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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,iW/iiNT4/iwas shown to be involved in the selection and development of chicken follicles by upregulating the expression of follicle-stimulating hormone receptors (iFSHR/i), stimulating the proliferation of follicular granulosa cells, and increasing the secretion of steroidal hormones. FSH also stimulates the expression ofiWNT4/i. To further explore the molecular mechanism by which FSH upregulatesiW/iiNT4/iand characterize the cis-elements regulatingiW/iiNT4/itranscription, in this study, we determined the critical regulatory regions affecting chickeniW/iiNT4/itranscription. 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 ofiW/iiNT4/ihad the strongest activity and greatest response to FSH stimulation, and we identified one SNP site in this segment, -3015 (Gamp;gt; 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 ofiWNT4/iin 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 (Gamp;gt; C) is involved in the regulation ofiW/iiNT4/igene 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.

Paternal nicotine exposure can alter phenotypes in future generations. The aim of this study is to explore whether paternal nicotine exposure affects the hepatic repair to chronic injury which leads to hepatic fibrosis in offspring. Our results demonstrate that nicotine down regulates mmu-miR-15b expression via the hyper-methylation on its CpG island shore region in the spermatozoa. This epigenetic modification imprinted in the liver of the offspring. The decreased mmu-miR-15b promotes the expression of Wnt4 and activates the Wnt pathway in the offspring mice liver. The activation of the Wnt pathway improves the activation and proliferation of hepatic stellate cells (HSCs) leading to liver fibrosis. Moreover, the Wnt pathway promotes the activation of the TGF-β pathway and the two pathways cooperate to promote the transcription of extracellular matrix (ECM) genes. In conclusion, this study found that nicotine promotes hepatic fibrosis in the offspring via the activation of Wnt pathway by imprinting the hyper-methylation of mmu-miR-15b.

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche., Short term systemic expression of the reprogramming factors Oct-3/4, Sox2, Klf4, c-Myc (OSKM) rejuvenates aging cells and promotes tissue regeneration. Here the authors show that myofiber-specific expression of OSKM accelerates muscle regeneration by reducing secretion of muscle stem cell quiescence promoting Wnt4.

Although intracellular Wnt signaling pathways need to be tightly regulated to promote hematopoietic stem cell self-renewal, the source and identity of important Wnt ligands in the bone marrow is still largely unknown. The noncanonical ligand Wnt4 is expressed in the bone marrow as well as in the stroma, and its overexpression in fetal liver cells facilitates thymic recovery; however, its impact on adult hematopoietic stem cell function remains unclear. Here, we report that the deletion of Wnt4 from hematopoietic cells in mice (Wnt4Δ/Δ) resulted in decreased lymphopoiesis at steady state. This was likely at least in part due to the increased proinflammatory environment present in the bone marrow of Wnt4Δ/Δ mice. Wnt4Δ/Δ hematopoietic stem cells displayed reduced reconstitution capacity in serial transplants, thus demonstrating defective self-renewal, and they expanded poorly in response to lipopolysaccharide stimulation. This appeared to be the result of the absence of Wnt4 in stem/progenitor cells, as myeloid-restricted Wnt4 deletion had no notable effect. Finally, we observed that Wnt4Δ/Δ stem/progenitor cells were more quiescent, presenting enhanced levels of stress-associated JNK phosphorylation and p16INK4a expression, likely contributing to the reduced expansion observed in transplants. In conclusion, our results identify a new, largely autocrine role for Wnt4 in hematopoietic stem cell self-renewal, suggesting that regulation of Wnt signaling in hematopoiesis may not need Wnt secretion and could be independent of morphogen gradients.

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 β2-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.

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.

(Pro)renin receptor [(P)RR] has multiple functions, but its regulation and role in the pathogenesis in glomerulonephritis (GN) are poorly defined. The aims of the present study were to determine the effects of direct renin inhibition (DRI) and demonstrate the role of (P)RR on the progression of crescentic GN. The anti-glomerular basement membrane nephritis rat model developed progressive proteinuria (83.64 ± 10.49 mg/day) and glomerular crescent formation (percent glomerular crescent: 62.1 ± 2.3%) accompanied by increased macrophage infiltration and glomerular expression of monocyte chemoattractant protein (MCP)-1, (P)RR, phospho-extracellular signal-regulated kinase (ERK)1/2, Wnt4, and active β-catenin. Treatment with DRI ameliorated proteinuria (20.33 ± 5.88 mg/day) and markedly reduced glomerular crescent formation (20.9 ± 2.6%), induction of macrophage infiltration, (P)RR, phospho-ERK1/2, Wnt4, and active β-catenin. Furthermore, primary cultured parietal epithelial cells stimulated by recombinant prorenin showed significant increases in cell proliferation. Notably, while the ERK1/2 inhibitor PD98059 or (P)RR-specific siRNA treatment abolished the elevation in cell proliferation, DRI treatment did not abrogate this elevation. Moreover, cultured mesangial cells showed an increase in prorenin-induced MCP-1 expression. Interestingly, (P)RR or Wnt4-specific siRNA treatment or the β-catenin antagonist XAV939 inhibited the elevation of MCP-1 expression, whereas DRI did not. These results suggest that (P)RR regulates glomerular crescent formation via the ERK1/2 signaling and Wnt/β-catenin pathways during the course of anti-glomerular basement membrane nephritis and that DRI mitigates the progression of crescentic GN through the reduction of (P)RR expression but not inhibition of prorenin binding to (P)RR.

Islets derived from stem cells hold promise as a therapy for insulin-dependent diabetes, but there remain challenges towards achieving this goal1-6. Here we generate human islet-like organoids (HILOs) from induced pluripotent stem cells and show that non-canonical WNT4 signalling drives the metabolic maturation necessary for robust ex vivo glucose-stimulated insulin secretion. These functionally mature HILOs contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic NOD/SCID mice. Overexpression of the immune checkpoint protein programmed death-ligand 1 (PD-L1) protected HILO xenografts such that they were able to restore glucose homeostasis in immune-competent diabetic mice for 50 days. Furthermore, ex vivo stimulation with interferon-γ induced endogenous PD-L1 expression and restricted T cell activation and graft rejection. The generation of glucose-responsive islet-like organoids that are able to avoid immune detection provides a promising alternative to cadaveric and device-dependent therapies in the treatment of diabetes.

A maternal low protein (LP) diet in rodents often results in low nephron endowment and renal pathophysiology in adult life, with outcomes often differing between male and female offspring. Precisely how a maternal LP diet results in low nephron endowment is unknown. We conducted morphological and molecular studies of branching morphogenesis and nephrogenesis to identify mechanisms and timepoints that might give rise to low nephron endowment. Sprague-Dawley rats were fed a normal protein (19.4% protein, NP) or LP (9% protein) diet for 3 weeks prior to mating and throughout gestation. Embryonic day 14.25 (E14.25) kidneys from males and females were either cultured for 2 days after which branching morphogenesis was quantified, or frozen for gene expression analysis. Real-time PCR was used to quantify expression of key nephrogenesis and branching morphogenesis genes at E14.25 and 17.25. At E17.25, nephron number was determined in fixed tissue. There was no effect of either maternal diet or sex on branching morphogenesis. Nephron number at E17.25 was 14% lower in male and female LP offspring than in NP controls. At E14.25 expression levels of genes involved in branching morphogenesis (Gfrα1, Bmp4, Gdnf) and nephrogenesis (Hnf4a, Pax2, Wnt4) were similar in the dietary groups, but significant differences between sexes were identified. At E17.25, expression of Gfrα1, Gdnf, Bmp4, Pax2 and Six2 was lower in LP offspring than NP offspring, in both male and female offspring. These findings provide new insights into how a LP diet leads to low nephron endowment and renal sexual dimorphism.

Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8‐Br‐cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock‐down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up‐regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.

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).

Occlusal trauma is one of the most important local contributing factors of periodontitis. It has been reported that Wnt4, a noncanonical Wnt ligand, can inhibit osteoclast formation and inflammation and promote bone formation in vivo. However, the prospects of Wnt4 application in occlusal trauma and periodontitis have not yet been described. This study aimed to investigate the function and the corresponding mechanism of Wnt4 to regulate bone metabolism in occlusal trauma and periodontitis.Osteogenic-induced MC3T3-E1 cells were treated with or without Porphyromonas gingivalis lipopolysaccharide (Pg. LPS) under cyclic uniaxial compressive stress. After treatment with mouse recombinant protein Wnt4 (rWnt4), the expression of osteogenic markers and activation of the IKK-NF-κB signaling pathway were evaluated in vitro. To investigate whether Wnt4 can promote osteogenesis via the ROCK signaling pathway, the expression of RhoA was evaluated in vitro. Finally, we evaluated the change in bone quantity and the activation of the IKK-NF-κB and ROCK signaling in mice with occlusal trauma and periodontitis to demonstrate the therapeutic efficacy of rWnt4 injection.Stimulation of traumatic force and Pg. LPS stimulation suppressed the expression of osteoblast markers, but their expression was rescued after rWnt4 treatment in vitro. In addition, the inhibition of the ROCK signaling pathway induced by force loading was reversed when rWnt4 was applied in vitro. Micro-CT, HE, and TRAP staining of the mandibles showed increased bone loss in the occlusal trauma-aggravated periodontitis group, whereas it was rescued after rWnt4 injection. The expression levels of IκBα and p65 were upregulated in occlusal trauma and periodontitis-bearing mice, whereas the expression levels of Runx2 and RhoA were downregulated. After rWnt4 injection, remarkably upregulation of Runx2 and RhoA expression was observed in occlusal trauma and periodontitis- bearing mice.Wnt4 not only inhibits IKK-NF-κB signaling but also activates ROCK signaling to inhibit osteoclast formation and promote bone regeneration in occlusal trauma and periodontitis-bearing mice.

The WNT signaling pathway contributes to renal fibrosis, which is a hallmark of chronic kidney disease (CKD). Serum concentration of WNT4 could be used to monitor the kidney disease; however, no data have yet been published on the subject.This study measures WNT4 protein in serum of CKD patients depending on the stage, type of nephropathy, the non-nephrotic (NNP) or nephrotic proteinuria (NP), inflammatory cell infiltration in kidney parenchyma (IIKP), interstitial fibrosis in biopsy and serum creatinine. We also evaluated the usefulness of the serum WNT4 as a marker of fibrosis and IIKP.The WNT4 protein level in serum of CKD patients and healthy individuals was measured using enzyme-linked immunoassay (ELISA). Patients' blood biochemical profiles and kidney biopsies were evaluated with common laboratory methods.The serum level of WNT4 protein was higher in CKD patients (i) regardless of the underlying etiology and at early stages of disease; (ii) with lupus nephritis and Immunoglobulin A (IgA) nephropathy; (iii) without or with a small area of IIKP; and (iv) with a small area covered with fibrosis. No difference was observed between NNP and NP patients. The utility of serum WNT4 as a marker of IIKP and fibrosis was not confirmed. Negative correlations with total and low-density lipoprotein (LDL)-cholesterol were found in CKD and IIKP patients. In patients with serum WNT4 above the median value, serum creatinine was higher. However, no correlation between serum WNT4 and creatinine level was found.The observed increase in serum WNT4 protein in the early stages of CKD and in patients diagnosed with immune-mediated glomerular disease may suggest that WNT4 may act as a mediator of inflammation. A certain association with the dysregulation of serum lipid metabolism can also be suspected. Serum WNT4 protein may be considered as the indicator of chronic glomerulonephritis, but not a diagnostic marker of IIKP and fibrosis.

International audience; Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

Uterine fibroids (UFs) or leiomyoma are frequently associated with somatic mutations in the mediator complex subunit 12 (MED12) gene; however, the function of these mutations in human UF biology is yet to be determined. Herein, we determined the functional role of the most common MED12 somatic mutation in the modulation of oncogenic Wnt4/β-catenin and mammalian target of rapamycin (mTOR) signaling pathways. Using an immortalized human uterine myometrial smooth muscle cell line (UtSM), we constitutively overexpressed either MED12-Wild Type or the most common MED12 somatic mutation (c.131G>A), and the effects of this MED12 mutation were compared between these cell lines. This immortalized cell line was used as a model because it expresses wild type MED12 protein and do not possess MED12 somatic mutations. By comparing the effect between MED12-WT and MED12-mutant (mut) stable cell populations, we observed increased levels of protein expression of Wnt4 and β-catenin in MED12-mut cells as compared with MED12-WT cells. MED12-mut cells also expressed increased levels of mTOR protein and oncogenic cyclin D1 which are hallmarks of cell growth and tumorigenicity. This somatic mutation in MED12 showed an effect on cell-cycle progression by induction of S-phase cells. MED12-mut cells also showed inhibition of autophagy as compared with MED12-WT cells. Together, these findings indicate that the MED12 somatic mutation has the potentials for myometrial cell transformation by dysregulating oncogenic Wnt4/β-catenin and its downstream mTOR signaling which might be associated with autophagy abrogation, cell proliferation, and tumorigenicity.

Our understanding of the relationship between stress-derived epinephrine and early pregnancy failure remains incomplete. Here, we explored the effect of epinephrine exposure on early pregnancy and pseudopregnancy in mice. Increased expression of adrenergic receptors Adra1b, Adra2b and Adrb2 was observed during decidualization and post-implantation embryogenesis was delayed or survival impaired. Epinephrine treatment also impaired decidualization in both the gravid and pseudopregnant uterus, suggesting the effect on decidualization was independent of the conceptus. This included a suppression of endometrial stroma cell proliferation and of key decidualization regulators, including COX2, BMP2 and WNT4. Collectively, these data demonstrate that maternal epinephrine exposure during early pregnancy impairs uterine decidualization and embryo development, underlying early pregnancy failure.

Background: Recurrent implantation failure (RIF) remains a critical and challenging problem in assisted reproductive technology mainly because of impaired decidualization. Endocytic and transcytotic activity in the endometrium are crucial for impaired decidualization. The most representative endocytic genes is the C-terminal Eps15 homology domain-containing 1 (EHD1). Whether the EHD1-mediated endocytic function is responsible for embryo implantation during decidualization remains to be determined. Methods: Endometrial tissues were collected from RIF patients (n=27) and fertile controls (n=27) between July 2017 and December 2018. Meanwhile, endometrial biopsies were performed on tissues from 10 women with infertility secondary to tubal factor in the late proliferative and mid-secretory phases of the menstrual cycle. Primary HESCs isolated from eutopic endometrial tissues were used to investigate the role of EHD1 in decidualization. Findings: RNA-seq analysis demonstrated that EHD1 expression was significantly higher in the mid-secretory endometrium of the RIF group than in that of the fertile control group. During the menstrual cycle, the expression of EHD1 increased in the mid-proliferative phase and decreased periodically in the mid-secretory phase and decidual phase. Furthermore, EHD1 overexpression impaired decidualization by suppressing the expression of prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) and the formation of the cytoskeleton. Mechanistically, Wingless-related MMTV integration site 4 (Wnt4) and its downregulated signaling showed significant changes following adenovirus-mediated overexpression of EHD1 in HESCs by RNA-seq analysis. The endocytosis pathway of the low-density-lipoprotein receptor-related proteins 5 and 6 (LRP5/6) co-receptor was decreased by EHD1 overexpression. Co-IP and confocal microscopy analysis showed that EHD1 bound to the Wnt4 protein, contributing to the decrease in Wnt4/β-catenin signaling during the decidualization of HESCs. Additionally, the Wnt4 agonist improved the impaired decidualization. Interpretation: Regulation of the EHD1-Wnt4 pathway may serve as a promising therapeutic strategy to improve endometrial receptivity in RIF women. Funding Statement: This work was supported by the National Key Research and Development Program of China (2018YFC1004401), the National Natural Science Foundation of China (81871165, 31872846, 81571402 and 31571189 ), a special grant for principal investigators from the Health Department of Jiangsu Province (ZDRCA2016070), and a special fund for the Jiangsu Province Social Development Project (NO.BE2018602). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: This study was carried out according to the Recommendations of Guidelines for Clinical Trials by the Ethics Committee of the Drum Tower Hospital. Informed consent was obtained from all participants before any study-related procedure was performed. This study was carried out according to the approval of Construction and Management of the Nanjing Multic-center Biobank Project (No. 2013-081-01).

Abnormal activation of Wnt signaling has been demonstrated in the wound healing process and the pathogenesis of fibrotic disorders, with Wnt4 specifically identified as having a key role in the pathogenesis of renal, pulmonary and liver fibrosis. Wnt4 also was found to be upregulated by transforming growth factor-β1 (TGF-β1) in fetal and postnatal murine fibroblasts and bone marrow mesenchymal cells, suggesting an underlying cooperation between Wnt4 and TGF-β1 in fibrosis. However, the specific roles of Wnt4 in TGF-β1-induced skin myofibroblast transition and hypertrophic scar formation remain unclear. In the present study, we first observed reduced Wnt4 expression in hypertrophic scar tissue compared with that in normal skin tissue. Following upregulation by TGF-β1, Wnt4 inhibited the TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts. Using fibroblast-populated collagen lattice contraction assays, we showed that the increased contractility induced by TGF-β1 was significantly blocked by exogenous Wnt4 and the α-smooth muscle actin (α-SMA) expression was decreased in fibroblasts in the collagen lattices. In addition, knockdown of Wnt4 resulted in further increases in α-SMA and collagen I expressions. Further investigation showed that Wnt4 could inhibit the autocrine effect of TGF-β1 as well as block the phosphorylation of Smad3 and ERK but not of AKT or JNK. Lastly, using hypertrophic scar-derived fibroblasts, we showed that the elevated α-SMA and collagen I levels were markedly reduced after treatment with Wnt4. Taken together, our results suggest that Wnt4 negatively regulates TGF-β1-induced fibroblast activation, which may represent a novel therapeutic strategy for the treatment and prevention of hypertrophic scars.

Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction, yet there are no effective therapies currently due to the failure of reconstructing the interruption of the neuroanatomical circuit. While neural stem cell (NSC) transplantation has been considered a potential strategy to repair the neural circuit after SCI, the efficacy of this strategy remains unproven. The main reason is that most of the transplanted NSC differentiates into astrocyte rather than neuron in the microenvironment of SCI. Our results demonstrated that Wnt4 significantly promotes the differentiation of NSC into neuron by activating both β-catenin and MAPK/JNK pathways and suppressing the activation of Notch signaling, which is acknowledged as prevention of NSC differentiation into neuron, through downregulating NICD expression, translocating and preventing the combination of NICD and RbpJ in nucleus. In addition, Wnt4 rescues the negative effect of Jagged, the ligand of Notch signaling, to promote neuronal differentiation. Moreover, in vivo study, transplantation of Wnt4-modified NSC efficaciously repairs the injured spinal cord and recovers the motor function of hind limbs after SCI. This study sheds new light into mechanisms that Wnt4-modified NSC transplantation is sufficient to repair the injured spinal cord and recover the motor dysfunction after SCI.

The functions of Wnt signalling in crab limb regeneration are poorly understood. Herein, we isolated and characterised the full-length cDNA of WNT4 from Portunus trituberculatus, designated PtWNT4. The 4831 bp cDNA encodes 323 amino acid polypeptide. Protein structure prediction showed that PtWNT4 has a conserved WNT domain. The PtWNT4 gene was expressed in all regenerative limb stages, and was upregulated from stage I, with highest expression in stage III, and expression then declined in stages IV and V. Immunohistochemistry revealed strong localisation at loose connective tissue during limb regeneration, but PtWNT4 protein levels decreased upon formation of muscle fibers. Injection of WNT4 dsRNA into regenerative limbs significantly decreased PtWNT4 mRNA levels from 36 h to 5 days after injection, indicating successful gene silencing. RNA interference knockdown of PtWNT4 expression greatly retarded limb regeneration compared with the control group. Blastema emergence phenotype analysis revealed limb regeneration rates of 0, 31.5% and 40.5% for the dsWNT4 group at 36 h, 3 days and 5 days, respectively, compared with 29.95%, 83.0% and 92.5% for the saline-injected control groups (p

Background/Aims: WNT4 protein is important for kidney development. Its expression was found to be altered in experimental models of chronic kidney disease (CKD). However, the expression of the WNT4 gene has yet not been studied in human renal biopsy samples from patients with broad spectrum of glomerular disease and at different stages of CKD. Thus, the aim of the study was to assess the WNT4 gene expression in renal biopsies of 98 patients using the real-time PCR technique. Materials: In order to assess the relative amounts of mRNA, in samples of patients with manifestation of different renal diseases and separately at different stages of CKD, by QPCR, total RNA was isolated from human kidney tissues collected during renal biopsies. Results of blood and urine samples assessment were used to calculate the correlations of biochemical parameters with WNT4 gene expression in both studied groups. Results: After pathomorphological evaluation, 49 patients were selected as presenting the most common cases in the studied group. Among the patients who developed focal segmental glomerulosclerosis (FSGS; n = 13), IgA nephropathy (IgAN; n = 10), IgAN with morphological presentation of focal segmental glomerulosclerosis (IgAN/FSGS; n = 8), membranous nephropathy (MN; n = 12), and lupus nephritis (LN; n = 6) were included in the analysis. We found that the level of WNT4 mRNA was higher in kidney specimens obtained from patients with MN as compared to those diagnosed with LN or IgAN. A correlation between WNT4 gene expression and serum albumin and cholesterol levels was observed in patients with FSGS, while WNT4 mRNA levels correlated with plasma sodium in patients diagnosed with LN. After consideration of 98 patients, based on the KDIGO classification of CKD, 20 patients were classified as CKD1 stage, 23 as stage 2, 13 as stage 3a, 11 as stage 3b, 13 as stage 4, and 18 as stage 5. WNT4 gene expression was lower in the CKD patients in stage 2 as compared to CKD 3a. Correlations of WNT4 mRNA level at different stages of CKD with indices of kidney function and lipid metabolism such as serum levels of HDL and LDL cholesterol, TG, urea, creatinine, sodium, and potassium were also found. Conclusions: Our results suggest that altered WNT4 gene expression in patients with different types of glomerular diseases and patients at different stages of CKD may play a role in kidney tissue disorganization as well as disease development and progression.

The diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signaling in vitro. We used a primary tissue culture system to generate a murine p53 null gastric tissue model containing both epithelium and mesenchymal stroma. To characterize the microenvironment and niche signaling, we used single cell RNA sequencing (scRNA-Seq) to determine the transcriptomes of 4,391 individual cells. Based on specific markers, we identified epithelial cells, fibroblasts and macrophages in initial tissue explants during organoid formation. The majority of macrophages were polarized towards wound healing and tumor promotion M2-type. During the course of time, the organoids maintained both epithelial and fibroblast lineages with the features of immature mouse gastric stomach. We detected a subset of cells in both lineages expressing Lgr5, one of the stem cell markers. We examined the lineage-specific Wnt signaling activation, and identified that Rspo3 was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that this primary tissue culture system enables one to study gastric tissue niche signaling and immune response in vitro.

Background To investigate the genetic contribution of copy number variations (CNVs) in Wingless-type MMTV integration site family, member 4 (WNT4), in a Chinese population with Müllerian anomalies (MA), copy number analysis of WNT4 by Multiplex ligation‐dependent probe amplification (MLPA) was performed on 248 female patients. Some studies have shown that heterozygous missense mutation of WNT4 can lead to MA. However, few studies on the relationship between WNT4 CNVs and MA have been performed. Results Among the 248 Chinese women affected by MA in this study, heterozygous deletion of WNT4 was detected in a single patient. Conclusions MLPA identified one heterozygous deletion in WNT4 in a single female patient among 248 Chinese women affected by MA. This study firstly reports CNVs of WNT4 in a large sample of MA patients from the Chinese population, which suggests that CNVs of WNT4 cannot be excluded in the occurrence of MA. This provides a genetic basis for precise treatment in the future.

Human umbilical cord blood-derived MSCs (hUC-MSCs) have the potential to differentiate into osteoblasts. This study investigated the function and potential mechanisms of a novel lncRNA LINC02381 in hUC-MSC osteogenic differentiation. hUC-MSCs were maintained in osteogenic differentiation medium. RT-qPCR assay was performed to assess LINC02381 expression. Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining were performed to evaluate osteogenic differentiation. The interaction between miR-21 and LINC0238/KLF12 was determined by luciferase reporter and RNA immunoprecipitation (RIP) assays. Chromatin immunoprecipitation (ChIP) assay was used to confirm the transcriptional regulation of KLF12 on Wnt4 promoter. The nuclear translocation of β-catenin was evaluated using immunofluorescence. hUC-MSCs seeded on Bio-Oss Collagen scaffolds were transplanted into nude mice to assess in vivo osteogenesis. Bone formation was observed by H&E and Masson's trichrome staining. OSX and OPN levels were assessed by immunohistochemistry. LINC02381 was up-regulated in the clinical samples of osteoporotic patients. However, LINC02381 expression was reduced during osteogenic differentiation of hUC-MSCs. Enforced expression of LINC02381 suppressed the osteogenic differentiation of hUC-MSCs. Mechanistically, LINC02381 sponged miR-21 to enhance KLF12 expression, which led to the inactivation of Wnt/β-catenin signaling pathway. Furthermore, miR-21 mimics or KLF12 silencing counteracted LINC02381-induced inhibition of osteogenic differentiation, whereas IWP-4 (an inhibitor of Wnt pathway) abolished this effect. In summary, LINC02381 repressed osteogenic differentiation of hUS-MSCs through sponging miR-21 to enhance KLF12-mediated inactivation of Wnt/β-catenin pathway, indicating that LINC02381 might be a therapeutic target for osteoporosis.

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.

The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5'-UTR and a 672 bp 3'-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N-glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene.

OBJECTIVE To identify key determinants of the interactive pathophysiologic processes in subchondral bone and cartilage in osteoarthritis (OA). METHODS We performed RNA sequencing on macroscopically preserved and lesional OA subchondral bone from patients in the Research Arthritis and Articular Cartilage study who underwent joint replacement surgery due to OA (n = 24 sample pairs: 6 hips and 18 knees). Unsupervised hierarchical clustering and differential expression analyses were conducted. Results were combined with data on previously identified differentially expressed genes in cartilage (partly overlapping samples) as well as data on recently identified OA risk genes. RESULTS We identified 1,569 genes that were significantly differentially expressed between lesional and preserved subchondral bone, including CNTNAP2 (fold change [FC] 2.4, false discovery rate [FDR] 3.36 × 10-5 ) and STMN2 (FC 9.6, FDR 2.36 × 10-3 ). Among these 1,569 genes, 305 were also differentially expressed, and with the same direction of effect, in cartilage, including the recently recognized OA susceptibility genes IL11 and CHADL. Upon differential expression analysis with stratification for joint site, we identified 509 genes that were exclusively differentially expressed in subchondral bone of the knee, including KLF11 and WNT4. These genes that were differentially expressed exclusively in the knee were enriched for involvement in epigenetic processes, characterized by, e.g., HIST1H3J and HIST1H3H. CONCLUSION IL11 and CHADL were among the most consistently differentially expressed genes OA pathophysiology-related genes in both bone and cartilage. As these genes were recently also identified as robust OA risk genes, they classify as attractive therapeutic targets acting on 2 OA-relevant tissues.

MicroRNAs (miRNAs) have been regarded as modulators in vascular pathologies, including hypertension. Dysregulated proliferation and migration of VSMCs (vascular smooth muscle cells) contributes to vascular remodeling during hypertension. miR-634 was reported to be dysregulated in hypertensive patients. The involvement of miR-634 in hypertension and the role of miR-634 on VSMCs proliferation and migration were then evaluated. Firstly, HASMCs (human aortic smooth muscle cells) were incubated with 2 μM angiotensin (Ang) II for 12 hours to establish the cell model of Ang II-induced hypertension. Results showed that Ang II treatment promoted proliferation and migration of HASMCs. Secondly, miR-634 was down-regulated in the hypertensive patients, and reduced in Ang II-induced HASMCs in a time dependent manner. Functional assays revealed that Ang II promoted proliferation and migration of HASMCs were suppressed by miR-634 mimic. Lastly, miR-634 targeted 3' untranslated region (UTR) of Wnt4, and reduced Wnt4 expression in HASMCs. miR-634 inhibited β-catenin nuclear translocation. Over-expression of Wnt4 counteracted the suppressive effects of miR-634 on Ang II-induced proliferation and migration of HASMCs. In conclusion, miR-634 inhibited HASMCs proliferation and migration through inactivation of Wnt4/β-catenin pathway.

The WNT family of proteins is crucial in numerous developmental pathways and tissue homeostasis. WNT4, in particular, is uniquely implicated in the development of the female phenotype in the fetus, and in the maintenance of müllerian and reproductive tissues. WNT4 dysfunction or dysregulation can drive sex-reversal syndromes, highlighting the key role of WNT4 in sex determination. WNT4 is also critical in gynecologic pathologies later in life, including several cancers, uterine fibroids, endometriosis, and infertility. The role of WNT4 in normal decidualization, implantation, and gestation is being increasingly appreciated, while aberrant activation of WNT4 signaling is being linked both to gynecologic and breast cancers. Notably, single-nucleotide polymorphisms (SNPs) at the WNT4 gene locus are strongly associated with these pathologies and may functionally link estrogen and estrogen receptor signaling to upregulation and activation of WNT4 signaling. Importantly, in each of these developmental and disease states, WNT4 gene expression and downstream WNT4 signaling are regulated and executed by myriad tissue-specific pathways. Here, we review the roles of WNT4 in women’s health with a focus on sex development, and gynecologic and breast pathologies, and our understanding of how WNT4 signaling is controlled in these contexts. Defining WNT4 functions provides a unique opportunity to link sex-specific signaling pathways to women’s health and disease.

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., Summary: Luminal mammary epithelial cells produce basement membrane laminin α5 necessary for mammary epithelial growth and differentiation. Laminin α5 loss compromises hormone receptor-positive luminal cell function and Wnt4-mediated crosstalk between epithelial cells.

Cardiac septum malformations account for the largest proportion in congenital heart defects. The transcription factor Sox7 has critical functions in the vascular development and angiogenesis. It is unclear whether Sox7 also contributes to cardiac septation development. We identified a de novo 8p23.1 deletion with Sox7 haploinsufficiency in an atrioventricular septal defect (AVSD) patient using whole exome sequencing in 100 AVSD patients. Then, multiple Sox7 conditional loss-of-function mice models were generated to explore the role of Sox7 in atrioventricular cushion development. Sox7 deficiency mice embryos exhibited partial AVSD and impaired endothelial to mesenchymal transition (EndMT). Transcriptome analysis revealed BMP signaling pathway was significantly downregulated in Sox7 deficiency atrioventricular cushions. Mechanistically, Sox7 deficiency reduced the expressions of Bmp2 in atrioventricular canal myocardium and Wnt4 in endocardium, and Sox7 binds to Wnt4 and Bmp2 directly. Furthermore, WNT4 or BMP2 protein could partially rescue the impaired EndMT process caused by Sox7 deficiency, and inhibition of BMP2 by Noggin could attenuate the effect of WNT4 protein. In summary, our findings identify Sox7 as a novel AVSD pathogenic candidate gene, and it can regulate the EndMT involved in atrioventricular cushion morphogenesis through Wnt4–Bmp2 signaling. This study contributes new strategies to the diagnosis and treatment of congenital heart defects.

Objective To investigate the functional interaction between the Wnt/β-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). Design Laboratory study. Setting Research laboratory. Patient(s) Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. Intervention(s) None. Main Outcome Measure(s) Gene expression, protein phosphorylation, and cell proliferation. Result(s) Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/β-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/β-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated β-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. Conclusion(s) Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent β-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

To explore the effect of miR-144 to the biological behavior of multiple myeloma cells and its mechanism.RT-PCR was used to detect the expression of miR-144 in multiple myeloma cells and plasma of MM patients. MTT assay was used to detect the proliferation and cloning ability of myeloma cells transfected by miR-144. Flow cytometry was used to detect the cell cycle distribution of myeloma cells with over-expression of miR-144. Apoptosis of myeloma cells with over-expression of miR-144 was detected by TUNEL assay. Transwell cell invasion and migration assay was used to detect the invasion and migration ability of myeloma cells with overexpressing on miR-144.Western blot analysis was used to detect the protein expression levels of MMP-9 and MMP-2 in myeloma cells with over expression of miR-144, as well as the expression levels of proteins related to Wnt/β-catenin signaling pathway.The expression level of miR-144 in MM cell lines and blood of MM patients was significantly lower than that in control group (P＜0.05). The proliferation, invasion and migration of myeloma cells with over-expression of miR-144 were significantly decreased (P＜0.05), and the apoptosis level was increased (P＜0.05). The expression levels of MMP-9, MMP-2, Wnt/β-catenin signaling pathway in myeloma cells with over-expression of miR-144 were significantly lower than those in control group (P＜0.05).MiR-144 can inhibit the proliferation, migration and invasion of multiple myeloma cells and induce cell apoptosis. The specific mechanism may be related with the activity of inhibiting Wnt/β-catenin signaling pathway.MiR-144调控Wnt4/β-catenin信号通路抑制多发性骨髓瘤 细胞恶性生物学行为的研究.探讨miR-144对多发性骨髓瘤细胞生物学行为的影响及机制.RT-PCR检测miR-144在多发性骨髓瘤细胞及多发性骨髓瘤（MM）患者血浆中的表达情况；MTT法检测miR-144模拟物转染至骨髓瘤细胞后细胞的增殖情况及细胞克隆形成能力；流式细胞术检测过表达miR-144的骨髓瘤细胞周期分布情况；Tunel法检测过表达miR-144的骨髓瘤细胞的凋亡情况；Transwell细胞侵袭实验与迁移实验检测过表达miR-144的骨髓瘤细胞的侵袭及迁移能力；Western blot法检测过表达miR-144的骨髓瘤细胞的MMP-9、MMP-2的蛋白表达水平及Wnt4/β-catenin信号通路相关蛋白的表达水平.MM细胞株及MM患者血中miR-144表达水平明显低于正常对照组（P＜0.05）。过表达miR-144的骨髓瘤细胞的增殖、侵袭、迁移能力明显降低（P＜0.05），细胞凋亡水平增加（P＜0.05）。过表达miR-144的骨髓瘤细胞的MMP-9、MMP-2、Wnt4/β-catenin信号通路相关蛋白的表达水平均明显低于对照组（P＜0.05）.MiR-144能抑制多发性骨髓瘤细胞的增殖、迁移和侵袭，诱导其凋亡，其具体机制可能与抑制Wnt/β-catenin信号通路的活性有关.

Background Wingless and Int-related protein (Wnt) ligands are aberrantly expressed in patients with colorectal cancer (CRC). However, the aberrant level of Wnt ligands in serum have not been explored. Here, we aimed to identify the levels of WNT4 in serum and explored its oncogenic role in CRC. Methods The Oncomine database was used to analyze the relationship between WNT4 and the prognosis of CRC. ELISA was performed to measure WNT4 levels in serum and conditioned medium from fresh CRC tissues and adjacent normal tissues. Western blot and immunohistochemistry were carried out to measure the expression of WNT4 in human CRC tissues and adjacent normal tissues. The migration and invasion of CRC cells were determined by trans-well assay, and the effects of WNT4 on CRC invasion and metastasis in vivo were verified by tumor xenograft in nude mice. Cancer-associated fibroblasts (CAFs) and angiogenesis in subcutaneous nodules were detected by immunofluorescence (IF). In addition, the suspended spheres formation and tube formation assay were performed to explore the effects of WNT4 on CAFs and angiogenesis respectively. Results WNT4 was significantly upregulated in serum of CRC patients, and CRC tissues were identified as an important source of elevated WNT4 levels in CRC patients. Interestingly, elevated levels of WNT4 in serum were downregulated after tumor resection. Furthermore, we found that WNT4 contributed to epithelial-to-mesenchymal transition (EMT) and activated fibroblasts by activating the WNT4/β-catenin pathway in vitro and in vivo. Moreover, angiogenesis was induced via the WNT4/β-catenin/Ang2 pathway. Those effects could be reversed by ICG-001, a β-catenin/TCF inhibitor. Conclusion Our findings indicated that serum levels of WNT4 may be a potential biomarker for CRC. WNT4 secreted by colorectal cancer tissues promote the progression of CRC by inducing EMT, activate fibroblasts and promote angiogenesis through the canonical Wnt/β-catenin signalling pathway.

Gastrointestinal stromal tumor (GIST) is a refractory malignant tumor without satisfactory therapy. In recent years, aberrant gene methylation has been highlighted as an inducer for tumor progression. In this study, we explored whether enhancer of zeste homolog 2 (EZH2)-mediated paired box 8 (PAX8) methylation affects GIST development through regulation of Wnt4. A total of 50 cases of GIST tissues were collected and the human GIST cell lines were cultured. PAX8 methylation was examined using MS-PCR. Following loss- and gain-function approaches, GIST cell proliferation, migration, invasion, and apoptosis were examined by CCK-8 assay, Transwell assay and flow cytometry. The expression of proliferation related factors and apoptosis related factors was determined. Finally, xenograft tumors in nude mice were observed to examine in vivo tumorigenicity of GIST cells. Downregulated PAX8 and upregulated EZH2 expression was found in GIST tissues. Overexpression of PAX8 or suppression of PAX8 methylation using DNA methyltransferase inhibitor 5-Aza-dC inhibited the proliferation, migration, and invasion of GIST cells while promoting their apoptosis (diminished PCNA, Ki67 and Bcl-2, elevated Bax, and cleaved caspase-3). EZH2 promoted PAX8 methylation to inhibit its expression. Downregulated PAX8 decreased Wnt4 expression to accelerate GIST progression both in vitro and in vivo. Collectively, EZH2 inhibits PAX8 expression by promoting its methylation, which thus downregulates Wnt4 expression, thereby promoting the development of GIST.

Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.

Background The increasing incidence of reproductive disorders in humans has been attributed to in utero exposure to estrogenic endocrine disruptors. In particular, exposure of the developing testis to exogenous estrogen can negatively impact male reproductive health. To determine how estrogens impact human gonad function, we treated the human testis-derived cell line NT2/D1 with estrogen and examined its impact on SOX9 and the expression of key markers of granulosa (ovarian) and Sertoli (testicular) cell development. Results Estrogen successfully activated its cognate receptor (estrogen receptor alpha; ESR1) in NT2/D1 cells. We observed a significant increase in cytoplasmic SOX9 following estrogen treatment. After 48 h of estrogen exposure, mRNA levels of the key Sertoli cell genes SOX9, SRY, AMH, FGF9 and PTGDS were significantly reduced. This was followed by a significant increase in mRNA levels for the key granulosa cell genes FOXL2 and WNT4 after 96 h of estrogen exposure. Conclusions These results are consistent with estrogen's effects on marsupial gonads and show that estrogen has a highly conserved impact on gonadal cell fate decisions that has existed in mammals for over 160 million years. This effect of estrogen presents as a potential mechanism contributing to the significant decrease in male fertility and reproductive health reported over recent decades. Given our widespread exposure to estrogenic endocrine disruptors, their effects on SOX9 and Sertoli cell determination could have considerable impact on the adult testis.

Publisher's version (útgefin grein), Pelvic organ prolapse (POP) is a downward descent of one or more of the pelvic organs, resulting in a protrusion of the vaginal wall and/or uterus. We performed a genome-wide association study of POP using data from Iceland and the UK Biobank, a total of 15,010 cases with hospital-based diagnosis code and 340,734 female controls, and found eight sequence variants at seven loci associating with POP (P 5%) and one with minor allele frequency of 4.87%. Some of the variants associating with POP also associated with traits of similar pathophysiology. Of these, rs3820282, which may alter the estrogen-based regulation of WNT4, also associates with leiomyoma of uterus, gestational duration and endometriosis. Rs3791675 at EFEMP1, a gene involved in connective tissue homeostasis, also associates with hernias and carpal tunnel syndrome. Our results highlight the role of connective tissue metabolism and estrogen exposure in the etiology of POP., This research has been conducted using the UK Biobank Resource under Application Number 24898 and 24711. We thank the individuals who participated in the study and whose contribution made this work possible. Folkert Asselbergs is supported by UCL Hospitals NIHR Biomedical Research Centre.

As a single cardiac malformation, ventricular septal defect (VSD) is the most common form of congenital heart disease. However, the precise molecular mechanisms underlying VSD are not completely understood. Numerous microRNAs (miRs/miRNAs) are associated with ventricular septal defects. miR-29c inhibits the proliferation and promotes the apoptosis and differentiation of P19 embryonal carcinoma cells, possibly via suppressing Wnt4 signaling. However, to the best of our knowledge, no innbsp;vivo studies have been published to determine whether overexpression of miR-29c leads to developmental abnormalities. The present study was designed to observe the effect of miRNA-29c on cardiac development and its possible mechanism innbsp;vivo. Zebrafish embryos were microinjected with different doses (1, 1.6 and 2nbsp;micro;mol) miR-29c mimics or negative controls, and hatchability, mortality and cardiac malformation were subsequently observed. The results showed that in zebrafish embryos, miR-29c overexpression attenuated heart development in a dose-dependent manner, manifested by heart rate slowdown, pericardial edema and heart looping disorder. Further experiments showed that overexpression of miR-29c was associated with the Wnt4/beta;-catenin signaling pathway to regulate zebrafish embryonic heart development. In conclusion, the present results demonstrated that miR-29c regulated the lateral development and cardiac circulation of zebrafish embryo by targeting Wnt4.

Thymic epithelial cells (TECs) are essential regulators of T-cell development and selection. miRNAs play critical roles in regulating TEC proliferation during the process of thymic aging. Our previous studies revealed that miR-199b-5p was upregulated in TECs from 1- to 3-month-old mice. But its function and potential mechanism are not clear. We hypothesized that miR-199b-5p may play an important role in age-related thymus involution via targeting some genes. To confirm it, the murine thymic epithelial cell line 1 (MTEC1) cells were used. Our results showed that overexpression of miR-199b-5p can enhance MTEC1 cell proliferation. On the contrary, repression of miR-199b-5p can inhibit MTEC1 cell proliferation. Meanwhile, it was confirmed that frizzled receptor 6 (Fzd6) is the direct target gene of miR-199b-5p. Furthermore, overexpression of miR-199b-5p can upregulate the expressions of β-catenin, Tcf7, Wnt4, and C-myc to activate Wnt signaling and cell cycle signaling. Silence of Fzd6 and co-transfection with siFzd6 and miR-199b-5p mimic/inhibitor confirmed that the biological function of miR-199b-5p is indeed by targeting Fzd6 in medullary TECs. Overall, miR-199b-5p is an important regulator in medullary TEC proliferation through targeting Fzd6 to activate Wnt signaling and cell cycle signaling. Our data indicate that miR-199b-5p may block the process of thymic aging and be a potential therapeutic target for thymus involution.

In China, epithelial ovarian cancer (EOC) patients account for the majority of ovarian cancer patients. The pathogenesis of EOC, one of the most lethal gynecological malignancies, remains unclear. Recently, the role of WNT4 in gynecological disease and tumor development was reported, and a suspicious association of WNT4 with EOC was identified in Europeans. However, the contributions of the WNT4 gene to EOC and the underlying molecular mechanisms remains largely unknown. To determine whether the WNT4 gene is associated with EOC, this study investigated polymorphisms of the WNT4 gene in Han Chinese individuals.We designed a case/control study with 707 EOC patients and 1563 unrelated healthy controls of Han Chinese descent. A total of eight tag single-nucleotide polymorphisms (SNPs) were genotyped successfully, and both single SNP and haplotype analyses were performed to detect the potential association of variations in the WNT4 gene with EOC.The SNP rs56318008 was found to be strongly associated with EOC risk. In the serous EOC subgroup, individuals harboring the T allele of rs56318008 exhibited a higher risk of EOC than individuals harboring the C allele. Moreover, the odds ratios and 95% confidence intervals revealed an increased risk of EOC in individuals with the T allele of the SNP, and haplotypic analyses confirmed the results, showing a similar pattern.Our results show that the WNT4 gene is associated with EOC risk, indicating that this gene may be a potential genetic risk factor for developing EOC.

Hypoxia is the most common characteristic of solid tumours driving cancer metastasis. Cancer cells release exosomes with various functions into the tumour microenvironment during cancer progression. However, the roles and associated mechanisms of hypoxic colorectal cancer (CRC) cell-derived exosomes remain poorly understood. Here, we found that exosomes secreted by hypoxic CRC cells promoted the migration and invasion abilities of normoxic CRC cells. Inhibition of exosome secretion by GW4869 reduced hypoxic exosome-mediated migration and invasion of normoxic CRC cells. Furthermore, we found that these hypoxic exosomes contained Wnt4 depending on HIF1α. Exosomal Wnt4 mediated hypoxic exosome-mediated migration and invasion of normoxic CRC cells. Moreover, exosomal Wnt4 enhanced β-catenin translocation to the nucleus in normoxic CRC cells. The activation of β-catenin signalling was important for the migration and invasion of normoxic CRC cells, which was eliminated by treatment with the β-catenin inhibitor ICG-001. Taken together, the results of our study indicate that hypoxia may stimulate tumour cells to release Wnt4-rich exosomes that are delivered to normoxic cells to enhance prometastatic behaviours, which might provide new targets for CRC treatment.

Wnt4 is a key regulator of ovary development in mammals, but its role in other vertebrates is unknown. Here, Kossack et al. show that zebrafish wnt4a is the ortholog of mammalian Wnt4 and is expressed...., In laboratory strains of zebrafish, sex determination occurs in the absence of a typical sex chromosome and it is not known what regulates the proportion of animals that develop as males or females. Many sex determination and gonad differentiation genes that act downstream of a sex chromosome are well conserved among vertebrates, but studies that test their contribution to this process have mostly been limited to mammalian models. In mammals, WNT4 is a signaling ligand that is essential for ovary and Müllerian duct development, where it antagonizes the male-promoting FGF9 signal. Wnt4 is well conserved across all vertebrates, but it is not known if Wnt4 plays a role in sex determination and/or the differentiation of sex organs in nonmammalian vertebrates. This question is especially interesting in teleosts, such as zebrafish, because they lack an Fgf9 ortholog. Here we show that wnt4a is the ortholog of mammalian Wnt4, and that wnt4b was present in the last common ancestor of humans and zebrafish, but was lost in mammals. We show that wnt4a loss-of-function mutants develop predominantly as males and conclude that wnt4a activity promotes female sex determination and/or differentiation in zebrafish. Additionally, both male and female wnt4a mutants are sterile due to defects in reproductive duct development. Together these results strongly argue that Wnt4a is a conserved regulator of female sex determination and reproductive duct development in mammalian and nonmammalian vertebrates.

The pharyngeal pouches are a series of epithelial outgrowths of the foregut endoderm. Pharyngeal pouches segment precursors of the vertebrate face into pharyngeal arches and pattern the facial skeleton. These pouches fail to develop normally in zebrafish foxi1 mutants, yet the role Foxi1 plays in pouch development remains to be determined. Here we show that ectodermal Foxi1 acts downstream of Fgf8a during the late stage of pouch development to promote rearrangement of pouch-forming cells into bilayers. During this phase, foxi1 and wnt4a are coexpressed in the facial ectoderm and their expression is expanded in fgf8a mutants. foxi1 expression is unaffected in wnt4a mutants; conversely, ectodermal wnt4a expression is abolished in foxi1 mutants. Consistent with this, foxi1 mutant pouch and facial skeletal defects resemble those of wnt4a mutants. These findings suggest that ectodermal Foxi1 mediates late-stage pouch morphogenesis through wnt4a expression. We therefore propose that Fox1 activation of Wnt4a in the ectoderm signals the epithelial stabilization of pouch-forming cells during late-stage of pouch morphogenesis.