effects on in vitro decidualization in mice. However, the obvious differences in HDC protein levels between day 4 of pregnancy and day 4 of pseudopregnancy, as well as between delayed and activated implantation, suggested that the blastocyst may be involved in regulating HDC expression. Furthermore, blastocyst-derived tumor necrosis factor α (TNFα) significantly increased HDC levels in the luminal epithelium. Histamine increased the levels of amphiregulin (AREG) and disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) proteins, which was abrogated by treatment with famotidine, a specific histamine type 2 receptor (H2R) inhibitor, or by TPAI-1 (a specific inhibitor of ADAM17). Intraluminal injection of urocanic acid (HDC inhibitor) on day 4 of pregnancy significantly reduced the number of implantation sites on day 5 of pregnancy. TNFα-stimulated increases in HDC, AREG and ADAM17 protein levels was abrogated by urocanic acid, a specific inhibitor of HDC. Additionally, AREG treatment significantly promoted in vitro decidualization. Collectively, our data suggests that blastocyst-derived TNFα induces luminal epithelial histamine secretion, and histamine increases mouse decidualization through ADAM17-mediated AREG release.

Areca nut-induced AREG promote oral epithelial cell proliferation, migration, and EMT.

OBJECTIVE: To analyze the biological effect and mechanism of areca nut extract (ANE) on human oral keratinocyte (HOK) cells. MATERIALS AND METHODS: The effect of gradient concentration of ANE on the proliferation activity of HOK cells was analyzed by cell counting kit-8 (CCK-8) assays. The differentially expressed genes between the ANE group and control group HOK cells were analyzed by second-generation transcriptome sequencing. Real-time PCR and western blot were, respectively, used to analyze the expression of AREG gene and protein in HOK cells. After AREG gene overexpression or knockdown, the proliferation, migration, and expression of proteins related to epithelial-mesenchymal transformation (EMT), MAPK signal pathway in HOK cells were, respectively, detected by CCK-8, wound healing, transwell, and western blot assays. RESULTS: ANE (500 µg/mL) promoted the proliferation and migration of HOK cells, ANE (2 mg/mL) promoted the EMT of HOK cells, and ANE (50 mg/mL) inhibited the proliferation of HOK cells. AREG knockdown inhibited ANE-induced proliferation and migration of HOK cells, while AREG overexpression promoted the proliferation and migration of HOK cells. Western blot assay showed that ANE activated MAPK signal pathway by upregulating AREG protein in HOK cells. CONCLUSIONS: ANE promoted HOK cell proliferation, migration, and EMT by mediating AREG-MAPK signaling pathway.

Effects of N361 Glycosylation on Epidermal Growth Factor Receptor Biological Function.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that is frequently modified by glycosylation post-translationally. In cancer, EGFR amplifications and hotspot mutations such as L858R that promote proliferation have been detected in a significant fraction of non-small cell lung carcinomas and breast adenocarcinomas. Molecular dynamic simulations suggested that glycosylation at asparagine residue 361 (N361) promotes dimerization and ligand binding. We stably expressed glycosylation-deficient mutant EGFR N361A, with or without the oncogenic mutation L858R. Immunofluorescence and flow cytometry demonstrated that the mutants were each well expressed at the cell membrane. N361A decreased proliferation relative to wild-type EGFR as well as decreased sensitivity to ligands. Proximity ligation assays measuring co-localization of EGFR with its binding partner HER2 in cells revealed that N361A mutations increased co-localization. N361A, located near the binding interface for the EGFR inhibitor necitumumab, desensitized cells expressing the oncogenic EGFR L858R to antibody-based inhibition. These findings underline the critical relevance of post-translational modifications on oncogene function.

Amphiregulin-producing TH2 cells facilitate esophageal fibrosis of eosinophilic esophagitis.

Background: Massive eosinophil infiltration into the esophagus is associated with subepithelial fibrosis and esophageal stricture in patients with eosinophilic esophagitis (EoE). However, the pathogenesis of esophageal fibrosis remains unclear. Objective: We sought to elucidate the cellular and molecular mechanisms underlying the induction of esophageal fibrosis. Methods: We established a murine model of EoE accompanied by fibrotic responses following long-term intranasal administration of house dust mite antigen. Using this murine model, we investigated the characteristics of immune cells infiltrating the fibrotic region of the inflamed esophagus using flow cytometry and histological analyses. We also analyzed the local inflammatory sites in the esophagus of patients with EoE using single-cell RNA sequencing, flow cytometry, and immunohistochemistry. Results: Enhanced infiltration of both amphiregulin-producing and IL-5-producing TH2 cells was detected in the fibrotic area of the esophagus in mice subjected to repeated house dust mite exposure. Deletion of amphiregulin in CD4+ T cells ameliorates esophageal fibrosis. An analysis of human esophageal biopsy samples showed that the infiltration of amphiregulin-producing CD4+ T cells was higher in patients with EoE than in control patients. Furthermore, the number of infiltrated amphiregulin-producing CD4+ T cells was associated with the degree of esophageal fibrosis in patients with EoE. Conclusions: Amphiregulin, produced by TH2 cells, contributes to esophageal fibrosis in EoE and may be a therapeutic target.

Enhanced amphiregulin exposure promotes modulation of the high grade serous ovarian cancer tumor immune microenvironment.

High grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Furthermore, HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies. Amphiregulin (AREG), a low affinity epidermal growth factor receptor ligand is known to be significantly upregulated in HGSOC patient tumors following neoadjuvant chemotherapy exposure. While much is known about AREG's role in oncogenesis and classical immunity, it is function in tumor immunology has been comparatively understudied. Therefore, the objective of this present study was to elucidate how increased AREG exposure impacts the ovarian tumor immune microenvironment (OTIME). Using NanoString IO 360 and protein analysis, it was revealed that treatment with recombinant AREG led to prominent upregulation of genes associated with ovarian pathogenesis and immune evasion (CXCL8, CXCL1, CXCL2) along with increased STAT3 activation in HGSOC cells. In vitro co-culture assays consisting of HGSOC cells and peripheral blood mononuclear cells (PBMCs) stimulated with recombinant AREG (rAREG) led to significantly enhanced tumor cell viability. Moreover, PBMCs stimulated with rAREG exhibited significantly lower levels of IFNy and IL-2. In vivo rAREG treatment promoted significant reductions in circulating levels of IL-2 and IL-5. Intratumoral analysis of rAREG treated mice revealed a significant reduction in CD8+ T cells coupled with an upregulation of PD-L1. Finally, combinatorial treatment with an AREG neutralizing antibody and carboplatin led to a synergistic reduction of cell viability in HGSOC cell lines OVCAR8 and PEA2. Overall, this study demonstrates AREG's ability to modulate cytotoxic responses within the OTIME and highlights its role as a novel HGSOC immune target.

Particulate matter facilitates amphiregulin-dependent lung cancer proliferation through glutamine metabolism.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) causes lung cancer, the molecular mechanisms underlying the PM-induced increases in lung cancer progression remain unclear. We applied the lung cancer cell line A549 (Parental; A549.Par) to PM for an extended period to establish a mimic PM-exposed lung cancer cell line, A549.PM. Our results indicate that A549.PM exhibits higher cell growth and proliferation abilities compared to A549.Par cells in vitro and in vivo. The RNA sequencing analysis found amphiregulin (AREG) plays a critical role in PM-induced cell proliferation. We observed that PM increases AREG-dependent lung cancer proliferation through glutamine metabolism. In addition, the EGFR/PI3K/AKT/mTOR signaling pathway is involved in PM-induced solute carrier family A1 member 5 (SLC1A5) expression and glutamine metabolism. Our findings offer important insights into how lung cancer proliferation develops upon exposure to PM.

The growth factors amphiregulin and epiregulin are novel stimulators of feline ovarian granulosa cell functions.

This study aimed to investigate the impact of the epidermal growth factor receptor ligands amphiregulin (AREG) and epiregulin (EREG) on the fundamental functions of feline ovarian granulosa cells. Granulosa cells isolated from feline ovaries were incubated with AREG and EREG (0, 0.1, 1 or 10 ng/mL). The effects of these growth factors on cell viability, proliferation (assessed through BrdU incorporation), nuclear apoptosis (evaluated through nuclear DNA fragmentation) and the release of progesterone and estradiol were determined using Cell Counting Kit-8 assays, BrdU analysis, TUNEL assays and ELISAs, respectively. Both AREG and EREG increased cell viability, proliferation and steroid hormone release and reduced apoptosis. The present findings suggest that these epidermal growth factor receptor ligands may serve as physiological stimulators of feline ovarian cell functions.

Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

BACKGROUND: Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. METHODS: Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. RESULTS: We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. CONCLUSION: Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

The protective roles of integrin α4ß7 and Amphiregulin-expressing innate lymphoid cells in lupus nephritis.

Type 2 innate lymphoid cells (ILC2s) have emerged as key regulators of the immune response in renal inflammatory diseases such as lupus nephritis. However, the mechanisms underlying ILC2 adhesion and migration in the kidney remain poorly understood. Here, we revealed the critical role of integrin α4ß7 in mediating renal ILC2 adhesion and function. We found that integrin α4ß7 enables the retention of ILC2s in the kidney by binding to VCAM-1, E-cadherin, or fibronectin on structural cells. Moreover, integrin α4ß7 knockdown reduced the production of the reparative cytokine amphiregulin (Areg) by ILC2s. In lupus nephritis, TLR7/9 signaling within the kidney microenvironment downregulates integrin α4ß7 expression, leading to decreased Areg production and promoting the egress of ILC2s. Notably, IL-33 treatment upregulated integrin α4ß7 and Areg expression in ILC2s, thereby enhancing survival and reducing inflammation in lupus nephritis. Together, these findings highlight the potential of targeting ILC2 adhesion as a therapeutic strategy for autoimmune kidney diseases.

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells.

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Hepatocellular RECK as a Critical Regulator of Metabolic Dysfunction-associated Steatohepatitis Development.

BACKGROUND & AIMS: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is an extracellular matrix regulator with anti-fibrotic effects. However, its expression and role in metabolic dysfunction-associated steatohepatitis (MASH) and hepatic fibrosis are poorly understood. METHODS: We generated a novel transgenic mouse model with RECK overexpression specifically in hepatocytes to investigate its role in Western diet (WD)-induced liver disease. Proteomic analysis and in vitro studies were performed to mechanistically link RECK to hepatic inflammation and fibrosis. RESULTS: Our results show that RECK expression is significantly decreased in liver biopsies from human patients diagnosed with MASH and correlated negatively with severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and fibrosis. Similarly, RECK expression is downregulated in WD-induced MASH in wild-type mice. Hepatocyte-specific RECK overexpression significantly reduced hepatic pathology in WD-induced liver injury. Proteomic analysis highlighted changes in extracellular matrix and cell-signaling proteins. In vitro mechanistic studies linked RECK induction to reduced ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and ADAM17 activity, amphiregulin release, epidermal growth factor receptor activation, and stellate cell activation. CONCLUSION: Our in vivo and mechanistic in vitro studies reveal that RECK is a novel upstream regulator of inflammation and fibrosis in the diseased liver, its induction is hepatoprotective, and thus highlights its potential as a novel therapeutic in MASH.

The ST2+ Treg/amphiregulin axis protects from immune-mediated hepatitis.

Introduction: The alarmin IL-33 has been implicated in the pathology of immune-mediated liver diseases. IL-33 activates regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) expressing the IL-33 receptor ST2. We have previously shown that endogenous IL-33/ST2 signaling activates ILC2s that aggravate liver injury in murine immune-mediated hepatitis. However, treatment of mice with exogenous IL-33 before induction of hepatitis ameliorated disease severity. Since IL-33 induces expression of amphiregulin (AREG) crucial for Treg function, we investigated the immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis. Methods: C57BL/6, ST2-deficient (Il1rl1-/-) and Areg-/- mice received concanavalin A to induce immune-mediated hepatitis. Foxp3Cre+ x ST2fl/fl mice were pre-treated with IL-33 before induction of immune-mediated hepatitis. Treg function was assessed by adoptive transfer experiments and suppression assays. The effects of AREG and IL-33 on ST2+ Tregs and ILC2s were investigated in vitro. Immune cell phenotype was analyzed by flow cytometry. Results and discussion: We identified IL-33-responsive ST2+ Tregs as an effector Treg subset in the murine liver, which was highly activated in immune-mediated hepatitis. Lack of endogenous IL-33 signaling in Il1rl1-/- mice aggravated disease pathology. This was associated with reduced Treg activation. Adoptive transfer of exogenous IL-33-activated ST2+ Tregs before induction of hepatitis suppressed inflammatory T-cell responses and ameliorated disease pathology. We further showed increased expression of AREG by hepatic ST2+ Tregs and ILC2s in immune-mediated hepatitis. Areg-/- mice developed more severe liver injury, which was associated with enhanced ILC2 activation and less ST2+ Tregs in the inflamed liver. Exogenous AREG suppressed ILC2 cytokine expression and enhanced ST2+ Treg activation in vitro. In addition, Tregs from Areg-/- mice were impaired in their capacity to suppress CD4+ T-cell activation in vitro. Moreover, application of exogenous IL-33 before disease induction did not protect Foxp3Cre+ x ST2fl/fl mice lacking ST2+ Tregs from immune-mediated hepatitis. In summary, we describe an immunoregulatory role of the ST2+ Treg/AREG axis in immune-mediated hepatitis, in which AREG suppresses the activation of hepatic ILC2s while maintaining ST2+ Tregs and reinforcing their immunosuppressive capacity in liver inflammation.

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration.

BACKGROUND & AIMS: Isthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. METHODS: We recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. Bromodeoxyuridine incorporation was used to trace newly generated cells during the injury and recovery phases. To define the epithelial lineage commitment process during recovery, we performed single-cell RNA-sequencing on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, using recombinant AREG treatment or AREG-deficient mice. RESULTS: We determined that an epidermal growth factor receptor ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through single-cell RNA-sequencing and bromodeoxyuridine incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, transforming growth factor-α did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration but increases surface cell commitment. CONCLUSIONS: These data demonstrate that different epidermal growth factor receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

The muscle regeneration marker FOXP3 is associated with muscle injury in Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), the immune system cells (ISC) synthesize molecules to regulate inflammation, a process needed to regenerate muscle. The relationship between those molecules and the muscle injury is unknown. Monocytes belonging to ISC are regulated by omega-3 fatty acids (ω-3 LCPUFAs) in DMD, but whether those fatty acids influence other ISC like T-cells is unknown. OBJECTIVE: We analyzed the expression of the muscle regeneration markers (FOXP3 and AREG) in circulating leukocytes of DMD patients with different lower limb muscle functions and whether ω-3 LCPUFAs regulate the expression of those markers, and the populations of circulating T-cells, their intracellular cytokines, and disease progression (CD69 and CD49d) markers. METHODS: This placebo-controlled, double-blind, randomized study was conducted in DMD boys supplemented with ω-3 LCPUFAs (n = 18) or placebo (sunflower oil, n = 13) for six months. FOXP3 and AREG mRNA expression in leukocytes, immunophenotyping of T-cell populations, CD49d and CD69 markers, and intracellular cytokines in blood samples were analyzed at baseline and months 1, 2, 3, and 6 of supplementation. RESULTS: Patients with assisted ambulation expressed higher (P = 0.015) FOXP3 mRNA levels than ambulatory patients. The FOXP3 mRNA expression correlated (Rho = -0.526, P = 0.03) with the Vignos scale score at month six of supplementation with ω-3 LCPUFAs. CD49d + CD8 + T-cells population was lower (P = 0.037) in the ω -3 LCPUFAs group than placebo at month six of supplementation. CONCLUSION: FOXP3 is highly expressed in circulating leukocytes of DMD patients with the worst muscle function. Omega-3 LCPUFAs might modulate the synthesis of the adhesion marker CD49d + CD8 + T-cells, but their plausible impact on FOXP3 needs more research.

Amphiregulin improves ventricular remodeling after myocardial infarction by modulating autophagy and apoptosis.

Myocardial infarction (MI) is defined as sudden ischemic death of myocardial tissue. Amphiregulin (Areg) regulates cell survival and is crucial for the healing of tissues after damage. However, the functions and mechanisms of Areg after MI remain unclear. Here, we aimed to investigate Areg's impact on myocardial remodeling. Mice model of MI was constructed and Areg-/- mice were used. Expression of Areg was analyzed using western blotting, RT-qPCR, flow cytometry, and immunofluorescence staining. Echocardiographic analysis, Masson's trichrome, and triphenyltetrazolium chloride staining were used to assess cardiac function and structure. RNA sequencing was used for unbiased analysis. Apoptosis and autophagy were determined by western blotting, TUNEL staining, electron microscopy, and mRFP-GFP-LC3 lentivirus. Lysosomal acidity was determined by Lysotracker staining. Areg was elevated in the infarct border zone after MI. It was mostly secreted by macrophages. Areg deficiency aggravated adverse ventricular remodeling, as reflected by worsening cardiac function, a lower survival rate, increased scar size, and interstitial fibrosis. RNA sequencing analyses showed that Areg related to the epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/protein kinase B (PI3K-Akt), mammalian target of rapamycin (mTOR) signaling pathways, V-ATPase and lysosome pathways. Mechanistically, Areg exerts beneficial effects via increasing lysosomal acidity to promote autophagosome clearance, and activating the EGFR/PI3K/Akt/mTOR signaling pathway, subsequently inhibiting excessive autophagosome formation and apoptosis in cardiomyocytes. This study provides a novel evidence for the role of Areg in inhibiting ventricular remodeling after MI by regulating autophagy and apoptosis and identifies Areg as a potential therapeutic target in ventricular remodeling after MI.

Clinical significance of amphiregulin in patients with chronic kidney disease.

BACKGROUND: Amphiregulin (AREG) is a ligand of epidermal growth factor receptor (EGFR), which plays an important role in injury-induced kidney fibrosis. However, the clinical significance of serum soluble AREG in chronic kidney disease (CKD) is unclear. In this study, we elucidated the clinical significance of serum soluble AREG in CKD by analyzing the association of serum soluble AREG levels with renal function and other clinical parameters in patients with CKD. METHODS: In total, 418 Japanese patients with CKD were enrolled, and serum samples were collected for the determination of soluble AREG and creatinine (Cr) levels, and other clinical parameters. Additionally, these parameters were evaluated after 2 and 3 years. Moreover, immunohistochemical assay was performed ate AREG expression in the kidney tissues of patients with CKD. RESULTS: Soluble AREG levels were positively correlated with serum Cr (p < 0.0001). Notably, initial AREG levels were positively correlated with changes in renal function (ΔCr) after 2 (p < 0.0001) and 3 years (P = 0.048). Additionally, soluble AREG levels were significantly higher (p < 0.05) in patients with diabetic nephropathy or primary hypertension. Moreover, AREG was highly expressed in renal tubular cells in patients with advanced CKD, but only weakly expressed in patients with preserved renal function. CONCLUSION: Serum soluble AREG levels were significantly correlated with renal function, and changes in renal function after 2 and 3 years, indicating that serum soluble AREG levels might serve as a biomarker of renal function and renal prognosis in CKD.