Targeting cancer-associated fibroblast-secreted WNT2 restores dendritic cell-mediated antitumour immunity.

OBJECTIVE: Solid tumours respond poorly to immune checkpoint inhibitor (ICI) therapies. One major therapeutic obstacle is the immunosuppressive tumour microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a key component of the TME and negatively regulate antitumour T-cell response. Here, we aimed to uncover the mechanism underlying CAFs-mediated tumour immune evasion and to develop novel therapeutic strategies targeting CAFs for enhancing ICI efficacy in oesophageal squamous cell carcinoma (OSCC) and colorectal cancer (CRC). DESIGN: Anti-WNT2 monoclonal antibody (mAb) was used to treat immunocompetent C57BL/6 mice bearing subcutaneously grafted mEC25 or CMT93 alone or combined with anti-programmed cell death protein 1 (PD-1), and the antitumour efficiency and immune response were assessed. CAFs-induced suppression of dendritic cell (DC)-differentiation and DC-mediated antitumour immunity were analysed by interfering with CAFs-derived WNT2, either by anti-WNT2 mAb or with short hairpin RNA-mediated knockdown. The molecular mechanism underlying CAFs-induced DC suppression was further explored by RNA-sequencing and western blot analyses. RESULTS: A negative correlation between WNT2+ CAFs and active CD8+ T cells was detected in primary OSCC tumours. Anti-WNT2 mAb significantly restored antitumour T-cell responses within tumours and enhanced the efficacy of anti-PD-1 by increasing active DC in both mouse OSCC and CRC syngeneic tumour models. Directly interfering with CAFs-derived WNT2 restored DC differentiation and DC-mediated antitumour T-cell responses. Mechanistic analyses further demonstrated that CAFs-secreted WNT2 suppresses the DC-mediated antitumour T-cell response via the SOCS3/p-JAK2/p-STAT3 signalling cascades. CONCLUSIONS: CAFs could suppress antitumour immunity through WNT2 secretion. Targeting WNT2 might enhance the ICI efficacy and represent a new anticancer immunotherapy.

ERO1L promotes the proliferation and metastasis of lung adenocarcinoma via the Wnt2/ß-catenin signaling pathway.

PURPOSE: This study aimed to explore the role and underlying mechanism of action of Endoplasmic reticulum oxidoreductin-1 L (ERO1L) in lung adenocarcinoma (LUAD). MATERIALS AND METHODS: The Gene expression profiling interactive analysis database was used to analyze the expression of ERO1L in LUAD cases. The expression of ERO1L and Wnt2 in LUAD tissue was evaluated using immunohistochemistry. We also used western blotting to assess the expression of ERO1L or Wnt2 and the phosphorylation of ß-catenin in LUAD cell lines. Plasmid transfection and small interfering RNA were used for overexpression and knockdown of ERO1L in LUAD cells, respectively. The proliferation, invasion and migration of LUAD cells were analyzed using cell viability, Transwell invasion and wound healing assays. The growth of LUAD tumors in animal models was assessed using tumor xenograft experiments. RESULTS: This study revealed that elevated ERO1L expression was associated with a poor prognosis in LUAD patients. In addition, ERO1L expression was significantly associated with lymph-node metastasis, TNM stage and tumor size. The expression of Wnt2 was positively associated with ERO1L expression in LUAD tissue samples and cell lines. ERO1L overexpression upregulated the expression of Wnt2 and ß-catenin phosphorylation in vitro. Additionally, ERO1L was essential for the ubiquitination of Wnt2. Last, ERO1L promoted the proliferation and metastasis of LUAD via the Wnt2 signaling pathway in vitro and in vivo. CONCLUSION: These findings suggest that ERO1L was highly expressed in LUAD tissue, and it promoted the proliferation and metastasis of LUAD by activating the Wnt2/ß-catenin signaling pathway.

High COL10A1 expression potentially contributes to poor outcomes in gastric cancer with the help of LEF1 and Wnt2.

BACKGROUND: COL10A1 is a secreted, short-chain collagen found in several types of cancer. Studies have shown that COL10A1 aberrant expression is considered an oncogenic factor. However, its underlying mechanisms and regulation of gastric cancer remain undefined. METHODS: The data on the expression of COL10A1, clinicopathological characteristics, and outcome of patients with GC were obtained from The Cancer Genome Atlas. The ALGGEN-PROMO database defined the related transcription factors. Quantitative real-time reverse transcription-polymerase chain reaction and western blotting analysis were used to identify the differential expression levels of COL10A1 and related transcription factors. RESULTS: We found that high COL10A1 expression is an independent risk factor for gastric cancer. Upregulation of LEF1 and Wnt2 was also observed in gastric cancer, suggesting a potential correlation between LEF1/COL10A1 regulation in the Wnt2 signaling pathway. CONCLUSION: High COL10A1 expression may contribute to poor outcomes via upregulation of LEF1 and Wnt2 in gastric cancer.

SNHG12 promotes carcinogenesis of human renal cell cancer via functioning as a competing endogenous RNA and sponging miR-30a-3p.

Small nucleolar RNA host gene 12 (SNHG12) has been indicated in the tumorigenesis of various human cancers, including clear cell renal cell carcinoma (ccRCC). However, the underlying mechanisms of SNHG12 driving progression of ccRCC remain incompletely understood. In the present study, we discovered that SNHG12 is up-regulated in ccRCC and that overexpression of SNHG12 predicted poor clinical outcome of ccRCC patients. SNHG12 knockdown notably inhibited proliferation and migration of RCC cells. Furthermore, we discovered that miR-30a-3p, a putative ccRCC inhibitor, was competitively sponged by SNHG12. Via the crosstalk network, SNHG12 was capable of up-regulating multiple target genes of miR-30a-3p, namely, RUNX2, WNT2 and IGF-1R, which have been identified to facilitate tumorigenesis of ccRCC. Taken together, our present study suggested a novel ceRNA network, in which SNHG12 could promote the malignancy of ccRCC although competitively binding with miR-30a-3p and consequently release the expression of its downstream cancer-related genes.

Loss of FLCN inhibits canonical WNT signaling via TFE3.

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt-Hogg-Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/ß inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/ß inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression-two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/ß inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/ß inhibition. Finally, we found that a constitutively active ß-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

Distinguishing colorectal adenoma from hyperplastic polyp by WNT2 expression.

BACKGROUND: Colorectal adenoma (CRA) is a classical premalignant lesion, with high incidence and mainly coexisting with hyperplastic polyp (HPP). Hence, this study aimed to distinguish CRA from HPP by molecular expression profiling and advance the prevention of CRA and its malignance. METHODS: CRA and paired HPP biopsies were collected by endoscopy. Through RNA-sequencing (RNA-seq), the differentially expressed genes (DEGs) were obtained. Functional enrichment analysis was performed based on the DEGs. The STRING database and Cytoscape were used to construct the protein-protein interaction (PPI) network and perform module analysis. Hub genes were validated by real-time quantitative PCR (RT-qPCR) and immunohistochemistry. The ROC curve was drawn to establish the specificity of the hub genes. RESULTS: 485 significant DEGs were identified including 133 up-regulated and 352 down-regulated. The top 10 up-regulated genes were DLX5, MMP10, TAC1, ACAN, TAS2R38, WNT2, PHYHIPL, DKK4, DUSP27, and ABCA12. The top 10 down-regulated genes were SFRP2, CHRDL1, KBTBD12, RERGL, DPP10, CLCA4, GREM2, TMIGD1, FEV, and OTOP3. Wnt signaling pathway and extracellular matrix (ECM) were up-regulated in CRA. Three hub genes including WNT2, WNT5A, and SFRP1 were filtered out via Cytoscape. Further RT-qPCR and immunohistochemistry confirmed that WNT2 was highly expressed in CRA. The area under the ROC curve (AUC) at 0.98 indicated the expression level of WNT2 as a candidate to differ CRA from HPP. CONCLUSION: Our study suggests Wnt signaling pathway and ECM are enriched in CRA, and WNT2 may be used as a novel biomarker for distinguishing CRA from HPP and preventing the malignance of CRA.

The diagnosis and prognosis values of WNT mRNA expression in colon adenocarcinoma.

WNT family genes have participated in the progression and development of many cancers, however, the association between colon adenocarcinoma (COAD) and WNTs have been rarely reported. This study investigated the significance of WNT genes expression in COAD from the standpoint of diagnosis and prognosis. The RNA-sequencing dataset of COAD was downloaded from The Cancer Genome Atlas and University of California, Santa Cruz Xena browser. The biology functions of WNT genes were investigated by biological analysis. Biological analysis of WNT family genes indicated that WNT genes were noticeably enriched in the complex process of WNT signaling pathway. The Pearson correlation analysis suggested WNT1 and WNT9B had a strong correlation. And receiver operating characteristic curves suggested that most of the genes could serve as a significant diagnostic makers in COAD (P < .05), especially WNT2 and WNT7B had high diagnostic values that the area under curve were 0.997 (95% confidence interval [0.994-1.000]) and 0.961 (95%CI [0.939-0.983]), respectively. And our multivariate survival analysis suggested the downregulated of WNT10B (P < .05) showed a favor prognosis in COAD overall survival. And the risk score model predicted that the upregulated expression of WNT10B might increase the risk of death. The very study we had conducted suggested that WNT genes had a certain connection with the diagnosis and prognosis of COAD. The messenger RNA expression of WNT2 and WNT7B might become potentially diagnostic biomarkers, and WNT10B might serve as an independent prognosis indicator for COAD.

Cancer-associated fibroblast-derived WNT2 increases tumor angiogenesis in colon cancer.

WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.

A wnt2 ortholog in the sea urchin Paracentrotus lividus.

Members of the wnt gene family encode secreted glycoproteins that mediate critical intercellular communications in metazoans. Large-scale genome and transcriptome analyses have shown that this family is composed of 13 distinct subfamilies. These analyses have further established that the number of wnt genes per subfamily varies significantly between metazoan phyla, highlighting that gene duplication and gene loss events have shaped the complements of wnt genes during evolution. In sea urchins, for example, previous work reported the absence of representatives of both the WNT2 and WNT11 subfamilies in two different species, Paracentrotus lividus and Strongylocentrotus purpuratus. Recently, however, we identified a gene encoding a WNT2 ortholog in P. lividus and, based on that finding, we also reanalyzed the genome of S. purpuratus. Yet, we found no evidence of a bona fide wnt2 gene in S. purpuratus. Furthermore, we established that the P. lividus wnt2 gene is selectively expressed in vegetal tissues during embryogenesis, in a pattern that is similar, although not identical, to that of other P. lividus wnt genes. Taken together, this study amends previous work on the P. lividus wnt complement and reveals an unexpected variation in the number of wnt genes between closely related sea urchin species.

Wnt/ß-catenin signalling plays diverse functions during the process of fibrotic remodelling in the exocrine pancreas.

BACKGROUND/OBJECTIVES: Wnt/ß-catenin signalling plays vital roles in tissue homeostasis. Dysregulation of the pathway has been implicated in the pathogenesis of cancer and fibroses in numerous tissues, including the pancreas. We studied the effect of microenvironmental changes pertaining to fibrotic tissue remodelling on the expression of selected Wnt/ß-catenin pathway proteins in the human exocrine pancreas. The role of acinar/stellate cross-talk on the expression of the proteins was elucidated in a long-term mouse co-culture system. METHODS: Expression of ß-catenin, Wnt2, Wnt5a and SFRP4 was analysed immunohistochemically in normal and moderately or highly fibrotic human pancreata (n = 8). The effect of humoral interactions on the expression of the proteins was studied by immunocytochemical means in parallel mono- and co-cultures of mouse acinar and stellate cells (PSCs). RESULTS: In human pancreatic tissue, fibrotic microenvironment was associated with redistribution of the proteins in and between epithelial and stromal compartments, compared to acinar-rich tissue. In non-fibrotic and moderately fibrotic tissue the proteins appeared only in acinar cells whereas in highly fibrotic tissue stromal fibroblastoid/stellate cells and macrophages were their predominant locations. Subcellular changes in the expression of ß-catenin and Wnt5a were detected. Our in vitro data suggest potential involvement of acinar cell/PSC cross-talk in mediating the changes observed in tissue specimens. CONCLUSIONS: Wnt/ß-catenin pathway-associated proteins are abundantly expressed in the exocrine pancreas with prominent changes in their cellular and subcellular expression patterns along with increasing levels of fibrosis. Diverse functions for Wnt/ß-catenin signalling during the course of fibrotic remodelling in the exocrine pancreas are suggested.

Wnt-2b in the intermediate hyperpallium apicale of the telencephalon is critical for the thyroid hormone-mediated opening of the sensitive period for filial imprinting in domestic chicks (Gallus gallus domesticus).

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2-3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3'-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis.

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.

Knockdown of mediator subunit Med19 suppresses bladder cancer cell proliferation and migration by downregulating Wnt/ß-catenin signalling pathway.

Mediator complex subunit 19 (Med19), a RNA polymerase II-embedded coactivator, is reported to be involved in bladder cancer (BCa) progression, but its functional contribution to this process is poorly understood. Here, we investigate the effects of Med19 on malignant behaviours of BCa, as well as to elucidate the possible mechanisms. Med19 expression in 15 BCa tissues was significantly higher than adjacent paired normal tissues using real-time PCR and Western blot analysis. Immunohistochemical staining of 167 paraffin-embedded BCa tissues was performed, and the results showed that high Med19 protein level was positively correlated with clinical stages and histopathological grade. Med19 was knocked down in BCa cells using short-hairpin RNA. Functional assays showed that knocking-down of Med19 can suppress cell proliferation and migration in T24, UM-UC3 cells and 5637 in vitro, and inhibited BCa tumour growth in vivo. TOP/FOPflash reporter assay revealed that Med19 knockdown decreased the activity of Wnt/ß-catenin pathway, and the target genes of Wnt/ß-catenin pathway were down-regulated, including Wnt2, ß-catenin, Cyclin-D1 and MMP-9. However, protein levels of Gsk3ß and E-cadherin were elevated. Our data suggest that Med19 expression correlates with aggressive characteristics of BCa and Med19 knockdown suppresses the proliferation and migration of BCa cells through down-regulating the Wnt/ß-catenin pathway, thereby highlighting Med19 as a potential therapeutic target for BCa treatment.

Deregulation of WNT2/FZD3/ß-catenin pathway compromises the estrogen synthesis in cumulus cells from patients with polycystic ovary syndrome.

Mechanistic insight into estrogen deficiency by polycystic ovary syndrome (PCOS) remains a longstanding challenge in reproductive medicine. Recent advance suggest that Wingless-type MMTV integration site family members (WNTs), in concert with its Frizzled (FZD) receptors, regulate normal folliculogenesis, luteogenesis and ovarian steroidogenesis. However, no studies have so far investigated any causality between WNT-FZDs interactions and disrupted estrogen synthesis under certain pathological conditions. Here, we show that (i) FZD3 expression was significantly up-regulated in the cumulus cells (CCs) from PCOS patients. This up-regulation, along with the activation of WNT2/ß-Catenin pathway, was tightly associated with insulin resistance and estrogen deficiency, two hallmarks of PCOS. (ii) Overexpression of exogenous FZD3 in human granulosa cell COV434 impaired long-term FSH incubation-induced CYP19A1 transactivation and the recruitment of ß-Catenin onto CYP19A1 promoter, and subsequently compromised FSH-stimulated estrogen production. (iii) Conversely, inhibition of FZD3 expression exhibited a therapeutic effect on estrogen synthesis in PCOS CCs. Thus, excessive FZD3 expression in CCs may act as a brake on steroidogenic activation that is normally overcome by FSH stimulation. Future endeavor in this field should help to elucidate the complicated crosstalk between energy metabolism and endocrine cells through WNT/FZD signaling molecules.

Decreased expression of WNT2 in villi of unexplained recurrent spontaneous abortion patients may cause trophoblast cell dysfunction via downregulated Wnt/ß-catenin signaling pathway.

WNT2 has been reported to be important for placental development, especially for the proper vascularization of the placenta. However, its precise role in first-trimester trophoblast cells is still unknown. WNT2 expression in the villous tissues of unexplained recurrent spontaneous abortion (URSA) patients was compared with that of healthy women by Western blot. The function of WNT2 in HTR-8/SVneo trophoblast cells was evaluated by altering the cellular WNT2 level through overexpression and shRNA knockdown. The molecular mechanism of the effect of WNT2 on trophoblast cells was investigated. The association of WNT2 with the Wnt/ß-catenin signaling pathway was studied through Western blot and immunofluorescence. Results showed that WNT2 protein expression was significantly decreased in villi of the URSA group compared with the control group. In vitro studies showed that WNT2 could promote human trophoblast cell proliferation and migration through activating the Wnt/ß-catenin signaling pathway. Moreover, upon the knockdown of WNT2, trophoblast cell proliferation and migration were significantly suppressed. In conclusion, our study indicated that WNT2 plays an important role in trophoblast function. WNT2 insufficiency might cause impaired trophoblast cell proliferation and migration via downregulation of Wnt/ß-catenin signaling pathway.

Wnt2 and WISP-1/CCN4 Induce Intimal Thickening via Promotion of Smooth Muscle Cell Migration.

OBJECTIVE: Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. APPROACH AND RESULTS: Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via ß-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. CONCLUSIONS: Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure.

Promoter Methylation Status of WNT2 in Placenta from Patients with Preeclampsia.

BACKGROUND Preeclampsia is a serious multisystem disorder of human gestation, affecting up to 10% of pregnant women worldwide, and results in maternal morbidity and mortality. The aim of this study was to determine the gene expression pattern and methylation status of the promoter of the WNT2 gene in placentas from patients with preeclampsia and to evaluate the potential role of the WNT2 pathway in the pathogenesis of preeclampsia. MATERIAL AND METHODS Real-time quantitative polymerase chain reaction (PT-PCR) was used to determine the WNT2 gene expression level. Western blot analysis was used to identify alterations in wnt2 protein expression. RESULTS The mRNA and protein expression levels of the WNT2 gene were reduced in placentas from patients with preeclampsia when compared with placentas from healthy women. The average methylation level of the promoter of the WNT2 gene was elevated in the placentas from patients with preeclampsia compared with the controls placentas from healthy women. CONCLUSIONS The findings of this study have shown that molecular mechanisms, including aberrant activation of the WNT2 gene signaling pathway, may be involved in the pathogenesis of preeclampsia. Promoter hypermethylation and reduced expression of the WNT2 gene requires further study to determine a potential role in the diagnosis and treatment of preeclampsia.

The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

Wnt4 is essential to normal mammalian lung development.

Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4(-/-) embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4(-/-) lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development.

MicroRNA MiR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting WNT2 signaling pathway.

MicroRNA miR-199a-5p impairs tight junction formation, leading to increased urothelial permeability in bladder pain syndrome. Now, using transcriptome analysis in urothelial TEU-2 cells, we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF, and WNT signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder, and we altered its levels in bladder smooth muscle cells (SMCs) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size, and up-regulated miR-199a-5p targets, including WNT2. Overexpression of WNT2 protein or treating SMCs with recombinant WNT2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of WNT2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM α-actin, and SM myosin heavy chain mRNA and protein levels. These changes as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of the smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor A downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of WNT-dependent inhibitory Krüppel-like transcription factor 4 in miR-199a-5p-overexpressing cells. In contrast, Krüppel-like transcription factor 4 was induced in antimiR-expressing cells following the activation of WNT2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the WNT2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, which is relevant for organ remodeling.