Wingless-type MMTV Integration Site Research Articles

DNA methylation profiles in preeclampsia and healthy control placentas.

Preeclampsia is a hypertensive disorder of pregnancy that affects 3-5% of all pregnancies. There is evidence to suggest that epigenetic mechanisms, such as DNA methylation, play a role in placental development and function. This study compared DNA methylation profiles of placentas from preeclampsia-affected pregnancies with placentas from healthy pregnancies to identify gene-specific changes in DNA methylation that may contribute to the development of preeclampsia. The methylation status of eight placental biopsies taken from preeclampsia-affected and 16 healthy pregnancies was analyzed using the Illumina Infinium Methylation 450 BeadChip array. Bisulfite pyrosequencing was used to confirm regions found to be differentially methylated between preeclampsia and healthy placentas. A total of 303 differentially methylated regions, 214 hypermethylated and 89 hypomethylated, between preeclampsia cases and controls were identified, after adjusting for gestational age (adjusted P < 0.05). Functional annotation found cell adhesion, wingless type MMTV Integration Site family member 2 (Wnt) signaling pathway, and regulation of transcription were significantly enriched in these gene regions. Hypermethylation of WNT2, sperm equatorial segment protein (SPESP1), NADPH oxidase 5 (NOX5), and activated leukocyte cell adhesion molecule (ALCAM) in preeclampsia placentas was confirmed with pyrosequencing. This study found differences in methylation in gene regions involved in cell signaling (WNT2), fertilization and implantation (SPESP1), reactive oxygen species signaling (NOX5), and cell adhesion (ALCAM). These results build on recently published studies that have reported significant differences in DNA methylation in preeclampsia placentas.

Pelacakan Protein Wingless-Type MMTV Integration Site Family Member-4 pada Uterus Mencit (DETECTION WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER-4 PROTEIN OF MOUSE UTERUS)

The aims of this study was to detect the expression of Wingless-type MMTV integration site familymember 4 (Wnt4) protein in the uterus of Swiss Webster mice. Laboratory animals that were used areadult Swiss Webster mice weighing 25-30 grams. Mice were kept and mated in a controlled laboratoryconditions. Pregnancy was determined by the presence of vaginal plug in female mice after breeding.Protein was isolated from the uterus at seven days of gestation. Immunoblotting was performed usingChemiluminescent Western Blot kit. The primary antibody used was anti Wnt-4 antibody with a 1: 1000dilution. The results showed protein bands with molecular weights ranging from 40 kDa showed a positivereaction to the primary antibody that were used so that it can be concluded that the protein is Wnt4protein.

Influence of an Allergen-Specific Th17 Response on Remodeling of the Airways.

We showed previously that sensitization of mice with dendritic cells (DCs) via the airways depends on activation of these cells with LPS. Allergen-pulsed DCs that were stimulated with low doses of LPS induce a strong Th2 response in vivo. Our objective was to investigate whether airway sensitization of mice by the application of DCs with a phenotype that is able to induce Th17 cells results in increased remodeling of the airways. We generated DCs from the bone marrow of mice and pulsed them with LPS-free ovalbumin. Subsequently, cells were activated with LPS with or without ATP for inflammasome activation. The activated cells were used to sensitize mice via the airways. Intranasal instillation of DCs that were activated with 0.1 ng/ml LPS induced a Th2 response with airway eosinophilia. High doses of LPS, particularly when given in combination with ATP, led to induction of a mixed Th2/Th17 response. Interestingly, we found a correlation between IL-17A production and the remodeling of the airways. Stimulation of mouse fibroblasts with purified IL-17A protein in vitro resulted in transforming growth factor-β1 secretion and collagen transcription. Interestingly, we found enhanced secretion of transforming growth factor-β1 by fibroblasts after costimulation with IL-17A and the profibrotic factor wingless-type MMTV integration site family, member 5A (Wnt5a). We showed that an allergen-specific Th17 response in the airway is accompanied by increased airway remodeling. Furthermore, we revealed that increased remodeling is not only based on neutrophilic inflammation, but also on the direct impact of IL-17A on airway structural cells.

Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus).

WNT4 (wingless-type MMTV integration site family, member 4) is regarded as a key regulator of gonad differentiation in mammalians. However, the potential role of wnt4 in teleosts during gonad differentiation and development is still unclear. The full-length cDNA sequence of wnt4 in olive flounder (Paralichthys olivaceus) was obtained using RACE (rapid amplification of cDNA ends) technique. The wnt4 ORF contains 1059 nucleotides, encoding a protein with a signal peptide domain and a wnt family domain. Expression in tissues of adult flounders was analyzed by real-time RT-PCR. The results showed that wnt4 was widely expressed in multiple tissues of flounders, and the expression level was significantly higher in ovary than in testis. Then wnt4 expression pattern was investigated during gonadal differentiation period and at gonadal development stages (I-V). The results showed the expression levels were significantly higher in testis than in ovary during gonadal differentiation. Notably, wnt4 expression had a very significant increase before testis differentiation. At gonad different developmental stages, there was no expression signal at stage I or stage II, and the expression of wnt4 was much stronger in ovary than in testis at stage III and stage IV, followed by a faint expression in stage V in both sexes. Our results imply that cloned wnt4 could be wnt4a. It is a sex-related gene and its expression pattern in gonadal differentiation period of flounder is different from that in mammalians or other teleosts. Flounder wnt4 might play more important role in testis than in ovary during gonadal differentiation.

Dual mechanisms of action of the RNA-binding protein human antigen R explains its regulatory effect on melanoma cell migration

Overexpression of wingless-type MMTV integration site family 5A (WNT5A) plays a significant role in melanoma cancer progression; however, the mechanism(s) involved remains unknown. In breast cancer, the human antigen R (HuR) has been implicated in the regulation of WNT5A expression. Here, we demonstrate that endogenous expression of WNT5A correlates with levels of active HuR in HTB63 and WM852 melanoma cells and that HuR binds to WNT5A messenger RNA in both cell lines. Although the HuR inhibitor MS-444 significantly impaired migration in both melanoma cell lines, it reduced WNT5A expression only in HTB63 cells, as did small interfering RNA knockdown of HuR. Consistent with this finding, MS-444-induced inhibition of HTB63 cell migration was restored by the addition of recombinant WNT5A, whereas MS-444-induced inhibition of WM852 cell migration was restored by the addition of recombinant matrix metalloproteinase-9, another HuR-regulated protein. Clearly, HuR positively regulates melanoma cell migration via at least 2 distinct mechanisms making HuR an attractive therapeutic target for halting melanoma dissemination.

A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma.

Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

Oncogenic Wnt3a expression as an estimable prognostic marker for hepatocellular carcinoma

To investigate member 3a of Wingless-type MMTV integration site family (Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of hepatocellular carcinoma (HCC) and Wnt3a expression. Wnt3a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25% (77 of 80), which was significantly higher (χ(2) = 48.818, P < 0.001) than that in the surrounding group (46.25%, 37 of 80). Brown Wnt3a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3a expression in HCC were related to poorly-differentiated grade (χ(2) = 20.211, P < 0.001), liver cirrhosis (χ(2) = 8.467, P < 0.004), hepatitis B virus (HBV) infection (χ(2) = 12.957, P < 0.001), higher tumor-node-metastasis stage (χ(2) = 22.960, P < 0.001), and 5-year survival rate (χ(2) = 15.469, P < 0.001). Oncogenic Wnt3a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.

WNT10A polymorphism may be a risk factor for non-syndromic hypodontia.

This study was designed to determine whether polymorphisms in the gene wingless-type MMTV integration site family, member 10A (WNT10A) are associated with non-syndromic hypodontia (tooth agenesis). A case-control study was performed involving 129 subjects with sporadic non-syndromic hypodontia (cases) and 218 healthy individuals (controls). DNA was obtained from whole blood and the ligase detection reaction method was used to analyze two single nucleotide polymorphisms (SNPs) of the WNT10A gene. A significant difference between cases and controls was observed in the allele and genotype frequencies of both SNPs (rs116998555 and rs147680216). For rs116998555, the presence of the T allele (the thymine variant) was associated with tooth agenesis [odds ratio (OR) = 5.722; 95% confidence interval (CI) = 3.053-10.727; P < 0.001], while for rs147680216, the A allele (the adenine variant) correlated with this condition (OR = 2.665; 95%CI = 1.512-4.695; P < 0.001). We provide here the first case-control study evidence that risk of hypodontia may be related to the WNT10A polymorphism. Our results also confirm the importance of the Wnt pathway in tooth development.

Expression of genes involved in adipogenesis and lipid metabolism in subcutaneous adipose tissue and longissimus muscle in low-marbled Pirenaica beef cattle

The ability to accumulate intramuscular fat (IMF) is a highly variable characteristic in beef cattle. In breeds with a low tendency to accumulate IMF, this can lead to compromised meat quality because of the contribution of fat to such organoleptic attributes as juiciness and taste. This study considered adiposity and gene expression of some of the main markers involved in adipogenesis and lipid metabolism in the subcutaneous (SC) adipose tissue (AT) and the longissimus thoracis muscle (LM) and investigated differences in adipogenic regulation between the tissues during growth and fattening under different conditions. Pirenaica beef cattle were chosen for the study due to the breed’s low tendency to accumulate IMF and the breed’s regional importance. The young Pirenaica bulls used (n=16) were allocated to four groups and slaughtered at 6, 12 and 18 months. From 12 months onwards the bulls slaughtered at 18 months were fed diets having different energy densities. Backfat thickness increased from 6 to 12 months (P<0.05) but then was unchanged, while other fattening parameters such as percentage chemical fat and marbling did not vary. The adipose cell size distribution displayed a bimodal distribution for SC adipocytes and a unimodal distribution for IMF cells, suggestive of tissue-specific hyperplasia. Gene expression of peroxisome proliferator-activated receptor γ (PPARG), CCAAT/enhancer-binding protein α (CEBPA), sterol regulatory element-binding transcription factor 1 (SREBF1), wingless-type MMTV integration site family 10B (WNT10B), fatty acid-binding protein 4 (FABP4), acetyl Co-A carboxylase α, lipoprotein lipase and fatty acid synthase (FASN) were determined by real-time quantitative PCR. Expression did not differ between the experimental groups within the tissues but did differ between the tissues: PPARG, FABP4 and FASN were upregulated in the SC AT, while CEBPA, WNT10B and SREBF1 were upregulated in the LM. Although age and diet energy density did not have a significant effect on increasing the amount of IMF, these factors could have influenced adipocyte development in this tissue differently than in the SC AT. This was evidenced by the different size distributions of the cells in the two tissues, and the differing expression patterns of certain markers in the SC AT and the LM, which may indicate a differential role of PPARG and WNT10B in triggering adipocyte proliferation and fat accumulation capacity.

WNT4-like protein is a cortical granule component in mouse oocytes and functions in regulating preimplantation embryogenesis

Mammalian cortical granules (CG) are membrane-bound organelles located in the cortex of the unfertilized oocytes. Upon fertilization, CG undergo exocytosis to function in blocking polyspermy. While cortical granules are important in fertilization, their exact biochemical composition and reproductive function have not been fully defined. In the present study, a 66 kDa wingless-type MMTV integration site family, member 4 (WNT4)-like protein, with mouse CG origin was identified. Oocytes that were double labeled with lectin Lens culinaris agglutinin (LCA) and WNT4 antibody showed colocalization of the WNT4 molecules and cortical granules. The disappearance of WNT4 molecules in the artificially activated oocytes that were devoid of cortical granules confirmed their granule origin. Following fertilization, WNT4 remained associated with zygotes and blastomeres of 2-cell and 8-cell embryos; however the amount of protein present was reduced more than 2-fold as embryos developed. Prior to implantation, WNT4 appeared to be detectable only in the trophoblast cells. Our functional study revealed that WNT4 molecules were involved in regulating zygotic cleavage and early embryogenesis. To our knowledge, this is the first study demonstrating mammalian cortical granules contain signaling molecules that are involved in the regulation of the first phase of embryonic development.

Expression of osterix Is Regulated by FGF and Wnt/β-Catenin Signalling during Osteoblast Differentiation.

Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF) and canonical Wingless-type MMTV integration site (Wnt/β-Catenin) signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx). We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/β-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation.

Molecular mechanisms underlying the exceptional adaptations of batoid fins

Extreme novelties in the shape and size of paired fins are exemplified by extinct and extant cartilaginous and bony fishes. Pectoral fins of skates and rays, such as the little skate (Batoid, Leucoraja erinacea), show a strikingly unique morphology where the pectoral fin extends anteriorly to ultimately fuse with the head. This results in a morphology that essentially surrounds the body and is associated with the evolution of novel swimming mechanisms in the group. In an approach that extends from RNA sequencing to in situ hybridization to functional assays, we show that anterior and posterior portions of the pectoral fin have different genetic underpinnings: canonical genes of appendage development control posterior fin development via an apical ectodermal ridge (AER), whereas an alternative Homeobox (Hox)-Fibroblast growth factor (Fgf)-Wingless type MMTV integration site family (Wnt) genetic module in the anterior region creates an AER-like structure that drives anterior fin expansion. Finally, we show that GLI family zinc finger 3 (Gli3), which is an anterior repressor of tetrapod digits, is expressed in the posterior half of the pectoral fin of skate, shark, and zebrafish but in the anterior side of the pelvic fin. Taken together, these data point to both highly derived and deeply ancestral patterns of gene expression in skate pectoral fins, shedding light on the molecular mechanisms behind the evolution of novel fin morphologies.

FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKD1

Fibroblast growth factors (FGFs) are required to specify hepatic fate within the definitive endoderm through activation of the FGF receptors (FGFRs). While the signaling pathways involved in hepatic specification are well understood, the mechanisms through which FGFs induce hepatic character within the endoderm are ill defined. Here we report the identification of genes whose expression is directly regulated by FGFR activity during the transition from endoderm to hepatic progenitor cell. The FGFR immediate early genes that were identified include those encoding transcription factors, growth factors, and signaling molecules. One of these immediate early genes encodes naked cuticle homolog 1 (NKD1), which is a repressor of canonical WNT (wingless-type MMTV integration site) signaling. We show that loss of NKD1 suppresses the formation of hepatic progenitor cells from human induced pluripotent stem cells and that this phenotype can be rescued by using a pharmacological antagonist of canonical WNT signaling. We conclude that FGF specifies hepatic fate at least in large part by inducing expression of NKD1 to transiently suppress the canonical WNT pathway.

Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance.

The canonical hedgehog (HH) pathway is a multicomponent signaling cascade (HH, protein patched homolog 1 (PTCH1), smoothened (SMO)) that plays a pivotal role during embryonic development through activation of downstream effector molecules, namely glioma-associated oncogene homolog 1 (GLI1), GLI2 and GLI3. Activation of GLIs must be tightly regulated as they modulate target genes which control tissue patterning, stem cell maintenance, and differentiation during development. However, dysregulation or mutations in HH signaling leads to genomic instability (GI) and various cancers, for example, germline mutation in PTCH1 lead to Gorlin syndrome, a condition where patients develop numerous basal cell carcinomas and rarely rhabdomyosarcoma (RMS). Activating mutations in SMO have also been recognized in sporadic cases of medulloblastoma and SMO is overexpressed in many other cancers. Recently, studies in several human cancers have shown that GLI1 expression is independent from HH ligand and canonical intracellular signaling through PTCH and SMO. In fact, this aberrantly regulated GLI1 has been linked to several non-canonical oncogenic growth signals such as Kirsten rat sarcoma viral oncogene homolog (KRAS), avian myelocytomatosis virus oncogene cellular homolog (C-MYC), transforming growth factor β (TGFβ), wingless-type MMTV integration site family (WNT) and β-catenin. Recent studies from our lab and other independent studies demonstrate that aberrantly expressed GLI1 influences the integrity of several DNA damage response and repair signals, and if altered, these networks can contribute to GI and impact tumor response to chemo- and radiation therapies. Furthermore, the ineffectiveness of SMO inhibitors in clinical studies argues for the development of GLI1-specific inhibitors in order to develop effective therapeutic modalities to treat these tumors. In this review, we focus on summarizing current understanding of the molecular, biochemical and cellular basis for aberrant GLI1 expression and discuss GLI1-mediated HH signaling on DNA damage responses, carcinogenesis and chemoresistance.

Abnormality of Wnt3a expression as novel specific biomarker for diagnosis and differentiation of hepatocellular carcinoma.

The member 3a of Wingless-type MMTV integration site family (Wnt3a) as an oncogene is overexpressed in many kinds of tumors with a worse outcome. However, the mechanism and alteration of Wnt3a expression in hepatocellular carcinoma (HCC) have not been clarified. In this study, the levels of Wnt3a expression were investigated in 80 HCC tissues or sera of 186 patients with chronic liver diseases. The incidence of hepatic Wnt3a expression in HCC tissues was 96.25% and significantly higher (χ (2) = 48.818, P < 0.001) than that in their surrounding tissues (46.25%). The higher level (>800ng/L) of circulating Wnt3a expression was found in 92.5% HCC patients and significantly related (P < 0.05) to alpha-fetoprotein (AFP) level, liver cirrhosis, hepatitis B virus infection, poor differentiation, tumor node metastasis, and extra-hepatic metastasis. The level of Wnt3a expression in HCC patients was obviously higher (P < 0.001) than that in any group of cases with benign liver diseases. The diagnostic specificity or the area under the receiver operating characteristic curve was 94.34% or 0.994 in Wnt3a and 69.81% or 0.710 in AFP for HCC, respectively. The present data suggested that Wnt3a expression associated with tumor progression should be a novel specific biomarker for diagnosis and differentiation of HCC.

The bone-sparing effects of estrogen and WNT16 are independent of each other

Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16(-/-) mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L3. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16(-/-) mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16(-/-) and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.

Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions.

The AHR is a ligand-activated transcription factor that mediates gene-environment interactions. Genome-wide expression profiling during differentiation of mouse ES cells into cardiomyocytes showed that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Dioxin (TCDD), its prototypical ligand, disrupted the expression of multiple homeobox transcription factors and inhibited cardiomyocyte contractility. Here we treated ES cells with TCDD at daily differentiation intervals to investigate whether TCDD-induced loss of contractility had a developmental window of sensitivity. Surprisingly, contractility was an AHR-dependent TCDD target solely between differentiation days 0 and 3 during the period of panmesoderm development, when TCDD also disrupted expression of genes in the TGFβ/BMP2/4 and wingless-type MMTV integration site (WNT)signaling pathways, suppressed the secretion of bone morphogenetic protein (BMP4), WNT3a, and WNT5a and elevated the secretion of Activin A, as determined by ELISA of the secreted proteins in the culture medium. Supplementing the culture medium with BMP4, WNT3a, or WNT5a during the first 3 days of differentiation successfully countered TCDD-induced impairment of contractility, while anti-WNT3a, or anti-WNT5a antibodies or continuous Noggin (a BMP4 antagonist) or Activin A treatment inhibited the contractile phenotype. In Ahr(+/+), but not in Ahr(-) (/) (-) ES cells, TCDD treatment significantly increased mitochondrial copy number, suggestive of mitochondrial stress and remodeling. Sustained AHR activation during ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and cause the loss of contractility in the resulting cardiomyocyte lineage.

Wnt signaling in age-related macular degeneration: human macular tissue and mouse model.

BackgroundThe wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.MethodsWe examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2−/−/Cx3cr1−/−/rd8 and Ccl2−/−/Cx3cr1gfp/gfp mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.ResultsWe found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2−/−/Cx3cr1−/−/rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2−/−/Cx3cr1−/−/rd8 and Ccl2−/−/Cx3cr1gfp/gfp mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2−/−/Cx3cr1−/−/rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2−/−/Cx3cr1−/−/rd8 retinas.ConclusionsWnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0683-x) contains supplementary material, which is available to authorized users.

Frizzled2 mediates the migration and invasion of human oral squamous cell carcinoma cells through the regulation of the signal transducer and activator of transcription-3 signaling pathway.

Frizzled2 (Fzd2) is a receptor for wingless-type MMTV integration site family members (Wnts), the aberrant overexpression of which has been noted to contribute to cancer metastasis. The present study was performed to characterize the role of Fzd2 in the migration and invasion of oral squamous cell carcinomas (OSCC) in vitro. Using TSCCa cells (a tongue SCC cell line) for loss- or gain-of-function of Fzd2, we found that a forced overexpression of Fzd2 promoted TSCCa cell migration and invasion, decreased the expression of epithelial‑cadherin (E-cadherin, an epithelial marker) and increased that of vimentin, Snail Slug, matrix metalloproteinases (MMPs)-2/-9/-13 and a-disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5). By contrast, RNA interference (RNAi)-mediated knockdown of Fzd2 had opposite effects on OSCC cells. In addition, we found that the phosphorylation of signal transducer and activator of transcription-3 (STAT3) was enhanced by Fzd2 overexpression, but suppressed by Fzd2 depletion, and that STAT3‑specific shRNA attenuated Fzd2 overexpression‑induced cell invasion. In summary, the present study demonstrated that Fzd2 contributes to the migration and invasion of OSCC cells, at least partly through regulation of the STAT3 pathway. These results suggest Fzd2 as a novel therapeutic target for OSCC.

Lgr4 controls specialization of female gonads in mice.

Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4(-/-) female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4(-/-) female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4(-/-) ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.