Calcium signaling induces partial EMT and renal fibrosis in a Wnt4mCherry knock-in mouse model.

The renal tubular epithelial cells (TEC) have a strong capacity for repair after acute injury, but when this mechanism becomes uncontrollable, it leads to chronic kidney diseases (CKD). Indeed, in progress toward CKDs, the TECs may dedifferentiate, undergo epithelial-to-mesenchyme transition (EMT), and promote inflammation and fibrosis. Given the critical role of Wnt4 signaling in kidney ontogenesis, we addressed whether changes in this signaling are connected to renal inflammation and fibrosis by taking advantage of a knock-in Wnt4mCh/mCh mouse. While the Wnt4mCh/mCh embryos appeared normal, the corresponding mice, within one month, developed CKD-related phenotypes, such as pro-inflammatory responses including T-cell/macrophage influx, expression of fibrotic markers, and epithelial cell damage with a partial EMT. The Wnt signal transduction component ß-catenin remained unchanged, while calcium signaling is induced in the injured TECs involving Nfat and Tfeb transcription factors. We propose that the Wnt4 signaling pathway is involved in repairing the renal injury, and when the signal is overdriven, CKD is established.

Nr4a1 enhances Wnt4 transcription to promote mesenchymal stem cell osteogenesis and alleviates inflammation-inhibited bone regeneration.

Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-ß1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-ß1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-ß1, was suppressed directly by TGF-ß1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-ß1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/ß-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.

A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis.

The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.

WNT4 (rs7521902 and rs16826658) polymorphism and its association with endometriosis - A systematic review and meta-analysis.

IMPORTANCE: This systematic review supports the involvement of the WNT4 gene in the pathophysiology of endometriosis. OBJECTIVE: To conduct a systematic review and meta-analysis on WNT4 rs7521902 and rs16826658 polymorphism associated with endometriosis based on multi-ethnic case-control studies. DATA SOURCES: Comprehensive searching was performed using Medline, Embase, and Google Scholar. STUDY SELECTION AND SYNTHESIS: Keywords used for searching using Boolean operators are endometriosis, WNT4, and polymorphism. This review followed PRISMA guidelines, and meta-analysis was conducted in STATA18. MAIN OUTCOMES: WNT4 polymorphisms identified in this review were rs7521902, rs16826658, rs2235529, rs3820282, and rs12037376. RESULTS: A total of 250 studies were identified through databases; 10 were eligible for this review, and eight were included in the meta-analysis. Two WNT4 polymorphisms (rs7521902 and rs16826658) were analysed in the meta-analysis. A lower risk of odds in having endometriosis was apparent in the CC genotype of rs7521092 polymorphism with a pooled OR of 0.86 (0.76, 0.99). Most articles were high-quality case-control studies and were at low risk of bias. CONCLUSION: This study highlighted the association of WNT4 polymorphisms (rs7521092) and endometriosis across Latin America, Europe, and Asian populations. RELEVANCE: Following the completion of the Human Genome Project, many genetic aspects of endometriosis were revealed, including the discovery of single nucleotide polymorphisms (SNPs). However, due to a lack of replications and conflicting results between studies, the conclusion of the endometriosis genetic pathway needed to be completed. This finding of WNT4 showed that its association with endometriosis was valid even in varied ethnicities, indicating a general genetic aspect of disease across populations. Nevertheless, further studies are needed to confirm this finding, including functional biological and longitudinal studies.

WYC-209 inhibited GC malignant progression by down-regulating WNT4 through RARα.

Gastric cancer (GC) has been a major health burden all over the world but there are fewer promising chemotherapeutic drugs due to its multidrug resistance. It has been reported that WYC-209 suppresses the growth and metastasis of tumor-repopulating cells but the effect on GC was not explored. MTT, colony formation, and transwell assays were performed to examine the effects of WYC-209 on the proliferation, colony growth, and mobility of GC cells. Western blotting and qRT-PCR were used to detect the expression of proteins and mRNA. RNA-seq and enrichment analyses were conducted for the differentially expressed genes and enriched biological processes and pathways. The rescue experiments were carried out for further validation. Besides, we constructed xenograft model to confirm the effect of WYC-209 in vivo. The dual-luciferase reporter and Chromatin immunoprecipitation were implemented to confirm the underlying mechanism. WYC-209 exerted excellent anti-cancer effects both in vitro and in vivo. Based on RNA-seq and enrichment analyses, we found that Wnt family member 4 (WNT4) was significantly down-regulated. More importantly, WNT4 overexpression breached the inhibitory effect of WYC-209 on GC progression. Mechanically, WYC-209 significantly promoted the binding between retinoic acid receptor α (RARα) and WNT4 promoter. WYC-209 exerts anti-tumor effects in GC by down-regulating the expression of WNT4 via RARα.

WntA and Wnt4 during the regeneration of internal organs in the holothurian Eupentacta fraudatrix.

BACKGROUND: Over the past few years, it has been established that wnt genes are involved in the regenerative processes of holothurians. The wnt4 gene was identified as one of the most active genes in Eupentacta fraudatrix regeneration using differential gene expression analysis and qPCR of individual genes. Also, the wntA gene was found in holothurians, which is present only in invertebrates and can perform unique functions. RESULTS: In this regard, both these genes and proteins were studied in this work. During regeneration, the Wnt4 protein is found in the cells of the coelomic and ambulacral epithelium, retractor muscles, and radial nerves. Single cells with this protein are also found in the connective tissue of the developing aquapharyngeal bulb and in the hypoderm of the body wall. Cells with WntA are found exclusively in the hypoderm of the body wall. CONCLUSION: We assume that both genes are involved in regeneration, but Wnt4 coordinates the formation of the epithelial tissue structure, while WntA maintains the state of the intercellular substance of the body wall.

WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers.

Wnt ligand WNT4 is critical in female reproductive tissue development, with WNT4 dysregulation linked to related pathologies including breast cancer (invasive lobular carcinoma, ILC) and gynecologic cancers. WNT4 signaling in these contexts is distinct from canonical Wnt signaling yet inadequately understood. We previously identified atypical intracellular activity of WNT4 (independent of Wnt secretion) regulating mitochondrial function, and herein examine intracellular functions of WNT4. We further examine how convergent mechanisms of WNT4 dysregulation impact cancer metabolism. In ILC, WNT4 is co-opted by estrogen receptor α (ER) via genomic binding in WNT4 intron 1, while in gynecologic cancers, a common genetic polymorphism (rs3820282) at this ER binding site alters WNT4 regulation. Using proximity biotinylation (BioID), we show canonical Wnt ligand WNT3A is trafficked for secretion, but WNT4 is localized to the cytosol and mitochondria. We identified DHRS2, mTOR, and STAT1 as putative WNT4 cytosolic/mitochondrial signaling partners. Whole metabolite profiling, and integrated transcriptomic data, support that WNT4 mediates metabolic reprogramming via fatty acid and amino acid metabolism. Furthermore, ovarian cancer cell lines with rs3820282 variant genotype are WNT4 dependent and have active WNT4 metabolic signaling. In protein array analyses of a cohort of 103 human gynecologic tumors enriched for patient diversity, germline rs3820282 genotype is associated with metabolic remodeling. Variant genotype tumors show increased AMPK activation and downstream signaling, with the highest AMPK signaling activity in variant genotype tumors from non-White patients. Taken together, atypical intracellular WNT4 signaling, in part via genetic dysregulation, regulates the distinct metabolic phenotypes of ILC and gynecologic cancers. SIGNIFICANCE: WNT4 regulates breast and gynecologic cancer metabolism via a previously unappreciated intracellular signaling mechanism at the mitochondria, with WNT4 mediating metabolic remodeling. Understanding WNT4 dysregulation by estrogen and genetic polymorphism offers new opportunities for defining tumor biology, precision therapeutics, and personalized cancer risk assessment.

MiRNA-21a, miRNA-145, and miRNA-221 Expression and Their Correlations with WNT Proteins in Patients with Obstructive and Non-Obstructive Coronary Artery Disease.

MicroRNAs and the WNT signaling cascade regulate the pathogenetic mechanisms of atherosclerotic coronary artery disease (CAD) development. OBJECTIVE: To evaluate the expression of microRNAs (miR-21a, miR-145, and miR-221) and the role of the WNT signaling cascade (WNT1, WNT3a, WNT4, and WNT5a) in obstructive CAD and ischemia with no obstructive coronary arteries (INOCA). METHOD: The cross-sectional observational study comprised 94 subjects. The expression of miR-21a, miR-145, miR-221 (RT-PCR) and the protein levels of WNT1, WNT3a, WNT4, WNT5a, LRP6, and SIRT1 (ELISA) were estimated in the plasma of 20 patients with INOCA (66.5 [62.8; 71.2] years; 25% men), 44 patients with obstructive CAD (64.0 [56.5; 71,0] years; 63.6% men), and 30 healthy volunteers without risk factors for cardiovascular diseases (CVD). RESULTS: Higher levels of WNT1 (0.189 [0.184; 0.193] ng/mL vs. 0.15 [0.15-0.16] ng/mL, p < 0.001) and WNT3a (0.227 [0.181; 0.252] vs. 0.115 [0.07; 0.16] p < 0.001) were found in plasma samples from patients with obstructive CAD, whereas the INOCA group was characterized by higher concentrations of WNT4 (0.345 [0.278; 0.492] ng/mL vs. 0.203 [0.112; 0.378] ng/mL, p = 0.025) and WNT5a (0.17 [0.16; 0.17] ng/mL vs. 0.01 [0.007; 0.018] ng/mL, p < 0.001). MiR-221 expression level was higher in all CAD groups compared to the control group (p < 0.001), whereas miR-21a was more highly expressed in the control group than in the obstructive (p = 0.012) and INOCA (p = 0.003) groups. Correlation analysis revealed associations of miR-21a expression with WNT1 (r = -0.32; p = 0.028) and SIRT1 (r = 0.399; p = 0.005) protein levels in all CAD groups. A positive correlation between miR-145 expression and the WNT4 protein level was observed in patients with obstructive CAD (r = 0.436; p = 0.016). Based on multivariate regression analysis, a mathematical model was constructed that predicts the type of coronary lesion. WNT3a and LRP6 were the independent predictors of INOCA (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Activation of the canonical cascade of WNT-ß-catenin prevailed in patients with obstructive CAD, whereas in the INOCA and control groups, the activity of the non-canonical pathway was higher. It can be assumed that miR-21a has a negative effect on the formation of atherosclerotic CAD. Alternatively, miR-145 could be involved in the development of coronary artery obstruction, presumably through the regulation of the WNT4 protein. A mathematical model with WNT3a and LRP6 as predictors allows for the prediction of the type of coronary artery lesion.

WNT4 promotes macrophage polarization via granulosa cell M-CSF and reduces granulosa cell apoptosis in endometriosis.

BACKGROUND: WNT4 gene polymorphism are common in endometriosis and may functionally link estrogen and estrogen receptor signaling. Previous study confirmed estrogen and estrogen receptor signaling recruit macrophage to promote the pathogenesis of endometriosis. To investigate the effect of WNT4 in endometriosis involved in macrophage polarization and whether WNT4 could reduce the apoptosis of granulosa cells. METHODS: An observational study consisting of 8 cases of women with endometriosis (diagnosed by surgery and histology) and 22 mice of endometriosis animal model was conducted. Granulosa cells were isolated from 16 patients with endometriosis and co-cultured with macrophage under WNT4 treatment using TUNEL assay, quantitative reverse transcription PCR, flow cytometry and ELISA analysis. 22 mice of endometriosis animal model confirmed the WNT4 treatment effects using histology and immunohistochemistry, Western blot and flow cytometry. RESULTS: We observed that the apoptotic proportion of granulosa cells was significantly decreased and M2 macrophage was significantly increased after WNT4 treatment during the granulosa cell and macrophage co-culture system. To reveal the underlying mechanism for this, we conducted a series of experiments and found that high expression of granulosa cell M-CSF led to the M2 polarization of macrophages. The animal model also suggested that the anti-apoptotic effect of WNT4 on granulosa cells were conducted by the M2 polarized macrophage. CONCLUSIONS: WNT4 could reduce granulosa cell apoptosis and improve ovarian reserve by promoting macrophage polarization in endometriosis. M-CSF secreted by granulosa cell after WNT4 treatment was the main mediator of macrophage polarization.

CDYL reinforces male gonadal sex determination through epigenetically repressing Wnt4 transcription in mice.

In mammals, male and female gonads initially develop from bipotential progenitor cells, which can differentiate into either testicular or ovarian cells. The decision to adopt a testicular or ovarian fate relies on robust genetic forces, i.e., activation of the testis-determining gene Sry, as well as a delicate balance of expression levels for pro-testis and pro-ovary factors. Recently, epigenetic regulation has been found to be a key element in activation of Sry. Nevertheless, the mechanism by which epigenetic regulation controls the expression balance of pro-testis and pro-ovary factors remains unclear. Chromodomain Y-like protein (CDYL) is a reader protein for repressive histone H3 methylation marks. We found that a subpopulation of Cdyl-deficient mice exhibited XY sex reversal. Gene expression analysis revealed that the testis-promoting gene Sox9 was downregulated in XY Cdyl-deficient gonads during the sex determination period without affecting Sry expression. Instead, we found that the ovary-promoting gene Wnt4 was derepressed in XY Cdyl-deficient gonads prior to and during the sex-determination period. Wnt4 heterozygous deficiency restored SOX9 expression in Cdyl-deficient XY gonads, indicating that derepressed Wnt4 is a cause of the repression of Sox9. We found that CDYL directly bound to the Wnt4 promoter and maintained its H3K27me3 levels during the sex-determination period. These findings indicate that CDYL reinforces male gonadal sex determination by repressing the ovary-promoting pathway in mice.

Mutation analysis of WNT4 gene in SRY negative 46,XX DSD patients with Mullerian agenesis and/or gonadal dysgenesis- An Indian study.

Developmental disruption of the Mullerian duct and gonads in females leads to Mullerian agenesis and gonadal dysgenesis, respectively. These two structural abnormalities are coming under the 46,XX DSD (Disorders of Sexual Development) classification, the majority of cases the aetiology remains elusive. Without the SRY gene, WNT4 plays a key role in female reproductive structure development. Since there are no studies that explored the involvement of the WNT4 gene in Indian 46,XX DSD patients, we analysed the role of WNT4 in Indian 46,XX DSD patients with Mullerian agenesis and/or Gonadal dysgenesis. In our study, we recruited 103 adolescent girls with primary amenorrhea. After the cytogenetic and SRY gene analysis, we included thirty-two 46,XX DSD patients with Mullerian agenesis and/or gonadal dysgenesis for WNT4 gene mutation analysis. PCR sequencing was performed for all the coding exons of the WNT4 gene. Bioinformatic tools like Mutation Taster, Human Splicing Finder, and miRDB were used. We observed single nucleotide variations in three patients. One patient showed a known synonymous polymorphism (c.861C > T; p.G287G, rs544988174). miRDB data revealed the absence of microRNA regulatory sites in this region. The other two cases carried a nucleotide substitution in intronic regions and did not affect the normal splicing mechanism. In conclusion, we could not find any indication about WNT4 involvement in the disease condition. In the future, WNT4 promoter analysis in these patients and molecular characterization of the WNT4 coding and promoter region in more patients are needed to link WNT4 variants with these structural abnormalities.

Loss of WNT4 in the gubernaculum causes unilateral cryptorchidism and fertility defects.

Undescended testis (UDT) affects 6% of male births. Despite surgical correction, some men with unilateral UDT may experience infertility with the contralateral descended testis (CDT) showing no A-dark spermatogonia. To improve our understanding of the etiology of infertility in UDT, we generated a novel murine model of left unilateral UDT. Gubernaculum-specific Wnt4 knockout (KO) mice (Wnt4-cKO) were generated using retinoic acid receptor ß2-cre mice and were found to have a smaller left-unilateral UDT. Wnt4-cKO mice with abdominal UDT had an increase in serum follicle-stimulating hormone and luteinizing hormone and an absence of germ cells in the undescended testicle. Wnt4-cKO mice with inguinal UDT had normal hormonal profiles, and 50% of these mice had no sperm in the left epididymis. Wnt4-cKO mice had fertility defects and produced 52% fewer litters and 78% fewer pups than control mice. Wnt4-cKO testes demonstrated increased expression of estrogen receptor α and SOX9, upregulation of female gonadal genes, and a decrease in male gonadal genes in both CDT and UDT. Several WNT4 variants were identified in boys with UDT. The presence of UDT and fertility defects in Wnt4-cKO mice highlights the crucial role of WNT4 in testicular development.

A Novel Single-Nucleotide Polymorphism in WNT4 Promoter Affects Its Transcription and Response to FSH in Chicken Follicles.

The signaling pathway of the wingless-type mouse mammary tumor virus integration site (Wnt) plays an important role in ovarian and follicular development. In our previous study, WNT4 was shown to be involved in the selection and development of chicken follicles by upregulating the expression of follicle-stimulating hormone receptors (FSHR), stimulating the proliferation of follicular granulosa cells, and increasing the secretion of steroidal hormones. FSH also stimulates the expression of WNT4. To further explore the molecular mechanism by which FSH upregulates WNT4 and characterize the cis-elements regulating WNT4 transcription, in this study, we determined the critical regulatory regions affecting chicken WNT4 transcription. We then identified a single-nucleotide polymorphism (SNP) in this region, and finally analyzed the associations of the SNP with chicken production traits. The results showed that the 5' regulatory region from −3354 to −2689 of WNT4 had the strongest activity and greatest response to FSH stimulation, and we identified one SNP site in this segment, −3015 (G > C), as affecting the binding of NFAT5 (nuclear factor of activated T cells 5) and respones to FSH stimulation. When G was replaced with C at this site, it eliminated the NFAT5 binding. The mRNA level of WNT4 in small yellow follicles of chickens with genotype GG was significantly higher than that of the other two genotypes. Moreover, this locus was found to be significantly associated with comb length in hens. Individuals with the genotype CC had longer combs. Collectively, these data suggested that SNP−3015 (G > C) is involved in the regulation of WNT4 gene expression by responding FSH and affecting the binding of NFAT5 and that it is associated with chicken comb length. The current results provide a reference for further revealing the response mechanism between WNT and FSH.

Wnt4 is crucial for cardiac repair by regulating mesenchymal-endothelial transition via the phospho-JNK/JNK.

Rational: Wnt4 plays a critical role in development and is reactivated during fibrotic injury; however, the role of Wnt4 in cardiac repair remains unclear. In this study, our aim was to clarify the pathophysiological role and mechanisms of Wnt4 following acute cardiac ischemic reperfusion injury. Methods and results: We investigated the spatio-temporal expression of Wnt4 following acute cardiac ischemic reperfusion injury and found that Wnt4 was upregulated as an early injury response gene in cardiac fibroblasts near the injury border zone and associated with mesenchymal-endothelial transition (MEndoT), a beneficial process for revascularizing the damaged myocardium in cardiac repair. Using ChIP assay and in vitro and in vivo loss- and gain-of-function, we demonstrated that Wnt4 served as a crucial downstream target gene of p53 during MEndoT. Wnt4 knockdown in cardiac fibroblasts led to decreased MEndoT and worsened cardiac function. Conversely, Wnt4 overexpression in cardiac fibroblasts induced MEndoT in these cells via the phospho-JNK/JNK signaling pathway; however, both the p53 and Wnt4 protein levels were dependent on the ß-catenin signaling pathway. JNK activation plays a critical role in the induction of MEndoT and is crucial for Wnt4 regulated MEndoT. Moreover, Wnt4 overexpression specifically in cardiac fibroblasts rescued the cardiac function worsening due to genetic p53 deletion by decreasing fibrosis and increasing MEndoT and vascular density. Conclusion: Our study revealed that Wnt4 plays a pivotal role in cardiac repair with involvement of phospho-JNK mediated MEndoT and is a crucial gene for cardiac fibroblast-targeted therapy in heart disease.

Mice Lacking Wnt9a or Wnt4 Are Prone to Develop Spontaneous Osteoarthritis With Age and Display Alteration in Either the Trabecular or Cortical Bone Compartment.

Osteoarthritis (OA) is a common degenerative disease of the joint, with a complex multifactorial not yet fully understood etiology. Over the past years, the Wnt signaling pathway has been implicated in osteoarthritis. In a recent genomewide association study (GWAS), the chromosomal location on chromosome 1, linked to the Wnt3a-Wnt9a gene locus, was identified as the most significant locus associated with a thumb osteoarthritis endophenotype. Previously, it was shown that WNT9a is involved in maintaining synovial cell identity in the elbow joint during embryogenesis. Here, we report that the conditional loss of Wnt9a in the Prx1-Cre expressing limb mesenchyme or Prg4-CreER expressing cells predispositions the mice to develop spontaneous OA-like changes with age. In addition, the trabecular bone volume is altered in these mice. Similarly, mice with a conditional loss of Wnt4 in the limb mesenchyme are also more prone to develop spontaneously OA-like joint alterations with age. These mice display additional alterations in their cortical bone. The combined loss of Wnt9a and Wnt4 increased the likelihood of the mice developing osteoarthritis-like changes and enhanced disease severity in the affected mice. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Wnt4, Wnt6 and ß-catenin expression in human placental tissue - is there a link with first trimester miscarriage? Results from a pilot study.

BACKGROUND: Demystifying the events around early pregnancy is challenging. A wide network of mediators and signaling cascades orchestrate the processes of implantation and trophoblast proliferation. Dysregulation of these pathways could be implicated in early pregnancy loss. There is accumulating evidence around the role of Wnt pathway in implantation and early pregnancy. The purpose of this study was to explore alterations in the expression of Wnt4, Wnt6 and ß-catenin in placental tissue obtained from human first trimester euploid miscarriages versus normally developing early pregnancies. METHODS: The study group consisted of first trimester miscarriages (early embryonic demises and incomplete miscarriages) and the control group of social terminations of pregnancy (TOPs). The placental mRNA expression of Wnt4, Wnt6 and ß-catenin was studied using reverse transcription PCR and real time PCR. Only euploid conceptions were included in the analysis. RESULTS: Wnt4 expression was significantly increased in placental tissue from first trimester miscarriages versus controls (p = 0.003). No significant difference was documented in the expression of Wnt6 (p = 0.286) and ß-catenin (p = 0.793). There was a 5.1fold increase in Wnt4 expression for early embryonic demises versus TOPs and a 7.6fold increase for incomplete miscarriages versus TOPs - no significant difference between the two subgroups of miscarriage (p = 0.533). CONCLUSIONS: This is, to our knowledge, the first study demonstrating significant alteration of Wnt4 expression in human placental tissue, from failed early pregnancies compared to normal controls. Undoubtedly, a more profound study is needed to confirm these preliminary findings and explore Wnt mediators as potential targets for strategies to predict and prevent miscarriage.

Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction.

BACKGROUND: Acute myocardial infarction (AMI)-induced excessive myocardial fibrosis exaggerates cardiac dysfunction. However, serum Wnt2 or Wnt4 level in AMI patients, and the roles in cardiac fibrosis are largely unkown. METHODS: AMI and non-AMI patients were enrolled to examine serum Wnt2 and Wnt4 levels by ELISA analysis. The AMI patients were followed-up for one year. MI mouse model was built by ligation of left anterior descending branch (LAD). FINDINGS: Serum Wnt2 or Wnt4 level was increased in patients with AMI, and the elevated Wnt2 and Wnt4 were correlated to adverse outcome of these patients. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial remodeling and cardiac dysfunction following experimental MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in neonatal rat cardiac myocytes (NRCMs) or fibroblasts (NRCFs). Mechanistically, the elevated Wnt2 or Wnt4 activated ß-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured NRCFs. In addition, Wnt2 or Wnt4 upregulated the expression of these Wnt co-receptors, frizzled (Fzd) 2, Fzd4 and (low-density lipoprotein receptor-related protein 6 (LRP6). Further analysis revealed that Wnt2 or Wnt4 activated ß-catenin /NF-κB by the co-operation of Fzd4 or Fzd2 and LRP6 signaling, respectively. INTERPRETATION: Elevated Wnt2 and Wnt4 activate ß-catenin/NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which contributes to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI.

A circular RNA, circUSP36, accelerates endothelial cell dysfunction in atherosclerosis by adsorbing miR-637 to enhance WNT4 expression.

Atherosclerosis is a fatal disorder that is fundamental to various cardiovascular diseases and severely threatens people's health worldwide. Several studies have demonstrated the role of circular RNAs (circRNAs) in the pathogenesis of atherosclerosis. circUSP36 acts as a key modulator in the progression of atherosclerosis, but the molecular mechanism underlying this role is as yet unclear. This study aimed to elucidate the mechanism by which circUSP36 exerts its function in an in vitro cell model of endothelial cell dysfunction, which is one of pathological features of atherosclerosis. The circRNA traits of circUSP36 were confirmed, and we observed high expression of circUSP36 in endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL). Functional assays revealed that overexpression of circUSP36 suppressed proliferation and migration of ox-LDL-treated endothelial cells. In terms of its mechanism, circUSP36 adsorbed miR-637 by acting as an miRNA sponge. Moreover, enhanced expression of miR-637 abated the impact of circUSP36 on ox-LDL-treated endothelial cell dysregulation. Subsequently, the targeting relationship between miR-637 and WNT4 was predicted using bioinformatics tools and was confirmed via luciferase reporter and RNA pull-down assays. Notably, depletion of WNT4 rescued circUSP36-mediated inhibition of endothelial cell proliferation and migration. In conclusion, circUSP36 regulated WNT4 to aggravate endothelial cell injury caused by ox-LDL by competitively binding to miR-637; this finding indicates circUSP36 to be a promising biomarker for the diagnosis and therapy of atherosclerosis.

Genetic analyses of gynecological disease identify genetic relationships between uterine fibroids and endometrial cancer, and a novel endometrial cancer genetic risk region at the WNT4 1p36.12 locus.

Endometriosis, polycystic ovary syndrome (PCOS) and uterine fibroids have been proposed as endometrial cancer risk factors; however, disentangling their relationships with endometrial cancer is complicated due to shared risk factors and comorbidities. Using genome-wide association study (GWAS) data, we explored the relationships between these non-cancerous gynecological diseases and endometrial cancer risk by assessing genetic correlation, causal relationships and shared risk loci. We found significant genetic correlation between endometrial cancer and PCOS, and uterine fibroids. Adjustment for genetically predicted body mass index (a risk factor for PCOS, uterine fibroids and endometrial cancer) substantially attenuated the genetic correlation between endometrial cancer and PCOS but did not affect the correlation with uterine fibroids. Mendelian randomization analyses suggested a causal relationship between only uterine fibroids and endometrial cancer. Gene-based analyses revealed risk regions shared between endometrial cancer and endometriosis, and uterine fibroids. Multi-trait GWAS analysis of endometrial cancer and the genetically correlated gynecological diseases identified a novel genome-wide significant endometrial cancer risk locus at 1p36.12, which replicated in an independent endometrial cancer dataset. Interrogation of functional genomic data at 1p36.12 revealed biologically relevant genes, including WNT4 which is necessary for the development of the female reproductive system. In summary, our study provides genetic evidence for a causal relationship between uterine fibroids and endometrial cancer. It further provides evidence that the comorbidity of endometrial cancer, PCOS and uterine fibroids may partly be due to shared genetic architecture. Notably, this shared architecture has revealed a novel genome-wide risk locus for endometrial cancer.

Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.