Scleroderma is a multisystem disease in which tissue fibrosis is caused by inflammation and vascular damage. The mortality of scleroderma has remained high due to a lack of effective treatments. However, exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs)-Ex have been regarded as potential treatments for various autoimmune diseases, and may also act as candidates for treating scleroderma.Mice with scleroderma received a single 50 μg HUMSCs-Ex. HUMSCs-Ex was characterized using transmission electron microscopy, nanoparticle tracking analysis and nanoflow cytometry. The therapeutic efficacy was assessed using histopathology, immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and western blot.HUMSCs-Ex ameliorated the deposition of extracellular matrix and suppressed the epithelial-mesenchymal transition process, and the effects lasted at least three weeks. In addition, HUMSCs-Ex promoted M1 macrophage polarization and inhibited M2 macrophage polarization, leading to the restoration of the balance of M1/M2 macrophages.We investigated the potential antifibrotic and anti-inflammatory effects of HUMSCs-Ex in a bleomycin-induced mouse model of scleroderma. So HUMSCs-Ex could be considered as a candidate therapy for scleroderma.The online version contains supplementary material available at 10.1007/s13770-021-00405-5. Scleroderma is an autoimmune tissue disease characterized by vascular dysfunction, inflammatory disorder and extensive fibrosis . AlthougMacrophages are involved in the process of scleroderma . After iStem cells are a kind of cell with the potential for self-renewal and multidirectional differentiation, so they have been used in the repair of various tissues and organs. Multiple studies have shown that mesenchymal stem cells (MSCs) mainly rely on paracrine factors to play a therapeutic role, and exosomes are an important component of paracrine factors , 10. Exo2 and 37 °C. The cells at passage four were used for experiments. The cell culture supernatant was centrifuged at 2000 × g for 30 min to eliminate cell debris. After centrifugation at 10,000 × g for 45 min, the supernatant was filtered through a 0.22-μm filter . The supernatant was transferred to new tubes and centrifuged at 100,000 × g for 70 min in an ultracentrifuge . After a second ultracentrifugation, exosomes were resuspended in 100 μL of cold PBS and taken for subsequent identification. The concentration of HUMSCs-Ex was measured using a BCA protein assay kit . Transmission electron microscopy (TEM)(Hitachi), nanoparticle tracking analysis (NTA) and nanoflow cytometry (nFCM)(NanoFCM) were used to analyze the morphology and surface markers of HUMSCs-Ex.Human umbilical cord mesenchymal stem cells were obtained from Shanghai Tenth People’s Hospital and cultured in DMEM/F12 complete medium  at 5% COFemale Balb/c mice aged 6 to 8 weeks were purchased from Shanghai SLAC Laboratory Animal Center. Female Balb/c mice aged 6 to 8 weeks were purchased from Shanghai SLAC Laboratory Animal Center. We did not explore the effects of the gender of the mice on results, but female mice are commonly used in the bleomycin-induced mouse model of scleroderma. Mice were randomly divided into the control group and the bleomycin-induced group. Mice in the bleomycin-induced group were subcutaneously injected with 100 μl bleomycin (Thermo Fisher Scientific) at 1 mg/mg once a day for 4 weeks. Mice in the control group were injected with 100 μL phosphate buffered saline (PBS) for 4 weeks. On day 28, the bleomycin-induced group was randomly divided into the exosome (Ex) group and the new bleomycin-induced (BLM) group. Mice in the Ex group were subcutaneously injected on their backs with 100 μL of PBS containing 50 μg of HUMSCs-Ex, while the control group and BLM group were injected with 100 μL PBS. Mice were randomly sacrificed, and skin and blood samples were collected at 1, 2, and 3 weeks after HUMSCs-Ex or PBS injection.Specimens from skin lesions were fixed overnight with 4% paraformaldehyde for 24 h at 4 °C, paraffin-embedded and sectioned at 4 μm. The sections were stained with hematoxylin & eosin (H&E) or Masson’s trichrome to assess the dermal thickness and collagen content, respectively. The histologic sections were scanned by pannoramic scan . Then the images were observed and photographed using CaseViewer software  at 100 × magnification and quantified in Image J software . Three microscopic fields were selected for each section. Dermal thickness was defined as the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction. Collagen content was defined as the percentage of collagen deposition area to total area.2O2 for 10 min and then 10% normal goat serum albumin  for 10 min. The sections were stained with mouse monoclonal antibodies against α-SMA , Vimentin , E-cadherin  and TGF-β1  overnight at 4 °C. The sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. Diaminobenzidine solution was used as a chromogen to visualize the immunostaining results. The immunohistochemistry sections were scanned by pannoramic scan. Then, the images were observed and photographed using CaseViewer software at 400 × magnification, and quantified in Image-Pro Plus software . Five microscopic fields were selected for each section, and the mean intensity optical density (IOD) was used to quantify the protein expression level.The paraffin-embedded skin sections were incubated with 3% HThe paraffin-embedded skin sections were blocked with 10% normal goat serum albumin and incubated with mouse anti-iNOS antibody  or rabbit anti-CD206 antibody , and rabbit anti-F4/80 antibody  overnight at 4 °C. The sections were incubated with Alexa Fluor 488 AffiniPure goat anti-rabbit IgG  or Alexa Fluor 488-conjugated AffiniPure goat anti-mouse IgG  and Cy3-conjugated AffiniPure goat anti-rabbit IgG  at for 1 h. Then nuclei were stained with 6-diamidino-2-phenylindole. The sections were observed by fluorescence microscopy  and scanned by pannoramic scan. Then the double positive cells were counted by CaseViewer software at 630 × magnification and five microscopic fields were selected for each section.The levels of IL-4, IL-6, IL-10 and IL-12p40 in serum were quantified by ELISA kits (BOSTER) according to the manufacturer’s instructions. The absorbance was determined at 450 nm by an ELISA reader  and the actual concentration was then calculated using the standard curve.Total RNA was extracted from skin samples using TRIzol reagent . Complementary DNAs (cDNAs) were reverse transcribed using the PrimeScript™ RT reagent Kit . qPCR was performed with AceQ Universal SYBR qPCR Master Mix  in a BIOER 9600 real-time PCR system. Target gene expression levels were calculated using the 2 − ΔΔCt method, and were normalized to ACTB. The primers used were as follows: col1α1,  5′-CTGGCGGTTCAGGTCCAAT-3′ (forward primer) and 5′-TTCCAGGCAATCCACGAGC-3′ (reverse primer); col3α1, 5′-CTGTAACATGGAAACTGGGGAAA-3′ (forward primer) and 5′-CCATAGCTGAACTGAAAACCACC-3′ (reverse primer); ACTB, 5′-GTGACGTTGACATCCGTAAAGA-3′ (forward primer) and 5′-GCCGGACTCATCGTACTCC-3′ (reverse primer).Skin samples were lysed with RIPA buffer (BOSTER) and PMSF (BOSTER) to extract protein. The concentration of protein was determined with a BCA protein assay kit (BOSTER). An equivalent amount of protein was loaded onto 10% SDS-PAGE gels (BOSTER), electrophoretically treated and then transferred to PVDF membranes (Millipore). The membranes were incubated with 5% nonfat milk in TBS-T for 2 h at room temperature, and then incubated with primary antibodies at 4 °C overnight. The membranes were incubated with secondary antibodies for 2 h at room temperature, and the protein bands were visualized using ECL reagents. Finally, the bands were photographed using gel image analysis system  and the intensity of the bands was quantified in Gel-Pro analyzer software (Media Cybernetics). The details of the primary and secondary antibodies are as follows: anti-iNOS , anti-Arg-1 , and horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG .P values < 0.05 were considered statistically significant. Data are presented as the mean ± SD.All statistical analyses were performed using GraphPad Prism software version 8.0.1 . One-way ANOVA was used to analyze the significant differences when multiple experimental groups were compared. TEM images showed that HUMSCs-Ex presented a globulare shape in Fig. As shown in Fig. + myofibroblasts was found in the BLM group than in the control group. HUMSCs-Ex reduced the number of α-SMA+ myofibroblasts. The above changes in Vimentin, E-cadherin and α-SMA were similar at 1 week, 2 weeks and 3 weeks  . HUMSCs-Ex reduced the protein expression levels of IL-4 and IL-10 . Compared with the control group, the number of F4/80+ iNOS+ positive cells (M1 macrophages) decreased in the BLM group. HUMSCs-Ex treatment increased the number of M1 macrophages  increased in the BLM group, and was attenuated by HUMSCs-Ex treatment  Fig. . The proAt present, combination treatment with methotrexate and corticosteroids is a conventional treatment for scleroderma, but it is associated with severe side effects such as pancytopenia . AlthougAlthough some studies have been conducted on MSCs for scleroderma, there are few reports of their use in the clinic. One of the existing studies shows that three months after a patient with refractory scleroderma injected with MSCs, the patient's ulcerations decreased and the blood circulation of their hands improved ; anotherStudies have shown that bone marrow mesenchymal stem cells or adipose mesenchymal stem cells  or theirWe observed that HUMSCs-Ex alleviated bleomycin-induced skin fibrosis for at least 3 weeks. Additionally, we identified the key proteins α-SMA, Vimentin, and E-cadherin, and found that HUMSCs-Ex inhibited the transition from the endothelial form to the mesenchymal form. Takahashi  analyzedActivated macrophages can trigger a cascade of immune responses, and the loss of the balance of M1/M2 macrophages can lead to the occurrence of diseases. The most typical examples are M1 macrophages, which can cause acute inflammation and tissue damage, and M2 macrophages, which are involved in wound healing and induce fibrosis . Skin CDIn conclusion, HUMSCs-Ex stimulated macrophage phenotypes from M2 to M1 and suppressed EMT, which might explain why HUMSCs-Ex played an antifibrotic role in scleroderma. For the first time, our study provided preliminary evidence for the clinical treatment of HUMSCs-Ex in scleroderma.Supplementary file1 (RAR 1213 kb)Below is the link to the electronic supplementary material.


Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active β-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/β-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors . Thus, the level of active nuclear β-catenin was increased, leading to increased levels of EMT inducers  and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active β-catenin and EMT inducers. The Wnt signaling pathway is a key molecular cascade that regulates cell fate in animals. In the canonical Wnt pathway, β-catenin is the key effector responsible for signal transduction to the nucleus and the activation of Wnt-specific genes responsible for the control of cell fate . When WnEMT is a key element of tumor progression that facilitates tumor cell migration and invasion, consequently supporting metastasis . InflammIn the present study, we show for the first time that the MCPIP1 protein is crucial for regulating the expression of negative regulators of canonical Wnt signaling , inhibiting the activation of β-catenin and maintaining the epithelial phenotype. We present evidence that the RNase activity of MCPIP1 might mediate tumor progression by controlling the transcriptional activation of β-catenin; promoting the expression of the transcription factors SNAI1, SNAI2, TWIST, and ZEB1; and inducing EMT.ITGA5, COL1A1 and FN1 and decreased levels of CLDN10, CDH1, TJP1 and OCLN, which are characteristic of the epithelial phenotype, in ccRCC patients versus healthy volunteers  or its RNase-depleted form (MCPIP1-D141) or silencing MCPIP1 (shMCPIP1)  Fig. . Tumors P1) Fig. . MesenchP1) Fig. . We obseP1) Fig. . Stable P1) Fig. . We obseP1) Fig. .Fig. 1InThe results regarding the impact of MCPIP1 on the regulation of the EMT process directed our interest toward the role of MCPIP1 in normal cells. As a model, we used an immortalized cell line derived from renal proximal tubule epithelial cells (RPTECs), which did not exhibit chromosomal abnormalities . SilenciOne hallmark of EMT is the loss of E-cadherin function, which results in the dissociation of β-catenin from the membrane and an increase in β-catenin-dependent transcription , 31. TheThe regulation of β-catenin occurs via its phosphorylation, which is crucial for its activity and degradation. β-Catenin can be phosphorylated on Ser45 and then on Ser33, Ser37, and Thr41, which mediates its degradation by the proteasome , 9. ImmuMoreover, tumors lacking MCPIP1 RNase activity had the highest level of active β-catenin (non-P S45) in the two investigated mouse models Fig. . Tumors SFRP4, ZNRF3, KREMEN1, CXXC4, and CSNK1A1 than overexpression of MCPIP1 lacking RNase activity  carcinogenesis and progression . We notiCF3 Fig. . MoreoveCF3 Fig. . It has CF3 Fig. . In addiCF3 Fig. . The RNaels Fig. . A similels Fig. .Fig. 6ThOur studies also showed that exposure of Caki-1 cells harboring mutations in the RNase domain of MCPIP1 to inhibitors of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p led to decreases in the levels of Wnt pathway inhibitors and increases in the expression of EMT inducers, confirming the importance of MCPIP1 in regulating the expression of transcription factors activating EMT Fig. .SFRP4, KREMEN1, ZNFR3, CXXC4 and CSNK1A1, which were considerably lower in grade III and IV tumors than in grade I and II tumors  , and we evaluated the correlations of the gene expression level of CTNNB1, which encodes β-catenin, with the gene expression levels of the transcription factors SNAI1, SNAI2 and ZEB1 , and correlations were also observed for SNAI2  and SNAI1 .Activated β-catenin has been found to be associated with advanced kidney cancers and lower patient survival . To asseors Fig. . Microarors Fig. . Moreove02) Fig. . Using TWe previously revealed that during ccRCC progression, the level of MCPIP1 decreases. Moreover, a low level of MCPIP1 in ccRCC cells increases the levels of vimentin and β-catenin and decreases that of E-cadherin, indicating that MCPIP1 may control the acquisition of mesenchymal features . In the SFRP4, ZNRF3, KREMEN1, CXXC4 and CSNK1A1, which act as negative regulators of Wnt signaling and reduce the level of active β-catenin. In addition, previous studies have shown that negative regulators of β-catenin and Wnt signaling play important roles in the development and progression of RCC . Mice were handled according to the regulations of national and local animal welfare bodies. Six-week-old female Foxn1t test, one-way ANOVA or two-way ANOVA as calculated with GraphPad Prism 7. P values for differentially expressed miRNAs were corrected for multiple comparisons using the Benjamini–Hochberg approach .Experiments were conducted with three independent replicates. The results are shown as the mean ± SD. Statistical significance was determined using two-tailed unpaired Student’s For the methods used for transduction, western blot analysis, mRNA extraction, real-time PCR analysis, miRNA validation, staining and microarray analysis, please see the “Supplementary MethodsSupplementary Figures


The Wnt/β-catenin pathway plays an important role in tumor progression and chemotherapy resistance and seems to be essential for the maintenance of cancer stem cells (CSC) in several tumor types. However, the interplay of these factors has not been fully addressed in bladder cancer. Here, our goal was to analyze the role of the Wnt/β-catenin pathway in paclitaxel resistance and to study the therapeutic efficacy of its inhibition in bladder cancer cells, as well as to determine its influence in the maintenance of the CSC-like phenotype in bladder cancer. Our results show that paclitaxel-resistant HT1197 cells have hyperactivation of the Wnt/β-catenin pathway and increased CSC-like properties compared with paclitaxel-sensitive 5637 cells. Paclitaxel sensitivity diminishes in 5637 cells after β-catenin overexpression or when they are grown as tumorspheres, enriched for the CSC-like phenotype. Additionally, downregulation of β-catenin or inhibition with XAV939 sensitizes HT1197 cells to paclitaxel. Moreover, a subset of muscle-invasive bladder carcinomas shows aberrant expression of β-catenin that associates with positive expression of the CSC marker ALDH1A1. In conclusion, we demonstrate that Wnt/β-catenin signaling contributes to paclitaxel resistance in bladder cancer cells with CSC-like properties. Bladder cancer is the tenth most commonly diagnosed cancer worldwide, with approximately 573,000 new cases and 213,000 deaths in 2020  and incubated for 10 min on ice. Then, 5 µL of allophycocyanin (APC)-conjugated anti-CD44 antibody  and 5 µL of phycoerythrin (PE)-conjugated anti-CD24 antibody  were added and incubated for 30 min on ice, then two washes with PBS were performed. Cells were centrifuged for 10 min at 3000 rpm and finally resuspended in 500 µL of PBS for examination using a FACS Canto II analytical flow cytometer . Samples without antibodies and single labeling with APC-CD44 or PE-CD24 were used as controls. The results were analyzed using Diva software .To study the expression of CD44 and CD24, 10Validated ON-TARGETplus SMART pools of CTNNB1 (L-003482) and non-targeting (D-001810) siRNA, as a negative control, were obtained from GE Dharmacon . Transfections were carried out using DharmaFECT 2 reagent  according to the manufacturer’s instructions. All siRNA pools were used at 50 nM. Cells were subjected to the different treatments 24 h after siRNA transfection. pCMV6-XL5 and pCMV6-XL5-CTNNB1 plasmids  were transiently transfected using the FuGENE reagent , according to the manufacturer’s instructions. Cells were subjected to the different treatments 24 h after plasmid transfection.Ser9 (1:1000) were from Cell Signaling ; and mouse monoclonal anti-active-β-catenin (1:500) was from Millipore .Mouse monoclonal anti-PARP (1:500), anti-β-catenin  and anti-E-cadherin (1:4000) were from BD Biosciences ; mouse monoclonal anti-ALDH1A1 (1:500), rabbit polyclonal anti-Sox-2 (1:3000) and anti-Oct-4 (1:2000) were from Santa Cruz ; mouse monoclonal anti-β-actin  was from Sigma-Aldrich ; rabbit polyclonal anti-cleaved caspase-3 (Asp175) (1:500) and anti-p-GSK3βCells were lysed in Nonidet P-40 (NP40) lysis buffer . Equal amounts of total protein, as determined by the BCA protein assay kit , were subjected to SDS-PAGE on 8% polyacrylamide gels and transferred to Hybond ECL nitrocellulose membranes . Blots were stained with Ponceau S to ensure protein amounts were equal. For immunodetection, blots were soaked in blocking buffer [1% Blocking Reagent  in 0.05% Tween 20-PBS] for 1 h and incubated with primary antibody in blocking buffer overnight at 4 °C. Blots were then washed in 0.05% Tween 20-PBS and incubated with either goat anti-mouse IgG  or goat anti-rabbit IgG  peroxidase-labeled antibodies in blocking buffer for 1 h. An enhanced chemiluminiscent ECL system  was applied according to the manufacturer’s protocol. All experiments were performed in triplicate. Scanning densitometry of blots was analyzed using QuantiScan software .2O2 aqueous solution for 30 min to exhaust endogenous peroxidase. Heat-induced epitope retrieval was performed with 1 mM EDTA (pH 9.0) in a microwave oven. Sections were incubated overnight at 4 °C with anti-β-catenin (1:1000), anti-ALDH1A1 (1:1000) and anti-Sox-2 (1:200) antibodies. Peroxidase-labeled secondary antibody and 3,3ʹ-diaminobenzidine were applied according to the manufacturer’s protocol . Slides were then counterstained with hematoxylin and mounted.Formalin-fixed, paraffin-embedded tissues from the transurethral resections of 140 patients with muscle-invasive bladder cancer were selected to make tissue microarrays using 1-mm-diameter tumor samples. The study was approved by the local ethical committee. Five-µm tissue sections were dewaxed, rehydrated and immersed in 3% Ht test. The association between the aberrant expression of β-catenin and low or high expression of ALDH1A1 was analyzed using Fisher’s exact test. Differences were considered significant when p < 0.05. Calculations were performed using Prism 6.0 .Data comparing differences between two conditions were statistically analyzed, when indicated, using the paired Student’s


Since the discovery of protein arginine methyltransferase 5 (PRMT5) and the resolution of its structure, an increasing number of papers have investigated and delineated the structural and functional role of PRMT5 in diseased conditions. PRMT5 is a type II arginine methyltransferase that catalyzes symmetric dimethylation marks on histones and non-histone proteins. From gene regulation to human development, PRMT5 is involved in many vital biological functions in humans. The role of PRMT5 in various cancers is particularly well-documented, and investigations into the development of better PRMT5 inhibitors to promote tumor regression are ongoing. Notably, emerging studies have demonstrated the pathological contribution of PRMT5 in the progression of inflammatory diseases, such as diabetes, cardiovascular diseases, and neurodegenerative disorders. However, more research in this direction is needed. Herein, we critically review the position of PRMT5 in current literature, including its structure, mechanism of action, regulation, physiological and pathological relevance, and therapeutic strategies. Arginine methylation is a ubiquitous post-translational modification (PTM) that occurs on both nuclear and cytoplasmic proteins . It is cNG-mono- methylarginine (MMA), which acts as an intermediate. Subsequently, type I PRMTs  catalyze the formation of asymmetric ω-NG, NG-dimethylarginine (ADMA), while type II PRMTs  catalyze the formation of symmetric ω-NG, N’G-dimethylarginine (SDMA) [Depending on the type of methylarginine that is introduced, PRMTs are classified into four types: type I, II, III, and IV. Type I, II, and III PRMTs transfer methyl groups from S-adenosylmethionine (SAM) to a terminal ω-guanidine nitrogen of protein arginine residue, generating methylarginine and another, product S-adenosylhomocysteine (SAH) . WhereasPRMTs are generally expressed in tissues and can methylate both histone and non-histone proteins. Methylation of arginine residues is critical to various biological processes, including cellular signaling transduction, mRNA splicing, transcription, DNA damage repair, cell proliferation and differentiation, and protein-protein interactions ,10,11,12Approximately 20 years ago, Pollack and colleagues studied the function of Janus kinase 2 (JAK2) in cell signaling, wherein they aimed to identify proteins that interact with JAK2. In this study, PRMT5, then known as Jak-binding protein 1 (JBP), was discovered and shown to possess methyltransferase activity . Ten yeaAs already mentioned, PRMT5 is a type II PRMT that symmetrically mono- or di-methylates its substrates by catalyzing the transfer of methyl groups from SAM to arginine residues on a substrate protein . This reC. elegans PRMT5 (cPRMT5), which has 34% sequence similarity with human PRMT5, showed that the presence of positively charged residues downstream of arginine is essential for high-affinity binding to a substrate [Similarly, with regard to sequence specificity, PRMT5 has an increased preference to methylate arginine residues that are sandwiched between two glycine residues (GRG motif) on RNA processing proteins . A separubstrate . These sGATA4, 6, and HOXD9. The knock-out of the PRMT5 gene in mice also causes early embryonic lethality as it prevents the pluripotency of blastocysts [The activity of PRMT5 is essential to a wide range of biological processes, including cellular growth and development, differentiation, chromatin regulation, splicing, translation, DNA damage response, protein trafficking, and cell signaling . PRMT5 istocysts . Becausestocysts . In addistocysts ,28. PRMTstocysts .In addition to histone methylation and its role in cellular development, PRMT5 coordinates other cellular processes through the methylation of diverse proteins. For example, PRMT5 activity is critical to hematopoiesis. It helps to maintain the viability and functions of hematopoietic stem cells (HSCs) through the repression of tumor protein p53 (p53), mechanistic target of rapamycin kinase (mTOR) signaling, and via regulation of the splicing of DNA repair genes . SimilarSince PRMT5 is highly involved in orchestrating several critical biological processes, its cellular function needs to be tightly regulated. Multiple sources of evidence in the literature have shown that the PRMT5’s activity is positively or negatively regulated via different mechanisms, including the alteration of its subcellular localization, turnover rate, and substrate specificity . These dPTM involves the addition or removal of distinct chemical groups on proteins to broaden proteins’ functions ,33. To dPRMT5 modulates the expression of several miRNA via its methyltransferase activity on histones at miRNA promoters . ConversARF and p16INK4a, thereby suggesting a role of MBD2/NuRD in regulating PRMT5 repressive activity on the endogenous inhibitors of the cell cycle [The substrate specificity and cellular function of PRMT5 are often directed by its associating, binding partner proteins . For exall cycle . Similarll cycle . Also, Bll cycle . In HeLall cycle . On the ll cycle . Also, tll cycle . Notablyll cycle . Nucleolll cycle . Thus, tAXIN2 and WIF1, both of which are negative regulators of wnt/β-catenin signaling [A growing number of studies have established the role of PRMT5 as a tumor promoting factor in several types of cancers. Owing to its methyltransferase activity on the histones and oncoproteins, PRMT5’s role in cancers is entrenched in distinct cellular processes like cell signaling, DNA damage response, gene regulation, and splicing, among others . Particuignaling . Similarignaling . In mantignaling . Anotherignaling . Taken tIn addition, PRMT5 promotes oncogenicity in various solid cancers, including colon, breast, prostate, lung, liver, bone, skin, ovarian, gastric, brain, and pancreatic cancers, among others . For insBeyond cancer, evidence suggests that PRMT5 plays an important role in diabetes. Type 2 diabetes mellitus (T2DM), which accounts for over 90% of diabetic patients, is mainly characterized by the dysfunction of pancreatic β-cells, resulting in defective insulin release and insulin resistance . Other aHOXA9, a gene that plays a critical role in the development of several cardiovascular diseases [GATA4, a transcription factor that regulates cardiac remodeling [Cardiovascular disease is a category of disease that occurs in the heart or blood vessels . The numdiseases . This fimodeling . Notablymodeling . Thus, tmodeling . A follomodeling . CollectThe role of PRMT5 in neurodegenerative diseases is not well studied. Given that a high expression of PRMT5 has been identified in the human brain, a few studies delineated the role of PRMT5 in neurodegenerative disorders . For insIn addition, the importance of PRMT5 in preventing Huntington’s disease (HD) has been suggested. HD is an autosomal dominant neurogenerative disease, the cause of which is often attributed to the presence of mutant polyglutamine sequence in the huntingtin (Htt) protein . Accordidel cancer cells, resulting in their targeting for destruction [Because PRMT5 is a critical regulator of several systems, it is unsurprising that the overexpression or dysregulation of PRMT5 is associated with several disease states, including several hematologic and solid-state cancers ,85,86. Ftruction . PRT543 truction . PR5-LL-truction . FurtherThe recent explosion of PRMT5 research in various disease model systems is indicative of its enormous potential as a drug target to inhibit the progression of human diseases. Undoubtedly, PRMT5 is an important type II arginine methyltransferase with diverse substrates and functions in humans. PRMT5’s unique heterooctameric structure facilitates its interaction with co-factors, partner proteins, and substrates, thus helping to maintain the genomic integrity, signal transduction, and development of cells. The clinical importance of PRMT5 is evident from its extensive role in driving or reducing inflammatory diseases, such as cancer, diabetes, neurodegeneration, and cardiovascular disease. Thus, the current knowledge on PRMT5 in the literature can be translated to improve the outcomes of patients with related diseases. Notably, our group was one of the first to establish the link of PRMT5 to inflammation through its methyltransferase activity on the NF-κB signaling pathway. Together with other groups, we have demonstrated the pathological relevance of the PRMT5/NF-κB axis in pancreatic cancer, colon cancer, and heart disease ,76. Howe


Both exendin-4 and CCK exhibited (p<0.001) proliferative and anti-apoptotic effects in BRIN BD11 beta-cells. Proliferative benefits were significantly (p<0.01) augmented by combined peptide treatment when compared to either parent peptide alone. These effects were linked to increases (p<0.001) in GLUT2 and glucokinase beta-cell gene expression, with decreased (p<0.05-p<0.001) expression of NFκB and BAX. [Lys12Pal]Ex-4/CCK exhibited prominent insulinotropic actions in vitro, coupled with beneficial (p<0.001) satiety and glucose homeostatic effects in the mice, with bioactivity evident 24 h after administration. Following twice daily injection of [Lys12Pal]Ex-4/CCK for 28 days in diabetic high fat fed (HFF) mice with streptozotocin (STZ)-induced compromised beta-cells, there were clear reductions (p<0.05-p<0.001) in energy intake and body weight. Circulating glucose was returned to lean control concentrations, with associated increases (p<0.001) in plasma and pancreatic insulin levels. Glucose tolerance and insulin secretory responsiveness were significantly (p<0.05-p<0.001) improved by hybrid peptide therapy. In keeping with this, evaluation of pancreatic histology revealed restoration of normal islet alpha- to beta-cell ratios and reduction (p<0.01) in centralised islet glucagon staining. Improvements in pancreatic islet morphology were associated with increased (p<0.05) proliferation and reduced (p<0.001) apoptosis of beta-cells. Together, these data highlight the effectiveness of sustained dual GLP-1 and CCK1 receptor activation by [Lys12Pal]Ex-4/CCK for the treatment of obesity-related diabetes.Combined activation of GLP-1 and CCK1 receptors has potential to synergistically augment the appetite-suppressive and glucose homeostatic actions of the individual parent peptides. In the current study, pancreatic beta-cell benefits of combined GLP-1 and CCK1 receptor upregulation were established, before characterising bioactivity and antidiabetic efficacy of an acylated dual-acting GLP-1/CCK hybrid peptide, namely [Lys The increasing use of GLP-1 mimetics to treat diabetes and obesity highlights the therapeutic importance of this class of drugs . Neverthvia CCK1 receptors (Cholecystokinin (CCK) is a hormone primarily secreted from gastrointestinal I-cells as well as neurons of the enteric and central nervous systems . The pepeceptors . CCK alseceptors , 15, witeceptors , 17. In eceptors  or overeeceptors  exhibit eceptors , which mvia phospholipase C or adenylate cyclase, respectively  mice with limited beta-cell compensation induced by low-dose STZ administration. Prior to this, bioactivity of [Lys12Pal]Ex-4/CCK was established in BRIN BD11 beta-cells, alongside assessment of acute and more prolonged in vivo effects on satiety and glucose homeostasis in mice. In addition, the positive interplay between GLP-1 and CCK receptor signalling on beta-cell growth and survival, with related impact on gene expression, was also studied. Ultimately, our data confirm that the profound beneficial pharmacological effects of combined activation GLP-1 and CCK receptor pathways in obesity-diabetes can be effectively harnessed within an appropriately designed single long-acting hybrid peptide.Our primary objective was to examine the therapeutic efficacy and islet cell morphology after 28 days twice daily treatment with [Lys12Pal]Ex-4/CCK (Ex-4/CCK on insulin secretion, BRIN-BD11 cells were seeded at a density of 150,000 cells per well into 24-well plates and allowed to attach overnight. Following a 40-minute preincubation (1.1 mM glucose KRB buffer), cells were incubated with KRB buffer (16.7 mM glucose) with [Lys12Pal]Ex-4/CCK or exendin-4 (10-6 M). In a second experiment cells were incubated with classical modulators of insulin secretion in combination with [Lys12Pal]Ex-4/CCK or exendin-4 (each at 10-6 M). For all experiments, BRIN-BD11 cells were incubated under test conditions for a 20-minute period at 37°C, with the resulting supernatant collected and insulin content assessed by radioimmunoassay . Mice were single-housed and kept in a temperature-controlled environment (22 ± 2°C) under a 12-hour light/dark cycle, with ad libitum access to drinking water and maintenance diet . All experiments were approved by Ulster University Animal Ethics Review Committee and conducted in accordance with the UK Animals (Scientific Procedures) Act 1986. To assess effects of [Lys12Pal]Ex-4/CCK on food intake in overnight (18 hour) fasted mice, mice (n=8) were administered with an intraperitoneal (i.p.) injection of saline vehicle (0.9% [w/v] NaCl) or [Lys12Pal]Ex-4/CCK (25 nmol/kg bw) and cumulative food consumption recorded at regular intervals over a 180 min observation period. Acute effects of [Lys12Pal]Ex-4/CCK on glucose homeostasis were also assessed in overnight fasted mice, following an i.p. injection of glucose alone (18 mmol/kg bw) or in combination with [Lys12Pal]Ex-4/CCK (25 nmol/kg bw). More persistent effects on feeding and glucose tolerance were examined as above, but with peptide injected 4, 8, 12 or 24 h prior to feeding or administration of glucose, as appropriate. In a separate series of experiments, the impact of established GLP-1 and CCK1 receptor antagonists (both at 25 nmol/kg bw), namely exendin(9-39) and SR27897  respectively, on [Lys12Pal]Ex-4/CCK induced (25 nmol/kg bw) satiety was examined.Acute 12Pal]Ex-4/CCK (25 nmol/kg bw) for 28 days. Mice were continued on high fat diet throughout the experiment. At regular intervals, cumulative energy intake and body weight were assessed, with circulating glucose, insulin and glucagon measured on day 28. At the end of the treatment period, glucose tolerance  and insulin sensitivity  tests were conducted. Terminal analyses involved dissection of pancreatic tissue, which was processed for quantification of hormone content or pancreatic islet morphology, following acid ethanol protein extraction or fixation in 4% PFA, respectively , were maintained for 3 weeks on high fat diet . After this period, they were administered three once weekly i.p. injections of streptozotocin  on weeks 3, 4 and 5. Starting on week 6, diabetic mice (non-fasting glycaemia >11.1 mmol/l) were grouped (n=8) and received twice-daily intraperitoneal injections (08:00 and 20:00) of saline vehicle (0.9% [w/v] NaCl) or Ex-4/CCK treated mice when compared to HFF-STZ controls, with values not significantly different from lean control mice  islet and beta-cell areas, that were not significantly affected by Ex-4/CCK was engineered as a long-acting GLP-1 and CCK1 receptor agonist .NI and PF contributed to the overall concept and experimental design, and reviewed the manuscript. AE, NT, and RL researched data, contributed to data interpretation, and edited the manuscript. NI, NT, and PF wrote the manuscript. All authors contributed to the article and approved the submitted version.These studies were supported by European Foundation for the Study of Diabetes (Lilly European Diabetes Research Programme) and Department for the Economy, Northern Ireland.PF and NI are named on patents filed by the University of Ulster for exploitation of peptide therapeutics.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Finally, there was significant association between higher β-catenin score and increased alkaline phosphatase, a marker of biliary injury and disease progression. Thus, β-catenin expression and activation changes during the progression of PSC, and its localization may be a useful prognostic tool for predicting recurrence of this disease.Primary sclerosing cholangitis (PSC) is a rare, chronic, cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts. We have previously demonstrated the importance of Wnt/β-catenin signaling in mouse models of PSC. In this study, we wished to determine the clinical relevance of β-catenin localization in patient samples. In livers explanted from patients diagnosed with PSC, the majority  lacked β-catenin protein expression. Biopsies from patients post-transplant were classified as recurrent or non-recurrent based on pathology reports and then scored for β-catenin activation as a function of immunohistochemical localization. Despite lack of statistical significance, patients with recurrent primary disease (n = 11) had a greater percentage of samples with nuclear, transcriptionally active β-catenin (average 58.8%) than those with no recurrence , while non-recurrence is correlated with β-catenin staining at the cell surface (average 52.63% for non-recurrent vs. 27.34% for recurrent), as determined by three different methods of analysis. β-catenin score and years-to-endpoint are both strongly associated with recurrence status ( Biliary fibrosis obstructs bile flow, which results in accumulation of “toxic bile” in the liver; this is thought to be a major contributor to the pathogenesis of this disease3.Primary sclerosing cholangitis (PSC) is a chronic, progressive, cholestatic liver disease that affects the intrahepatic and/or extrahepatic bile ducts. Continuous inflammation of the ducts causes concentric (“onion-skin”) periductal fibrosis, which eventually leads to obliteration and loss of bile ducts and the formation of a fibrous scar4. Risk factors for recurrent PSC include co-morbidities such as inflammatory bowel disease or cholangiocarcinoma5; however, there are currently no specific biochemical parameters that predict clinical outcome after transplantation. Currently, elevated levels of alkaline phosphatase (ALP), a serum marker of biliary injury, are associated with greater risk of negative outcomes, such as liver transplantation and death6. However, serum ALP levels tend to fluctuate over time due to transient blockage of strictured ducts and this may limit its usefulness as a surrogate marker for recurrence7. Histological staging of liver fibrosis in biopsies has also been used to predict whether patients will progress over time8, but this procedure is not favored due to its invasive nature and sampling variability7. Clearly, identification of biomarkers that help to predict which patients might develop recurrent PSC would have important clinical implications.There is no FDA-approved disease-altering treatment for PSC, and the only life extending treatment is liver transplantation. Almost half of patients with PSC will experience disease progression, with end stage liver disease or cancer requiring transplant. Despite transplant, the disease will recur in about 25% of patients9). Conversely, β-catenin activation also promotes the development and progression of liver diseases such as diet-induced steatohepatitis, fibrosis, and hepatocellular cancer (reviewed in10). β-catenin is expressed throughout the adult liver, and can be found at the cell membrane, where it exists as a part of adherens junctions, as well as inside the cell, where it is normally degraded in the absence of Wnts. Activation of this pathway occurs when Wnt protein binds to its receptor Frizzled and co-receptor low density lipoprotein–related protein 5 or 6 on the cell surface, resulting in a cascade of events that frees β-catenin from destruction, allowing it to accumulate in the cytoplasm and eventually translocate to the nucleus, where it activates target gene expression. In the normal adult liver, nuclear localization of β-catenin is found only in the pericentral hepatocytes, where it regulates metabolic zonation10. However, in pathological conditions such as hepatocellular carcinoma (HCC), β-catenin can become constitutively activated and translocate to the nucleus irrespective of location within the hepatic lobule.β-catenin is the effector of the Wnt signaling pathway, which has a well-described yet multifaceted role in liver physiology and pathology. Its normal activity is essential for hepatic development, biliary morphogenesis, postnatal hepatic growth, and hepatocyte maturation (reviewed in11. Cytochrome P450 (Cyp)7a1, the rate limiting enzyme in the synthesis of bile acids from cholesterol, is expressed in the pericentral zone of the liver, coincident with constitutive activation of β-catenin12. Furthermore, Cyp7a1 is suppressed in the absence of β-catenin, suggesting that this gene is either a direct or indirect target of Wnt signaling14. β-catenin also forms an inhibitory complex with farnesoid X receptor (FXR), the bile acid nuclear receptor that represses Cyp7a1. Loss of β-catenin from hepatocytes eliminates sequestration of FXR, which decreases bile acid synthesis and lowers the level of total hepatic bile acids, resulting in protection from bile duct ligation-induced liver injury, fibrosis, and ductular response13. That loss of β-catenin is associated with a protective phenotype suggests that alterations in β-catenin expression could have predictive and perhaps therapeutic implications for human cholestatic liver disease.In addition to its roles in zonation and injury repair, this signaling pathway has recently been shown to alter bile acid metabolism as well as the progression of cholestatic liver disease in animal models of PSCTo determine if these findings were relevant to PSC, we measured the expression of β-catenin protein in explanted livers from patients transplanted for PSC, as well as in post-transplant biopsies from PSC patients. The results indicate that β-catenin expression and/or localization is not only a useful marker of disease progression but is also a prognostic marker for disease recurrence.All experimental protocols were approved by the University of Pittsburgh Institutional Review Board (IRB) Office of Research Protections, and all methods were carried out in accordance with relevant guidelines and regulations. All tissues and materials used in this retrospective study were obtained under the IRB protocols listed below, and informed consent was waived by the University of Pittsburgh IRB.Frozen liver samples from 16 patients with a primary diagnosis of PSC at the time of explant, as well as liver samples from 7 control non-cholestatic patients, were obtained from the Pittsburgh Liver Research Center’s Clinical Biospecimen Repository and Processing Core  at the University of Pittsburgh, School of Medicine. The corresponding anonymous pathology reports were also obtained, and pertinent patient information is detailed in Supplemental Table For the biopsy studies, a database containing a list of patients in which the primary diagnosis is PSC and who have received orthotopic liver transplantation between 1984 and 2013 was obtained. Each patient in the database had n ≥ 1 post-transplant follow-up biopsies. Based on the pathology diagnosis that accompanied each biopsy, this database was stratified into patients with non-recurrent  and recurrent (obstruction/cholangitis or recurrent primary disease as primary diagnosis) disease. From these categories, n = 11 recurrent and n = 10 non-recurrent patients were randomly chosen for analysis. Biopsy tissues in paraffin were obtained from the Department of Pathology, School of Medicine, University of Pittsburgh . The pertinent patient information and corresponding anonymous pathology reports are detailed in Supplemental Table 15. Membranes were probed with β-catenin  from BD Biosciences, San Jose, CA; and glyceraldehyde-3-phosphate dehydrogenase  from Santa Cruz Biotechnology, Dallas, TX. Densitometry analysis of WBs was performed using ImageJ software (version 1.44o). A total of 4 WBs were run in order to assess the 7 normal and 16 PSC livers. Samples MA102, MA103, MA104, and C268, F138, G139, G336, and B805 were repeated on multiple blots, and the β-catenin/GAPDH ratios were normalized to the representative blot in order to maintain consistency between blots.Approximately 20 mg of tissue from explanted livers was extracted, resolved on Tris–HCl precast gels by SDS-PAGE, and transferred to polyvinylidene difluoride membranes as described previouslyRNA was extracted from frozen human livers, converted to cDNA, and assayed on the Applied Biosystems StepOnePlus Real-Time PCR System with StepOne v2.1 software. The comparative ΔΔCT method was used for analysis of the data and all data is presented normalized to NL at baseline. GAPDH expression was used as the internal control. The following primers were used: Cyp7a1-fwd: 5′-GGT GTT GTG CCA CGG AAA AT-3′; Cyp7a1-rev: 5′-TCC ATC CAT CGG GTC AAT GC-3′; SHP-fwd: 5′-GTG GCT TCA ATG CTG TCT GGA- ‘3; SHP-rev: 5′-ACT TCA CAC AGC ACC CAG TGA-3′; GAPDH-fwd: 5′-TCC AAA ATC AAG TGG GGC GA-3′; GAPDH-rev: 5′-TGA TGA CCC TTT TGG CTC CC-3′. For this assay, n = 16 PSC livers and n = 5 normal livers were analyzed due to insufficient sample quantity in 2 normal livers, which were used only for WB.Liver total bile acids were measured using a total bile acids kit from Crystal Chem , as per the manufacturer’s instructions. Total bile acid levels were normalized to protein concentration for each sample. For this assay, n = 16 PSC livers and n = 5 normal livers were analyzed due to insufficient sample quantity in 2 normal livers, which were used only for WB.16. A sample was considered positive for nuclear β-catenin when at least 10 positive hepatocyte nuclei were identified in a 200 × field.Paraffin sections from post-transplant biopsies were used for β-catenin IHC staining, which was performed by the BRPC. Tissue scoring was performed by an experienced pathologist (ADS) who was blinded to patient demographics and outcomes. β-catenin staining was categorized as 0, representing no stain/decreasing stain; 1, representing membranous β-catenin staining; or 2, representing membranous β-catenin with some weak nuclear staining, according to a similar classification published previouslyFibrosis scores were based on pathology reports for the amount of portal fibrosis and were scored as follows: none (0); mild (1); moderate (2); severe (3). In the few cases where classification was either mild-moderate or moderate-severe, the higher score was used.t test if p < 0.05. For analysis of post-transplant biopsies, β-catenin score distribution across recurrence and non-recurrence status was calculated. To check the association between these two variables, contingency tables were created and Fisher’s exact test was performed.For analysis of frozen liver samples, statistical analysis was performed with Prism version 7.0a (GraphPad Software), and comparisons between normal and PSC were deemed statistically significant by unpaired two-tailed Student’s To check the predictive value of β-catenin score and years-to-endpoint on the final recurrence status, a binomial logistic regression model was fitted. The β-catenin score served as the categorical variable  and years-to-endpoint was the continuous predictor variable. Binarized recurrence status (0 for non-recurrence and 1 for recurrence) was the outcome of the regression model. Once the coefficients were estimated, ANOVA test was performed on coefficient of β-catenin score, years-to-endpoint and the interaction between score and years-to-endpoint. The statistical figures and analysis were performed by R programming via publicly available packages. Regression model and ANOVA test was performed with default parameter settings.To test the association between β-catenin score and ALP, Wilcoxon Rank Sum test was applied, where p-values were calculated to test the difference in ALP between pairwise β-catenin scores. In addition, Fisher’s Exact test was applied to test the association between β-catenin score and fibrosis stage.13, we wanted to see if the same was true in human disease as well. Explanted liver tissue were obtained from sixteen patients with a primary diagnosis of PSC at the time of transplant, as well as from seven patients with non-cholestatic livers, and analyzed for β-catenin by Western blotting. Because advanced PSC causes liver fibrosis and can result in extensive loss of parenchyma, we standardized β-catenin expression to the expression of housekeeping gene GAPDH for accurate comparison. A representative Western blot is shown in Fig. Given our previous findings that loss of β-catenin allowed mouse livers to better adapt to chronic cholestatic injury13. However, SHP expression is not increased in PSC livers compared to NL, indicating lack of FXR activation in late-stage PSC  mRNA, a target of FXR signaling that negatively regulates Cyp7a1PSC Fig. C. β-cate18. We obtained a database containing a list of patients who received OLT for PSC and had n ≥ 1 post-transplant follow-up biopsies. This database was then stratified into patients with non-recurrent or recurrent disease as described in the Methods. β-catenin staining was performed on randomly selected patients from each group , and localization was categorized as absent (0), membranous only (1), or membranous/weak nuclear localization (2) as described in the “Given that β-catenin is absent in a majority of PSC livers that were analyzed at the time of orthotopic liver transplantation (OLT), we then wondered if β-catenin status may be a useful predictor for disease progression in PSC patients. To address this, we analyzed β-catenin expression in serial biopsies from a separate cohort of PSC patients that had received OLT. Follow-up needle biopsies are routinely done on PSC patients after transplant, and can be used to detect recurrent PSC, which occurs in 20–25% of recipient livers19. Non-mutated nuclear β-catenin can be difficult to detect by IHC, as it is lesser in quantity and intensity than in hepatic tumors with CTNNB1 mutations16. Therefore, biopsies with n > 10 positive hepatocyte nuclei per 200 × field were considered to have active β-catenin. Although PSC is a biliary disease, and β-catenin is strongly positive in cholangiocytes as well as in other non-parenchymal cells, we focused on hepatocyte localization because the role of activated β-catenin in cholangiocytes is unknown, whereas β-catenin activation in hepatocytes plays a variety of critical functions in cholestatic liver disease, including regulating bile acid metabolism and hepatocyte proliferation.In a normal liver, β-catenin is usually expressed at the hepatocyte membrane. However, the presence of nuclear β-catenin in hepatocytes indicates that this pathway is transcriptionally activeβ-catenin IHC from three representative non-recurrent patients is shown in Fig. The relationship of β-catenin localization to disease recurrence was analyzed in three different ways. Samples were first categorized as either recurrent or non-recurrent based on the final status of the patient; if the patient was classified as ‘recurrent’, then all the biopsies for that particular patient were counted as recurrent. The number of recurrent and non-recurrent samples were then distributed by β-catenin score Fig. A. In thiTo determine whether nuclear localization of β-catenin could be used as an indicator of disease recurrence, a binomial logistic regression was fitted to study the importance of β-catenin score and years-to-endpoint in predicting final recurrence status. We used a time cutoff of 10 years for the analysis in order to eliminate outliers  or membranous β-catenin ; mild (1); moderate 2); severe 3). The fibrosis scores were then distributed by β-catenin scores Fig. A. Figure. The fib; severe 23. In fact, the FXR agonist obeticholic acid  or INT-747) has been FDA approved for use in patients with primary biliary cholangitis (PBC)25. A phase II clinical trial has demonstrated that OCA also leads to a significant reduction in serum markers of liver injury and cholestasis in patients with PSC26. Like FXR agonism, loss of β-catenin markedly improves the cholestatic phenotype through suppression of bile acid synthesis in a mouse model of biliary injury. Therefore, we hypothesized that downregulation of β-catenin may be a possible mechanism by which the liver adapts to chronic cholestatic liver injury. Indeed, we found that β-catenin protein levels are significantly decreased in liver explants from adult PSC patients. On the other hand, in an analysis of liver biopsies post-transplant, nuclear β-catenin was more frequently associated with recurrent PSC, suggesting that activation of β-catenin may contribute to disease progression. This points to a dynamic process whereby β-catenin expression and/or activation changes during the progression of disease, indicating that it may be useful as a biomarker that could help with disease staging or prognosis.FXR agonists, which decrease bile acid biosynthesis and increase transport, have been touted as potential treatments for cholestatic liver disease associated with accumulation of toxic bile27. Thus, animal models are often used to study pathogenesis and evaluate novel therapeutic strategies. To date, no animal model fully recapitulates all of the clinical and pathological features of PSC; however, several exhibit certain characteristics resembling PSC28. Bile duct ligation (BDL) is a commonly used experimental model of “toxic bile” accumulation that results in cholestasis, portal inflammation, fibrosis, and proliferation of cholangiocytes29. Although BDL more closely resembles a model of acute biliary obstruction than sclerosing biliary disease, it is nonetheless useful in elucidating the pathways involved in the progression of cholestatic liver injury and peribiliary fibrosis. Mice with a disruption of the Mdr2 gene (Mdr2 KO) spontaneously develop sclerosing cholangitis, characterized by pericholangitis, atypical ductular proliferation, and onionskin-type periductal fibrosis, which mirrors some of the key features of human PSC31. The mechanism of liver injury is thought to be defective biliary phospholipid secretion, which results in production of “toxic bile” containing an increase in hydrophobic bile acids30. This in turn causes cholangiocyte injury leading to production of pro-fibrogenic cytokines. However, the role of Mdr3 (the human orthologue of mouse Mdr2) variants in the pathogenesis of human PSC is still unclear32. Since no single animal model exhibits all attributes of PSC, it may be relevant to study multiple models as complementary approaches to evaluate and validate the contribution of cell signaling pathways.Because of the low incidence of PSC, the difficulties in assessing cholangiocyte injury in humans, and the heterogeneity of the PSC population, the initiation and progression of PSC is not well understood13. This is due to suppression of bile acid synthesis which is enhanced by loss of β-catenin, providing evidence that inhibiting this pathway during cholestasis may alleviate injury and progression of disease. Interestingly, however, β-catenin suppression in the Mdr2 KO led to exacerbation of hepatic and biliary injury33. Since β-catenin plays an important role in the regenerative response, it is likely that the chronic injury resulting from loss of Mdr2 is worsened when the liver is unable to repair itself, leading to a more severe phenotype. This dichotomy is recapitulated in our explant studies. The majority of explanted liver tissue from end-stage PSC patients had decreased β-catenin coincident with decreased Cyp7a1 expression. The loss of β-catenin in these livers is global, affecting both β-catenin at the membrane surface and active β-catenin in the pericentral region, whereas loss of Cyp7a1 would most likely occur primarily in the pericentral region due to its regulation by β-catenin. As a consequence of decreased bile acid synthesis, hepatic bile acid levels were equivalent in both PSC and NL. Several other studies have shown no difference in biliary bile acid concentrations between PSC patients and controls, supporting this finding and suggesting that β-catenin suppression may be beneficial in preventing overproduction of harmful bile acids35. On the other hand, loss of β-catenin may also impede critical processes such as regeneration during ongoing injury, which may ultimately contribute to liver failure. In fact, the importance of β-catenin in prevention of liver failure was demonstrated in a study showing that increased nuclear β-catenin and hepatocyte proliferation was associated with spontaneous regeneration after acetaminophen overdose, alleviating the requirement for liver transplant36. Because the livers in our first cohort were explanted, it means that all attempts to compensate for injury had failed, and thus we were unable to address whether loss of β-catenin alleviates or exacerbates end-stage liver disease in PSC.In the mouse BDL model, we found that loss or inhibition of β-catenin led to decreased hepatobiliary injuryHowever, analysis of the biopsies allows us to look at β-catenin activation and expression as a function of time, which can potentially inform our interpretation of the explants. Notably, in our longitudinal studies of liver biopsies we found that activation of β-catenin (as defined by nuclear translocation) appears to predict PSC recurrence after transplant. It is not yet clear what role, if any, β-catenin signaling plays in a heterogeneous disease like PSC. However, one hypothesis supported by the biopsy data is that early activation of β-catenin in post-transplant livers might exacerbate disease progression through as-yet unknown mechanisms, while membranous β-catenin is indicative of a more mild phenotype. In light of these findings, the notable loss of β-catenin in the explanted livers indicates that in later stages of the disease, downregulating β-catenin expression may be a compensatory response by the liver to ameliorate chronic cholestatic disease.37. Whether β-catenin activation results in increased expression of Cyp7a1 or increased transcription of some other unidentified target is not known. Although evidence from mouse models suggests that this phenomenon may be attributable to increased bile acid synthesis, we were unable to verify that biopsies with nuclear β-catenin had more Cyp7a1 due to sample limitations. It should also be noted that β-catenin-driven HCC arises because of genetic mutations in members of the Wnt/β-catenin signaling pathway that result in stabilization of β-catenin. In this study, we do not propose that β-catenin translocates to the nucleus because it is mutated, as in HCC, but rather that it is activated through canonical Wnt signaling or other physiological mechanisms. Indeed, we and others have shown that several Wnts are upregulated in mouse models of cholestasis, resulting in activation of hepatocyte β-catenin39.Our findings are also in line with previous observations that active β-catenin is associated with cholestasis in HCC41. Thus, it is possible that we may have underestimated the number of β-catenin-positive hepatocyte nuclei in both recurrent and non-recurrent groups. Ideally, identification of a reliable marker of β-catenin activation that is specific for cholestatic liver disease would allow for a more accurate assessment of clinical specimens.Interestingly, 1 recurrent patient (4848) and one non-recurrent patient (1826) showed an absence of β-catenin in their final biopsy, despite having either nuclear or membranous staining in their previous biopsies. This loss may indicate a tendency to lose β-catenin expression during the later stages of disease as observed in the explanted livers; however, because the sample size is small, further studies with larger patient populations would be needed to strengthen this finding. Further confounding interpretation is the difficulty of detecting nuclear β-catenin by immunohistochemistry, as previously documented by us and othersBoth animal studies and the human studies here indicate that loss of β-catenin is protective against cholestatic liver injury and may be a way that the liver adapts to chronic cholestatic injury as a natural evolution of disease. The simultaneous downregulation of Cyp7a1 indicates a possible interaction of the Wnt/β-catenin pathway with nuclear receptors such as FXR that regulate bile acid synthesis, and although this seems to be the case in the BDL mouse model, the absence of SHP upregulation and lack of hepatic bile acid accumulation in the PSC explants suggest alternative or redundant mechanisms. Conversely, increased expression or activation of β-catenin is an indicator of worse prognosis. β-catenin localization also closely correlated with ALP level, which is also an independent predictor of recurrence in our study. Our studies revealed that the diagnostic capability of β-catenin localization is similar to that of ALP. While fibrosis may not be an accurate predictor of disease recurrence, it may be desirable to perform β-catenin IHC on biopsies taken during post-transplant follow-up in order to support a prediction of recurrence in conjunction with ALP levels. Extending this concept, it would be interesting to correlate β-catenin localization with patient response to therapeutic treatment such as OCA or ursodeoxycholic acid (UDCA), which would indicate whether β-catenin activation could be altered or even reversed. Unfortunately, data on what medication a patient received, if any, was unavailable, and thus we were not able to determine if there was any relationship between the variables.It it tempting to speculate that β-catenin is not only a biomarker for risk stratification but also a potential causative factor in PSC progression. However, as animal studies have demonstrated, β-catenin is pleiotropic and can likely have multiple functions during cholestatic liver disease, including reducing bile acid synthesis, inducing regeneration and hepatocyte proliferation, and reducing oxidative stress. Therefore, caution should be exercised when interpreting the results from these preliminary findings, especially in light of the limited size of the cohorts. Additional analyses with larger numbers of patient cases will be needed to substantiate the findings in this retrospective study and also address the mechanism by which the liver may be suppressing β-catenin during end-stage cholestatic liver disease. Nonetheless, these studies are proof-of-concept that β-catenin expression and localization may be a useful prognostic tool for PSC.Supplementary Information 1.Supplementary Information 2.


A brain region typically receives inputs from multiple upstream areas. However, currently, no method is available to selectively dissect neurons that receive monosynaptic inputs from two upstream regions. Here, we developed a method to genetically label such neurons with a single gene of interest in mice by combining the anterograde transsynaptic spread of adeno-associated virus serotype 1 (AAV1) with intersectional gene expression. Injections of AAV1 expressing either Cre or Flpo recombinases and the Cre/Flpo double-dependent AAV into two upstream regions and the downstream region, respectively, were used to label postsynaptic neurons receiving inputs from the two upstream regions. We demonstrated this labelling in two distinct circuits: the retina/primary visual cortex to the superior colliculus and the bilateral motor cortex to the dorsal striatum. Systemic delivery of the intersectional AAV allowed the unbiased detection of the labelled neurons throughout the brain. This strategy may help analyse the interregional integration of information in the brain. In this paper, a method is developed to genetically label neurons that receive monosynaptic inputs from two upstream regions of the brain. This could improve the analysis of interregional integration of information in neurons. The neurons that distribute information to two or more downstream areas through collateral projections can be readily labelled using multiple retrograde tracers4. Moreover, one can genetically target these neurons to monitor and manipulate their activity using several approaches, such as the dual retrograde viral vector infection5 and optogenetic identification of axonal projections during extracellular recordings9. In contrast, although a range of viral vectors and transgenic animals has been successfully used to genetically target specific circuit structures12, no method is currently available to selectively express a single gene of interest in neurons that receive monosynaptic inputs from multiple upstream regions, thereby limiting the study of the structure and function of such neurons.The brain consists of many distinct regions with corresponding functions. A single brain region generally receives synaptic inputs from multiple upstream areas and distributes the information processed within the region to multiple downstream regions. Such integration and distributions are fundamental interregional interactions that support a variety of brain functions14 with the previously developed intersectional expression system (INTRSECT)16. High titres of AAV1 exhibit anterograde transsynaptic spread, which depends on neurotransmitter release machinery14. Widely applied in mice22 and rats25, injection of the Cre recombinase-expressing AAV1 and AAV with a Cre-inducible expression cassette into brain regions containing presynaptic and postsynaptic neurons, respectively, allows the selective labelling of postsynaptic neurons receiving synaptic input from the presynaptic region. INTRSECT is an intersectional gene expression system that depends on multiple recombinases16. The Con/Fon expression cassette of the INTRSECT system turns on gene expression only when both Cre and Flpo recombinases are co-expressed in the target cells. We hypothesised that introducing the AAV1 expressing Cre and Flpo into two upstream brain regions and a Con/Fon expression cassette-containing AAV into the downstream region may aid in selectively labelling neurons that receive monosynaptic inputs from both upstream regions ons Fig. . As a pron/Fon cassette has been well-characterised16, this test was primarily conducted to verify the selective recombination with the newly constructed pAAV-hSyn-Flpo-3×FLAG plasmid, which expresses 3×FLAG-tagged Flpo. To do this, 293T cells were transfected with recombinase-expressing plasmids (pAAV-hSyn-Cre or pAAV-hSyn-Flpo-3×FLAG) and recombinase-inducible, enhanced yellow fluorescent protein (EYFP)-expressing plasmids (pAAV-EF1α-DIO-EYFP or pAAV-EF1α-fDIO-EYFP). Cre/DIO and Flpo/fDIO combinations selectively induced EYFP expression , unlike the cells transfected with the Cre- or Flpo-expressing plasmid alone  without back projection34. We injected either AAV1-Cre and AAV1-EF1α-DIO-EYFP or AAV1-Flpo and AAV1-EF1α-fDIO-EYFP locally into the presynaptic and postsynaptic regions, respectively , confirming recombinase-dependent EYFP expression. These results suggest that both AAV1-Cre and AAV1-Flpo exhibited anterograde transneuronal spread, inducing site-specific recombination in postsynaptic neurons in all pathways tested in this study. To the best of our knowledge, this is the first demonstration of the transneuronal spread of AAV1-Cre in the M2-DS pathway and AAV1-Flpo in the retina-SC and M2-DS pathways.We then tested the anterograde transneuronal spread of AAV1 expressing either Cre or Flpo under the hSyn promoter . AAV1 exhibits anterograde transneuronal spread in many, but not all, pathwaysely Fig. . In all ely Fig. . EYFP ex36, but whether individual sSC neurons receive monosynaptic inputs from both regions remains elusive37. We injected AAV1-Cre, AAV1-Flpo, and AAV1-hSyn-Con/Fon-EYFP into the right eye, left V1, and left SC, respectively , indicating that the injection of both vectors into the presynaptic regions  was necessary for EYFP labelling in the postsynaptic area .To genetically target neurons that receive monosynaptic inputs from two upstream regions, we performed intersectional, anterograde transneuronal labelling. The sSC receives synaptic inputs from both the retina and V1ely Fig. . Togetheely Fig.  c, many ely Fig. , d. EYFPon/Fon-EYFP expression cassette. To this end, we used AAV-PHP.eB, which crosses the blood-brain barrier and transduces the whole brain following intravenous administration38. We injected AAV1-Cre and AAV1-Flpo locally into the right eye and left V1, respectively, as well as AAV-PHP.eB-hSyn-Con/Fon-EYFP intravenously via the retroorbital sinus. EYFP-positive cells were observed in the left sSC  mainly project to the deep SC (dSC)dSC Fig. . Next, wely Fig. . As the  SC Fig. , and the34. The striatum is composed of medium spiny neurons, which are DARPP-32-positive principal cells comprising ~95% of the total striatal neuronal population, and interneurons including choline acetyltransferase (ChAT)-positive and parvalbumin (PV)-positive cells40. All these types of neurons receive monosynaptic inputs from M243. However, it remains unknown whether these individual DS neurons receive bilateral M2 monosynaptic inputs. To test this, we locally injected AAV1-Cre, AAV1-Flpo, and AAV1-hSyn-Con/Fon-EYFP into the left M2, right M2, and bilateral DS, respectively  from the AAV transfer plasmid, while maintaining a high AAV1 titre14. Although these limitations need to be addressed, our method provides a straightforward approach to genetically dissect neurons that directly receive various types of information from multiple brain regions.A potential drawback of our approach is the necessity for accurate local triple AAV injection. However, the combination with intravenous PHP.eB injection reduced the number of necessary local injections to two, and such double injection is routinely used in many studies. Moreover, several recent studies applied local tripleGuide for the Care and Use of Laboratory Animals published by the National Institutes of Health.All procedures related to animal care and use were approved by the Institutional Animal Care and Use Committee of Osaka City University (approved protocol #15030) and were performed in accordance with the on/Fon-EYFP (no. 55650), and pUCmini-iCAP-PHP.eB (no. 103005). hSyn and EF1α denote the human synapsin 1 promoter and elongation factor 1 alpha promoter, respectively. The DIO, fDIO, and Con/Fon denote Cre-dependent, Flpo-dependent, and Cre/Flpo double-dependent gene expression cassettes, respectively61. pXR1 and pHelper were obtained from the National Gene Vector Biorepository62 and the AAV Helper-Free System , respectively. During the AAV1 preparation described below, we were unable to obtain a high titre with pAAV-EF1α-mCherry-IRES-Flpo, which has a long insert (~5.0 kb) exceeding the AAV packaging limit of ~4.4 kb between inverted terminal repeats (ITRs). Therefore, we constructed a shorter plasmid with a ~3.0 kb insert between ITRs, pAAV-hSyn-Flpo-3×FLAG , which has the hSyn promoter followed by Flpo with 3×FLAG C-terminal tag . This plasmid was verified by sequencing and used throughout the study. Site-specific recombination of the DIO-, fDIO-, and Con/Fon-EYFP cassettes by Cre and/or Flpo was confirmed by transfecting the corresponding plasmids (0.26 µg/well) into 293T cells  seeded on 24-well tissue culture plates.The following plasmids were obtained from Addgene: pAAV-hSyn-Cre-WPRE (no. 105553), pAAV-EF1α-mCherry-IRES-Flpo (no. 55634), pAAV-EF1α-DIO-EYFP (no. 20296), pAAV-EF1α-fDIO-EYFP (no. 55641), pAAV-hSyn-C63. Briefly, AAV1 was produced by co-transfecting three plasmids  using PEI MAX  into 293T cells seeded on eight 15-cm tissue culture dishes. For AAV-PHP.eB38 production, the pXR1 plasmid was switched to the pUCmini-iCAP-PHP.eB plasmid (20 µg/dish). The cells were harvested 72 h after transfection, and AAV was purified using the AAVpro Purification Kit Maxi (All Serotypes)  according to the manufacturer’s instructions. The titre measurement, performed using qPCR , included the following: AAV1-Cre (1.8–2.3 × 1013 vg/mL), AAV1-Flpo (3.9 × 1013 vg/mL), AAV1-DIO-EYFP (1.4 × 1013 vg/mL), AAV1-fDIO-EYFP (1.3 × 1013 vg/mL), AAV1-Con/Fon-EYFP (1.7 × 1013 vg/mL), and PHP.eB-Con/Fon-EYFP (1.0 × 1012 vg/mL).AAV vectors were prepared as described previously with minor modifications64. Intravitreal injection65 was performed on the right eye to deliver AAV1-Cre into the retina. First, a small incision was made into the sclera using a 29-gauge needle . Then, a 33-gauge syringe  was inserted through the same incision to inject 1 µL of AAV1-Cre supplemented with 1:10 (v/v) 1% Fast Green FCF  at a speed of 3–6 µL/min into the vitreous. The needle was kept inside the vitreous for 1–2 min before removal. Retroorbital injection66 was performed for the systemic delivery of AAV-PHP.eB. A 29-gauge syringe  was inserted into the right retroorbital sinus, and 10 µL of PHP.eB-Con/Fon-EYFP  was slowly injected. Stereotaxic injections were performed to deliver AAV to specific brain areas. A craniotomy was performed above the injection site, and AAV was injected (5 µL/h) through a pulled glass pipette. Different AAVs were injected separately on different days with an interval of six or more days in the order of AAV1-Cre, AAV1-Flpo, and one of DIO-, fDIO-, or Con/Fon-EYFP. This multi-step stereotaxic injection was used to minimise any risk of contamination among the AAVs during surgery25. The coordinates67 and volumes are listed as follows: left V1 , left AC , left SC , M2 , and DS .Three types of AAV injections were performed using male C57BL/6 J mice  under anaesthesia 68. The sections and retinae were incubated sequentially with 5% bovine serum albumin (BSA)/0.3% Triton X-100 in PBS for either 30 min (for sections) or 60 min (for retinae) at room temperature, primary antibodies in 5% BSA/PBS overnight at 4 °C, and corresponding secondary antibodies with DAPI  in 5% BSA/PBS either for 2 h at room temperature or overnight at 4 °C. The samples were washed with PBS between incubations. The following primary antibodies were used: mouse anti-Cre recombinase , rabbit anti-FLAG (DYKDDDDK) , chicken anti-GFP , rabbit anti-DARPP-32 , goat anti-ChAT , and rabbit anti-PV . The following secondary antibodies  were used at a dilution of 1:800: goat anti-chicken IgY conjugated with Alexa Fluor 488 (A-11039), goat anti-mouse IgG with Alexa Fluor 594 (A-11032), goat anti-rabbit IgG with Alexa Fluor 594 (A-11037), donkey anti-goat IgG with Alexa Fluor 594 (A-11058), and goat anti-rabbit IgG with Alexa Fluor 647 (A-21245). The sections and retinae were mounted on coverslips with an antifade mountant , and fluorescent images were obtained using confocal microscopes  equipped with 10×  and 20×  objectives. Cells positive for EYFP, ChAT, or PV were detected from the fluorescent images using automated software , followed by manual correction of erroneous detections25. DARPP-32-positive cells were detected manually using the Fiji software (ver. 2.1.0)69.Mice were transcardially perfused with 0.9% saline and then 4% paraformaldehyde in 0.1 M phosphate buffer under anaesthesia after 18 ± 4 days following the last surgery. The brains and the eyes  were enucleated and stored in the same fixative overnight at 4 °C. The brains were then transferred and kept in 30% sucrose in PBS for more than 48 h at 4 °C and sectioned using a freezing microtome  at a thickness of 40 μm parallel to the coronal plane. Whole-mount retinae were prepared by removing the cornea, sclera, and lens from the eyes and making radial incisions into the retinaP < 0.05 obtained using the Mann–Whitney U test was considered as a significant difference. All data are reported as the mean ± standard deviation unless specified otherwise.Statistical analysis was performed using IBM SPSS Statistics . Further information on research design is available in the Peer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryEditorial RequestStyle and formatting checklistFile checklistRevised manuscript submission file checklist


Iron accumulates in the vital organs with aging. This is associated with oxidative stress, inflammation, and mitochondrial dysfunction leading to age-related disorders. Abnormal iron levels are linked to neurodegenerative diseases, liver injury, cancer, and ocular diseases. Canonical Wnt signaling is an evolutionarily conserved signaling pathway that regulates many cellular functions including cell proliferation, apoptosis, cell migration, and stem cell renewal. Recent evidences indicate that iron regulates Wnt signaling, and iron chelators like deferoxamine and deferasirox can inhibit Wnt signaling and cell growth. Canonical Wnt signaling is implicated in the pathogenesis of many diseases, and there are significant efforts ongoing to develop innovative therapies targeting the aberrant Wnt signaling. This review examines how intracellular iron accumulation regulates Wnt signaling in various tissues and their potential contribution in the progression of age-related diseases. Hence, abnormal Wnt signaling can be detrimental and is implicated in diseases across different tissue types. Interestingly, an overlap between dysregulated iron homeostasis and aberrant Wnt signaling exists in pathologies including colorectal carcinoma (CRC) [A multitude of age-related diseases are associated with disrupted cellular iron homeostasis. Wnt/ma (CRC) –3, diabema (CRC) , 5, and ma (CRC) –8. For tβ (IL-1β) secretion and NLRP3 inflammasome stimulation in human monocytes through the NF-κB pathway [κB pathway activation in activated rat stellate cells, leading to significant increases in inflammatory molecules such as IL-1β [Iron is a necessary dietary component, as it is crucial to processes ranging from oxygen transport by red blood cells  to fetal pathway . Similaras IL-1β . Hyperfeas IL-1β  and macras IL-1β  that areas IL-1β  and the as IL-1β  by upregβ-catenin signaling in several tissue-specific contexts.The rationale for much of the research discussed in the following sections is that oxidative stress –23, inflNematostella vectensis shares 11 of the 12 known Wnt gene subfamilies with humans [β-catenin research has led to a plethora of discoveries over the last four decades.The first Wnt gene was discovered in 1982 as an oncogene activated in mouse models of virally induced mammary carcinoma and was named Int-1 . Severalh humans . BecauseToday, we know of 19 Wnt genes in the mammalian genome that encode 12 subfamilies of Wnt proteins (Wnts) . These WThe trafficking and secretory pathways are two examples of conserved characteristics between the human Wnt proteins , 55. As β-catenin, as this is the best studied pathway in relation to iron homeostasis.The distance of Wnt secretion and subsequent signaling is a heavily debated topic, as there is evidence for Wnt targeting both local , 65 and β-catenin, as it is the primary regulator in Wnt target gene expression. With cytosolic accumulation of β-catenin, subsequent nuclear translocation occurs allowing the protein to bind to the T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factor family [In canonical Wnt signaling, there is a complex system of transducing factors that cooperate to instill cell responsiveness to the Wnt ligands. A central player in this cascade is r family , 71. Wntr family . FZD is r family , 72, 73,r family . LRP5 anr family , 75. It r family . While tr family , the FZDβ-catenin is bound by an intact destruction complex and targeted for ubiquitin-mediated degradation, preventing β-catenin accumulation and nuclear translocation. This ultimately results in Wnt target gene repression. The destruction complex consists of proteins Axin, Adenomatous polyposis coli (APC), and Wilms tumor gene on the X chromosome (WTX), casein kinase 1-α (CK1α), glycogen synthase kinase 3-β (GSK-3β), and other factors [β-catenin and other members of the destruction complex [α and GSK-3β are serine-threonine kinases that perform multiple functions related to Wnt signaling. In terms of the destruction complex, they phosphorylate Axin-bound β-catenin to target it for ubiquitination [β-Transducin repeat-containing protein (β-TrCP), a component of the E3 ubiquitin ligase complex, recognizes phosphorylated β-catenin and catalyzes the addition of ubiquitin polymers to direct β-catenin for degradation by proteasomes preventing their nuclear translocation [β-catenin in the nucleus leaves the repressive TCF/LEF complex active, which then permits Groucho/transducin-like enhancer (Gro/TLE) family proteins to recruit histone deacetylases that inhibit Wnt target gene expression [In the absence of Wnt, cytoplasmic  factors . Axin is complex . APC and complex . CK1α antination , 78. β-Tpression .β and CK1γ [β and CK1γ phosphorylate the LRP tail at the PPPSP motif [β-catenin destruction complex [β-Catenin then accumulates in the cytosol and subsequently translocates to the nucleus. Nuclear β-catenin interacts with the TCF/LEF interface discussed earlier to activate Wnt target gene expression [In the presence of Wnt, canonical Wnt stimulation begins with Wnt ligand facilitating the heterodimerization of FZD and its coreceptor, such as LRP5 or LRP6. Ligation induces a conformational change in the receptors and by a currently unclear mechanism, recruits, and activates GSK-3and CK1γ –82. At tSP motif  and in rSP motif , respect complex . β-Catenpression , 85, 86 β-catenin signaling plays a multitude of roles in a host of tissues throughout life. Wnts generally function as growth factors to cause proliferation and have more than 100 downstream target genes [β-catenin signaling would have a variety of negative implications. The nature of this aberrance, however, is not as intuitive. In some diseases, such as in cases of CRC [As previously discussed, Wnt/et genes –37, 87. et genes  and Cyclet genes . Others et genes , as wellet genes . Wnt siget genes , which is of CRC  or AMD [s of CRC  or osteos of CRC , Wnt sigβ-catenin signaling are those that lead to an increase in active β-catenin translocating to the nucleus and thus increasing the expression of Wnt target genes. In cases of chronic wounds [β-catenin inactivation, positive Wnt modulators can be protective. However, in the context of diseases characterized by Wnt/β-catenin activation, positive Wnt modulators may be harmful. For example, patients being treated with lithium, a canonical Wnt activator, were shown to have a significantly increased chance of developing renal carcinogenesis [Positive modulators of Wnt/c wounds , vitiligc wounds , 97, or ogenesis .One important positive regulation pathway of vertebrate Wnt signaling involves the four R-spondin (RSPO) proteins, which are characterized by two furin domains and a thrombospondin domain . In the β-catenin [β-catenin destruction complex and allowing Wnt target gene expression [C. elegans mitotic spindle orientation [In addition to the well-studied RSPO and Norrin positive modulators, there are several others that have been discovered over the years. Protein phosphatase-2A (PP2A) is a serine-threonine phosphatase composed of three subunits that can positively regulate the canonical Wnt pathway by dephosphorylating a variety of proteins including -catenin . Other epression . Still, entation , distal-entation , and neuentation .β-catenin signaling are those that lead to the destruction of β-catenin and a decrease in the expression of Wnt target genes. This may be protective in diseases characterized by Wnt/β-catenin activation, but deleterious during diseases characterized by Wnt/β-catenin inactivation [Negative modulators of Wnt/tivation . The pottivation . Specifitivation , 120.A prototypical downregulator of Wnt signaling is Notum, an extracellular enzyme that inhibits Wnt proteins by removing their vital palmitoylate residues , 122. Noβ-catenin degradation [In addition to the Notum feedback loop, there are several other negative feedback loops that regulate canonical Wnt signaling. An important one of these was mentioned in our discussion of positive Wnt regulation, with regard to the E3 ligases RNF43 and ZNRF3. These two ligases are known to control FZD membrane expression by mediating the ubiquitination of the FZD cytoplasmic loops, which leads to FZD lysosomal degradation , 137. A radation , 139, whβ-catenin signaling leading to multiple disorders as outlined in In the remainder of this review, we will discuss the recent findings on how iron modulates Wnt/β-catenin results in increased colonic cell proliferation, yielding a presentation of colonic polyps associated with increased colorectal cancer risk [The role of Wnt signaling in cancerous pathologies is historically validated, with the discovery of Wnt signaling based on experimentation involving mouse models of breast cancer and cancer-causing retrovirus mouse mammary tumor Virus (MMTV) . In expecer risk –147. In cer risk .With researchers investigating more on the connection between cancer and Wnt, iron has emerged as a prominent Wnt regulator in the context of proliferative pathologies. In studies using APC knockout cell lines Caco-2 and SW480, it was highlighted that growth on FeSO4-loaded media and hemin-loaded media, both rich in iron, increased Wnt signaling . Thus, iβ-catenin activation in several tissue-specific cancer cell lines confirming that the antitumor effect of HQBA is through iron chelation [β-catenin signaling by chelating iron. Upon treatment with acyl hydrazones, intracellular iron was chelated, Wnt/β-catenin activation was reversed, and cell proliferation significantly decreased in human CRC cell lines SW480 and DID-1 [β-catenin signaling in certain cancerous pathologies, and that iron chelation may be a potential therapeutic strategy to prevent cancer progression.Two recent reports revealed that iron chelators can reverse the Wnt activation during cancer. In the first study, a specific iron chelator HQBA inhibited Wnt signaling in a variety of cancer cell lines and inhibited growth of mammary tumors in MMTV-Wnt1 mouse models of Wnt-dependent breast cancer and in MMTV-PyMT mouse models of Wnt-independent breast cancer  indicatihelation . A seconnd DID-1 . Taken tβ-catenin signaling. The first study discusses posthemorrhagic chronic hydrocephalus (PHCH), a potentially fatal medical condition often arising after an intraventricular hemorrhage (IVH) [β-catenin signaling [β-catenin signaling seen in PHCH after an IVH. This served to broadly improve PHCH occurrence and severity [β-catenin signaling in neural progenitor cells (NPCs) could be normalized by DFX, leading to an increase in NPC differentiation and outgrowth [β-catenin activation, which can be therapeutically resolved with DFX treatment.The relationship between iron overload and aberrant Wnt signaling has been described in two studies that suggest excess iron in neuronal cells pathologically increases Wnt/ge (IVH) . PHCH isge (IVH)  and ferrge (IVH) . Additioignaling , 154. Asseverity . Similarutgrowth . These sβ-thalassemia and hemochromatosis are often associated with decreased bone density and integrity [β-thalassemia [in vivo iron-induced osteoporotic rat model demonstrated similar reduction of Wnt signaling that was recovered with DFX in bone tissues [in vivo mouse study suggests that hepcidin-induced osteoporosis, mediated through iron overload, may also be targeted via inhibition of Forkhead box O3a to recover canonical Wnt signaling [Iron , 158 andntegrity . Indeed,lassemia , suggestlassemia , 163. Inlassemia , is assolassemia . For thelassemia . The stulassemia . These rlassemia . Despite tissues . Anotherignaling .Interestingly, while iron overload leads to decreased activity of the Wnt pathway in osteoblasts, another study reported that iron overload induces ROS-mediated apoptosis and upregulation of the Wnt pathway in bone marrow mesenchymal cells of patients with myelodysplastic syndromes . This hiβ-catenin knockout (KO) mice are known to have similar hepatic pathology resulting from factors such as fibrosis and oxidative stress [β-catenin KO mice [β-catenin KO mice have increased steatohepatitis and fibrosis that is preceded by both inflammation and oxidative stress. Moreover, they demonstrate that treatment with an antioxidant can help prevent this disease progression. In effect, the absence of β-catenin exacerbates the detrimental effects of hepatic iron overload. The study also showed that iron overload led to a decrease in β-catenin in mice where it was not knocked out. This is postulated to be an initial protective mechanism, because these mice had less expression of Cype2e1, a β-catenin target associated with oxidative stress [β-catenin expression was proven to be detrimental in cases of β-catenin KO which increased inflammation and eventually lead to Cype2e1 reappearance, and, thus, further decrease in β-catenin due to prolonged iron overload could prove to be similarly detrimental in liver tissue. We recently showed that iron overload in normal mouse liver decreases Wnt pathway by suppressing Sirtuin 3 signaling [Chronic iron overload in hepatocytes, such as in cases of hemochromatosis, is associated with severe hepatic injury and cancer largely because of iron-induced steatosis, fibrosis, inflammation, and oxidative stress , 172. Ine stress , 174. Fo KO mice . This ste stress . Howeverignaling . Thus, sVisual impairment is a national and a global health concern that impairs physical and mental health in affected individuals. The retina is one of the highest energy consuming organs in the body. Impaired metabolic changes in eye diseases often drive neuronal and vascular pathologies , 178. ThRetina expresses many iron containing proteins like RPE65, an isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol in the visual cycle , fatty aβ-catenin signaling pathway are a hallmark of cancer, diabetes mellitus, and other degenerative disorders. Elucidating the complex interplay between iron and Wnt pathway could lead to new insights into the mechanisms of disease progression and enrich our understanding of the aging biology. Control of body iron stores and Wnt inhibitors can thus serve as promising clinical targets to overcome the myriad ramifications of aging.A multivariate genomic scan has revealed high levels of iron in the blood to be intimately associated with reduced healthspan . Also, i