Transcriptomic Analysis of Pubertal and Adult Virgin Mouse Mammary Epithelial and Stromal Cell Populations.

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and fibroblasts. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Inefficient Sox9 upregulation and absence of Rspo1 repression lead to sex reversal in the B6.XYTIR mouse gonad†.

Sry on the Y-chromosome upregulates Sox9, which in turn upregulates a set of genes such as Fgf9 to initiate testicular differentiation in the XY gonad. In the absence of Sry expression, genes such as Rspo1, Foxl2, and Runx1 support ovarian differentiation in the XX gonad. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. In the B6.YTIR mouse, carrying the YTIR-chromosome on the B6 genetic background, Sry is expressed in a comparable manner with that in the B6.XY mouse, yet, only ovaries or ovotestes develop. We asked how testicular and ovarian differentiation pathways interact to determine the gonadal sex in the B6.YTIR mouse. Our results showed that (1) transcript levels of Sox9 were much lower than in B6.XY gonads while those of Rspo1 and Runx1 were as high as B6.XX gonads at 11.5 and 12.5 days postcoitum. (2) FOXL2-positive cells appeared in mosaic with SOX9-positive cells at 12.5 days postcoitum. (3) SOX9-positive cells formed testis cords in the central area while those disappeared to leave only FOXL2-positive cells in the poles or the entire area at 13.5 days postcoitum. (4) No difference was found at transcript levels of all genes between the left and right gonads up to 12.5 days postcoitum, although ovotestes developed much more frequently on the left than the right at 13.5 days postcoitum. These results suggest that inefficient Sox9 upregulation and the absence of Rspo1 repression prevent testicular differentiation in the B6.YTIR gonad.

Exploring the Role of Wnt Ligands in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

OBJECTIVES: This study aimed to investigate the functions of 19 types of Wnt ligands during the process of osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4. MATERIALS AND METHODS: The expression levels of 19 types of Wnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14 days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability. RESULTS: During hPDLSCs osteogenic differentiation, the expression of 19 types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly. CONCLUSION: Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis. CLINICAL RELEVANCE: Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes.

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

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

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

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

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

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

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

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

Gamete-exporting organs of vertebrates: dazed and confused.

Mature gametes are transported externally for fertilization. In vertebrates, the gonads are located within the coelom. Consequently, each species has specific organs for export, which often vary according to sex. In most vertebrates, sperm ducts and oviducts develop from the Wolffian and Müllerian ducts, respectively. However, exceptions exist. Both sexes of cyclostomes, as well as females of basal teleosts, lack genital ducts but possess genital pores. In teleosts of both sexes, genital ducts are formed through the posterior extensions of gonads. These structures appear to be independent of both Wolffian and Müllerian ducts. Furthermore, the development of Wolffian and Müllerian ducts differs significantly among various vertebrates. Are these gamete-exporting organs homologous or not? A question extensively debated around the turn of the 20th century but now largely overlooked. Recent research has revealed the indispensable role of Wnt4a in genital duct development in both sexes of teleosts: zebrafish and medaka. wnt4a is an ortholog of mammalian Wnt4, which has functions in Müllerian duct formation. These results suggest a potential homology between the mammalian Müllerian ducts and genital ducts in teleosts. To investigate the homology of gamete-exporting organs in vertebrates, more detailed descriptions of their development across vertebrates, using modern cellular and genetic tools, are needed. Therefore, this review summarizes existing knowledge and unresolved questions on the structure and development of gamete-exporting organs in diverse vertebrate groups. This also underscores the need for comprehensive studies, particularly on cyclostomes, cartilaginous fishes, basal ray-finned fishes, and teleosts.

Effect of miR 27a targeting regulation of SFRP1 on biological behavior of colorectal cancer / 安徽医科大学学报

Objective @#To investigate the expression of miR-27a in colorectal cancer cell , and to analyze the effect of its targeted regulation of ( Secreted Frizzled Related Protein , SFRP1) on the biological behavior of colorectal cancer cells .@*Methods @#Real time fluorescent quantitative PCR(qRT-PCR) was employed to examine the expres sion of miR-27a and SFRP1 mRNA in colorectal cancer tissues and adjacent normal tissues . Western blot was used to detect the expression of SFRP1 protein in colorectal cancer tissues and adjacent normal tissues . TargetScan soft ware and dual luciferase reporter gene test were used to detect the targeted regulation of miR-27a on SFRP1 . HCT116 cells were transfected with miR-27a mimic , miR-27a inhibitor and negtive control (NC) . The expression of miR-27a and SFRP1 mRNA in each group was determined by qRT-PCR . MTT colorimetry was performed to eval uate the proliferation of each group cells . Transwell assay was used to evaluate the cell invasion and migration ability . Meanwhile , the protein expression levels of SFRP1 , key factors Wnt4 and β-catenin in the Wnt/β catenin signaling pathway were determined by Western blot.@*Results @#Compared with adjacent normal tissues , miR-27a was highly expressed in colorectal cancer tissues , while SFRP1 was low expressed in colorectal cancer tissues ( P < 0.05) . Target Scan software and dual luciferase reporter gene test showed that miR-27a targeted SFRP1 . Compared with NC group , the expression of miR-27a of miR-27a mimic group increased , the proliferation , invasion and migration ability enhanced , the expression of SFRP1 protein decreased , while Wnt4 and β-catenin protein expression increased ( P < 0.05 ) . Compared with miR-27a mimic group , the expression of miR-27a of miR-27a inhibitor group decreased , the proliferation , invasion and migration ability reduced , the expression of SFRP1 protein in creased , while Wnt4 and β-catenin protein expression decreased( P < 0.05) .@*Conclusion @#miR-27a can target SFRP1 , inhibit the proliferation , invasion and migration of colorectal cancer cells , mainly by up regulating SFRP1 and blocking the downstream Wnt/β-catenin signaling pathway , which provides a new direction for clinical treatment.

Effect of electroacupuncture at Wnt/β-catenin signaling pathway on inhibiting cartilage degeneration in rats with knee osteoarthritis / 中国针灸

OBJECTIVE@#To explore the action mechanism of electroacupuncture (EA) for knee osteoarthritis (KOA) based on Wnt/beta-catenin (Wnt/β-catenin) signaling pathway.@*METHODS@#Ten rats were randomly selected into a sham-operation group among 50 male 2-month-old SD rats, and the KOA model was established in the remaining 40 rats by modified Hulth method. Four weeks after the model establishment, the rats were randomly divided into a model group, an experimental A group, an experimental B group and an experimental C group, 10 rats in each group. The rats in the sham-operation group and model group did not receive any intervention. The rats in the experimental A group were treated with EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) for 15 min. The rats in the experimental B group were treated with EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) for 30 min. The rats in the experimental C group were treated with EA at non-acupoint for 15 min. EA intervention was given once a day, five times a week, and totally 12-week treatment was given. After 12 weeks, the knee cartilage tissues were stained and the morphological changes were observed under light microscopy; the severity of cartilage degeneration was evaluated by modified Mankin's score; the content of interleukin-1β (IL-1β) in synovium tissues was detected by ELISA method; the content of Wnt-4, β-catenin and matrix metalloprotein-13 (MMP-13) in cartilage tissues was detected by Western blot method.@*RESULTS@#Compared with the sham-operation group, in the model group the morphology and structure of cartilage were disordered, the number of cells was significantly reduced, the matrix was decontaminated and tidal line was incomplete; the Mankin's score was significantly increased (0.05).@*CONCLUSION@#EA at "Neixiyan" (EX-LE 4) and "Dubi"(ST 35) may reduce the expression of MMP-13 and the production of inflammatory factor IL-1β through Wnt/β-catenin signaling pathway, thus inhibit the degradation of cartilage matrix and the apoptosis of chondrocyte, and improve the morphology and structure of cartilage.

Expression of WNT4 and its inhibitory factor SFRP1 in renal tissue of diabetic nephropathy rats / 基础医学与临床

Objective To observe the change of expression of WNT4/β-catenin signaling pathway and its inhibitory factor secreted frizzled-related protein 1 (SFRP1) in renal tissue in diabetic nephropathy(DN) rats, and to explore its possible role in the development of renal fibrosis. Methods Rats were randomly divided into normal control(NC) group and DN group, and equipped with 8 in each group. The IDDM model was prepared by tail vein injection of STZ 55 mg/kg. Hemotoxyin and eosin、Periodic Acid-Schiff and Masson stain were used to observe the morphological structure and fibrotic lesions in renal tissue;Immunohistochemical analysis was used to observe the protein expression of WNT4 and β-catenin in renal tissue;Western blot was used to detect the protein expression changes of WNT4, SFRP1, β-catenin, p-GSK-3β, GSK-3β, Collagenl, a-SMA, E-cadherin in renal tissue in each group;The mRNA expression of WNT4 and SFRPl in renal tissues of rat was detected by realtime PCR. Results Compared with NC group, renal tissue fibrosis was obvious in DN group. Compared with NC group, the protein and mRNA expressions of WNT4 significantly increased (P＜0.05), the protein expressions of β-catenin, p-GSK-3β, α-SMA and collagen I significantly increased (P < 0.05), the protein expressions of Ecadherin significantly decreased (P＜0. 05), the protein and mRNA expression of SFRPl significantly decreased (P＜0.05). Conclusions In the case of DN, the signal pathway of WNT4/β-catenin is abnormal activation. The expression of SFRPl is decreased, and that may inhibit this pathway and promote the development of renal fibrosis in DN.

Effects of WNT4 gene silencing on migration and invasion of renal tubular epithelial cells of rats stably transfected with PAX2 gene / 吉林大学学报(医学版)

Objective; To investigate the effects of WNT4 gene silecing on the migration and invasion of renal tubular epithelial cells of the rats stably transfected with paired box gene 2 (PAX2) gene, and to clarify whether the PAX2 gene regulates the biological activities of renal tubular epithelial cells through WNT4 gene or not. Methods; The renal tubular epithelial cells transfected with PAX2 gene in the logarithmic phase were divided into stably transfected group and control group. The cells in stably transfected group were divided into stably transfected control group (without WNT4 siRNA silencing) and silencing 72 h group (with WNT4 siRNA silencing for 72 h). The relative expression amounts of PAX2 and WNT4 proteins in renal tubular epithelial cells of the rats in stably transfected group and control group were detected by Western blotting method. The relative expression amounts of WNT4 mRNA in renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group were detected by Real time PCR method. The migration rates of renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group were detected by cell wound scratch assay. The number of transmembrane renal tubular epithelial cells of the rats in stably transfected control group and silencing 72 h group was detected by Transwell experiment. Results: The Western blotting results showed that the relative expression amounts of PAX2 and WNT4 proteins in renal tubular epithelial cells of the rats in stably transfected group were higher than those in control group (P0. 05); the migration rate of renal tubular epithelial cells in silencing 72 h group after 18 h was lower than that in stably transfected control group (P<0. 05). The Transwell experiment results showed that the number of transmembrane renal tubular epithelial cells in silencing 72 h group was lower than that in stably transfected control group (P<0. 05). Conclusion: PAX2 gene can regulate the biological activities of renal tubular epithelial cells through WNT4 gene.

Identification and expression analysis of two Wnt4 genes in the spotted scat (Scatophagus argus)

Background: WNT4 is a protein that plays a crucial role in ovarian differentiation and development in mammals, with a relatively well understood function in mammalian gonadal differentiation. The role of WNT4 in teleost fish; however, remains unclear. In the present study, cDNAs of Wnt4a and Wnt4b were cloned and characterized in the spotted scat. The expression patterns of two Wnt4 genes in the gonads at different stages of development and in fish after treatment with 17a-methyltestosterone (MT) were investigated. Results: The tissue distribution showed that Wnt4a was expressed in various tissues, including the gonads, gills, spleen, brain, and fin. Interestingly, Wnt4b not only was expressed in the gills, brain, and spleen, but also was obviously expressed in the ovary. During gonad development, Wnt4a was highly expressed in the testis at stage I and Wnt4b was mainly expressed in the ovary at stages II-III. After MT treatment, the mRNA expression of Wnt4a increased significantly up to 40 d, and the transcript level of Wnt4b decreased at 20 d. Conclusions: These results suggest that Wnt4a may be involved in gonad development and plays a role in the process of spermatogonial proliferation. Our results also demonstrate that Wnt4b is not only expressed in the nervous system, but also in the ovary and it may be involved in ovary development of the spotted scat.

Sequence Analysis and Molecular Characterization of Wnt4 Gene in Metacestodes of Taenia solium

Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium.

Modulation of Wnt/β-catenin signaling pathway by irbesartan in highglucose-induced tubular epithelial-mesenchymal transition / 中国药理学通报

Aim To investigate the effects of irbesartan on Wnt/β-catenin signaling pathway in tubular epithelial-mesenchymal transition(EMT)in HKCs induced by high-glucose.Methods Human kidney proximal tubular epithelial cell line(HKCs)cultured in vitro was divided into four groups:normal-glucose group,mannitol control group,high-glucose group and high-glucose plus irbesartan group.Immunocytochemistry staining was used to observe the expression of β-catenin;the protein expression of Wnt4,β-catenin,E-cadherin and α-SMA was assessed by Western blot;Wnt4 and β-catenin mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with normal-glucose and mannitol control group,both the protein and the mRNA of Wnt4 were up-regulated in HKCs stimulated by high-glucose.α-SMA expression significantly increased but E-cadherin decreased in HG group.The cytoplastic and nuclear fraction of β-catenin enhanced with highglucose stimulation.But no difference of the total protein and mRNA of β-catenin was observed between highglucose-treatment and control groups.Highglucose induced Wnt4 and β-catenin expression in a time-dependent manner,both peaking at 24 h.Irbesartan reduced the promotional effect of HG on Wnt4 and α-SMA expression,and nuclear translocation of β-catenin.HG-mediated inhibition of E-cadherin was also restored by irbesartan.Conclusion These data supported a functional role for Wnt/β-catenin signaling pathway during epithelial-mesenchymal transition of HKCs induced by high glucose and suggested that irbesartan might reverse tubular EMT by regulating activity of Wnt/β-catenin pathway.

Expressão da proteína Wnt4 e seu possível papel como um antígeno associado ao câncer de próstata / [Expression of the Wnt4 protein and its possible role as an antigen associated with prostate cancer]

A ativação aberrante da via de sinalização de Wnt tem sido freqüentemente associada à tumorigênese do câncer de próstata (CaP). No entanto, pouco se sabe sobre o papel e perfil de expressão do membro Wnt4 no desenvolvimento deste tumor. Este trabalho apresenta dois principais objetivos: 1- Avaliar a expressão da proteína Wnt4 em amostras tumorais e não tumorais (hiperplasia prostática benigna - HPB) de próstata através de imunohistoquímica em microarranjos de tecido (TMA) e 2- Avaliar o potencial papel da proteína Wnt4 como antígeno associado ao CaP. A análise do perfil de expressão da proteína Wnt4 em amostras tumorais e de HPB apresentou marcação citoplasmática e de membrana, não havendo diferenças estatisticamente significativas no valor mediano da intensidade de marcação entre elas. Ressalta-se uma marcação mais intensa para Wnt4 na borda apical das células epiteliais voltadas para o lúmen do ácinos prostáticos. O perfil de expressão de Wnt4 não apresentou correlação com o Escore de Gleason, níveis de PSA, recidiva clínica e presença de um segundo tumor. No entanto, pacientes cujas amostras de tumor de próstata marcavam mais fortemente para a proteína Wnt4 apresentavam recorrência bioquímica da doença num tempo menor em relação a pacientes cujas amostras apresentavam marcação fraca ou ausente. A expressão das proteínas E-caderina e Topoisomerase II-α também foi analisada nas mesmas amostras do TMA e seus perfis de marcação também não apresentaram correlação com os parâmetros clínicopatológicos investigados. A expressão de Topoisomerase II-α foi maior em amostras de CaP quando comparadas com amostras de HPB. Neste estudo, mostramos haver correlação entre o perfil de marcação das proteínas Wnt4, Ecaderina e Topoisomerase II-α. Ensaios de imunoblot utilizando a proteína Wnt4 recombinante identificaram a presença de autoanticorpos contra esta proteína em uma mistura de soros de pacientes com CaP, mas não em amostras de soros de indivíduos controles sadios. Em conjunto nossos dados mostram que a proteína Wnt4 não é diferencialmente expressa entre tecidos de CaP e HPB e que quando superexpressa em amostras de tumores de CaP parece apresentar correlação com menor tempo de recorrência bioquímica. Este estudo também apresenta a primeira evidência de que a proteína Wnt4 é capaz de induzir resposta imune humoral em pacientes com CaP, podendo constituir, portanto, um potencial antígeno associado ao CaP.

Aberrant Wnt signaling pathway activation has been associated with prostate cancer (PCa) tumorigenesis. However, the role and expression profiling of Wnt4 member in PCa tumorigenesis is poorly understood. This work presents two main objectives: 1- Evaluate the expression of Wnt4 in tumor and non tumor prostate samples (benign prostate hyperplasia - BPH) through immunohistochemistry in tissue microarrays (TMA) and 2- Evaluate the potential role of Wnt4 as a tumor associated antigen in PCa. Wnt4 expression profiling in PCa and BPH presented a cytoplasmatic and membrane staining pattern, with no significant statistical difference at the median intensity staining values between these two samples. It is noteworthy the stronger Wnt4 staining at the apical border of epithelial cells toward the prostatic acini lumen. Wnt4 expression pattern did not present any correlation to Gleason score, PSA levels, clinical relapse and presence of a second tumor. However, PCa patients whose tumor samples were strongly stained for Wnt4 presented biochemical recurrence earlier than those whose tissues presented weak or absent Wnt4 staining. The expression of E-cadherin and Topoisomerase II-α were also analyzed in these same samples on this TMA and their expression profiling did not present any correlation to the clinical-pathological parameters analyzed as well. The expression of Topoisomerase II-α was higher in PCa tumor samples as compared to BPH tissues. In this study, we showed a correlation between the expression profiling of Wnt4, E-cadherin and Topoisomerase II-α. Immunoblot assays using the Wnt4 recombinant protein identified autoantibodies against this protein in a pool of serum samples from PCa patients, but not in a pool sample from healthy donor individuals. As a whole, our data show that Wnt4 protein is not differentially expressed between PCa e BPH tissues and that there is correlation between highly Wnt4 protein expression in PCa tumor tissues and an earlier biochemical recurrence. This study also provides the first clues that Wnt4 could trigger humoral immune response in PCa patients and hence corresponding to a potential PCa tumor associated antigen.