Wnt Signaling Members Research Articles

The scaffold protein IQGAP3 is highly upregulated in most epithelial cancers. While recent studies have highlighted its pivotal roles in cancer cell proliferation and metastasis, a deeper mechanistic understanding of IQGAP3 is currently lacking. We have here used TurboID to map IQGAP3 proximity partners and identified the Wnt signaling members Axin1 and CK1α as IQGAP3-interacting proteins. Our functional studies demonstrated that overexpression of IQGAP3 increases β-catenin levels, while IQGAP3 depletion reduces β-catenin levels in gastric cancer cells. Mechanistically, IQGAP3 disrupts Axin1-CK1α interaction, thereby inhibiting β-catenin phosphorylation and ultimately leading to its accumulation. Importantly, we discovered that IQGAP3 itself is regulated by Wnt signaling, suggesting its involvement in a positive feedback loop in Wnt/β-catenin signaling through interactions with Axin1 and CK1α. These findings identify IQGAP3 as a novel mediator of β-catenin stabilization and underscore its potential as a target for cancer therapy.

The blood-brain barrier (BBB), a highly regulated interface between the blood and the brain, prevents blood-borne substances and pathogens from entering the CNS. Nevertheless, pathogens like Neisseria meningitidis and Borrelia bavariensis can breach the BBB and infect the brain parenchyma. The self-assembling BBB-spheroids can simulate the cross talk occurring between the cells of the barrier and neuroinvasive pathogens. BBB spheroids were generated by co-culturing human brain microvascular endothelial cells (hBMECs), pericytes and astrocytes. The BBB attributes of spheroids were confirmed by mapping the localization of cells, observing permeability of angiopep2 and non-permeability of dextran. Fluorescent Neisseria, Borrelia or E. coli (non-neuroinvasive) were incubated with spheroids to observe the adherence, invasion and spheroid integrity. Transcriptome analysis with NGS was employed to investigate the response of BBB cells to infections. hBMECs were localized throughout the spheroids, whereas pericytes and astrocytes were concentrated around the core. Within 1 hr of exposure, Neisseria and Borrelia adhered to spheroids, and their microcolonization increased from 5 to 24 hrs. Integrity of spheroids was compromised by both Neisseria and Borrelia, but not by E. coli infection. Transcriptome analysis revealed a significant change in the expression of 781 genes (467 up and 314 down regulated) in spheroids infected with Neisseria, while Borrelia altered the expression of 621 genes (225 up and 396 down regulated). The differentially expressed genes could be clustered into various biological pathways like cell adhesion, extracellular matrix related, metallothionines, members of TGF beta, WNT signaling, and immune response. Among the differentially expressed genes, 455 (48%) genes were inversely expressed during Neisseria and Borrelia infection. The self-assembling spheroids were used to perceive the BBB response to neuroinvasive pathogens - Neisseria and Borrelia. Compromised integrity of spheroids during Neisseria and Borrelia infection as opposed to its intactness and non-adherence of E. coli (non-neuroinvasive) denotes the pathogen dependent fate of BBB. Genes categorized into various biological functions indicated weakened barrier properties of BBB and heightened innate immune response. Inverse expression of 48% genes commonly identified during Neisseria and Borrelia infection exemplifies unique response of BBB to varying neuropathogens.

Dysregulation of Wingless and Int-1 (Wnt) signaling has been strongly associated with development and progression of osteoarthritis (OA). Here, we set out to investigate the independent effects of either mechanical stress (MS) or inflammation on Wnt signaling in human neocartilage pellets, and to relate this Wnt signaling to OA pathophysiology. OA synovium-conditioned media (OAS-CM) was collected after incubating synovium from human end-stage OA joints for 24 h in medium. Cytokine levels in the OAS-CM were determined with a multiplex immunoassay (Luminex). Human neocartilage pellets were exposed to 20% MS, 2% OAS-CM or 1 ng/mL Interleukin-1β(IL-1β). Effects on expression levels of Wnt signaling members were determined by reverse transcription-quantitative polymerase chain reaction. Additionally, the expression of these members in articular cartilage from human OA joints was analyzed in association with joint space narrowing (JSN) and osteophyte scores. Protein levels of IL-1β, IL-6, IL-8, IL-10, tumor necrosis factor α, and granulocyte-macrophage colony-stimulating factor positively correlated with each other. MS increased noncanonical WNT5A and FOS expression. In contrast, these genes were downregulated upon stimulation with OAS-CM or IL-1β. Furthermore, Wnt inhibitors DKK1 and FRZB decreased in response to OAS-CM or IL-1β exposure. Finally, expression of WNT5A in OA articular cartilage was associated with increased JSN scores, but not osteophyte scores. Our results demonstrate that MS and inflammatory stimuli have opposite effects on canonical and noncanonical Wnt signaling in human neocartilage. Considering the extent to which MS and inflammation contribute to OA in individual patients, we hypothesize that targeting specific Wnt pathways offers a more effective, individualized approach.

Wnt signaling members are involved in the differentiation of cells associated with eyespot and band color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here, we explore the localization and function of Armadillo/β-catenin dependent (canonical) and Armadillo/β-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

Comparative studies on the effect of pig adipose-derived stem cells (pASCs) preconditioned with hypoxia or normoxia on skin wound healing in mice

Simple SummaryInvestigation into effective targets of drug resistance is important for identifying novel strategies in cancer therapy. The study aimed to determine the functional role of paclitaxel (PTX) resistance on MCF-7 cell survival related to PI3K/Akt and MAPK pathways. Therefore, we generated PTX-resistant (PTX-res) MCF-7 cells exposed to increasing concentrations of PTX (5–100 nM) over a period of 6 months. Increased cell survival, proliferation, and colony formations were observed in PTX-res MCF-7 cells, while survival inhibition was determined in non-resistant wt cells. PTX-res MCF-7 cells appeared morphologically different from wt cells with their star-like shape which showed the mesenchymal characteristics of cells. Active PI3K/Akt signaling and increased motility were confirmed by upregulation of the EMT pathway members in PTX-res MCF-7 cells. We suggested that the active Akt signaling was related to the upregulated stress-mediated activation of MAPK signaling members, as shown by the significant p38 and SAPK/JNK activation in our results. To sensitize PTX-res MCF-7 cells we treated wt and PTX-res MCF-7 cells with specific c-Jun N-terminal kinase inhibitor, JNK-IN-8, and significant suppression on p38, SAPK/JNK expression was observed. Wnt signaling was highly affected by JNK inhibition. We concluded that JNK inhibition is a potential target to reverse PTX-resistance related to Wnt signaling.Paclitaxel (PTX) is a widely used chemotherapeutic agent in the treatment of breast cancer, and resistance to PTX is a common failure of breast cancer therapy. Therefore, understanding the effective molecular targets in PTX-resistance gains importance in identifying novel strategies in successful breast cancer therapy approaches. The aim of the study was to investigate the functional role of PTX resistance on MCF-7 cell survival and proliferation related to PI3K/Akt and MAPK pathways. The generated PTX-resistant (PTX-res) MCF-7 cells showed enhanced cell survival, proliferation, and colony formation potential with decreased cell death compared to wt MCF-7 cells. PTX-res MCF-7 cells exhibited increased motility profile with EMT, PI3K/Akt, and MAPK pathway induction. According to the significant SAPK/JNK activation in PTX-res MCF-7 cells, specific c-Jun N-terminal kinase inhibitor, JNK-IN-8 is shown to suppress the migration potential of cells. Treatment of JNK inhibitor suppressed the p38 and SAPK/JNK and Vimentin expression. However, the JNK inhibitor further downregulated Wnt signaling members in PTX-res MCF-7 cells. Therefore, the JNK inhibitor JNK-IN-8 might be used as a potential therapy model to reverse PTX-resistance related to Wnt signaling.

Mdm4 controls ureteric bud branching via regulation of p53 activity

Autophagy negative-regulating Wnt signaling enhanced inflammatory osteoclastogenesis from Pre-OCs in vitro

Wnt family members have recently been distinguished in the adult ovary with potential roles in ovarian function. Though particular growth factors interact with Wnt signaling members in extraovarian cell types, it is unclear whether this interaction is applicable in the granulosa cells. Therefore, the current study aimed to determine the effect of insulin-like growth factor-1 (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-β) on Wnt ligands WNT2 and WNT4 and Wnt receptor Frizzled-4 (FZD4) protein levels in cultured mouse granulosa cells. Granulosa cells were isolated from antral follicles of adult Balb/C mice and cultured for 24 hours in the presence of 100 ng/mL of IGF-I, or EGF or FGF-β. WNT2, WNT4 and FZD4 protein levels were evaluated through western blotting after the culture process. IGF-I treated granulosa cells had significantly the highest level of WNT2 and WNT4 as well as FZD4 when compared to FGF-β and EGF groups. FGF-β group had a significantly higher level of WNT2, WNT4 and FZD4 expression when compared to EGF group. FZD4 expression was at the highest level in the IGF-I group and this difference was statistically significant for all groups including uncultured cells and vehicle group. In addition, FGF-β was shown to positively affect the adhesion of granulosa cells. This study demonstrates that IGF-I, FGF-β and EGF have differential effects on the expressions of WNT2, WNT4, and FZD4 in cultured mouse granulosa cells, suggesting that particular growth factors related to ovarian function might conduct their roles in the ovary through Wnt signaling.

Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C-8C) and sham groups (5S-8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

IntroductionThe importance of seminal vesicle secretion and uterine Wnt signaling for uterus preparation and embryo implantation has been described.Materials and MethodsIn this study, we evaluated the gene expression of Wnt ligands (Wnt4 and Wnt5a) and their corresponding receptors (Fzd2 and Fzd6) using qRT-PCR and active β-catenin protein levels using western blotting in the uterine tissue of female mice mated with intact and seminal vesicle-excised (SVX) males during the pre-implantation window. We evaluated the association between these factors and implantation rates and embryo spacing.ResultsmRNA expression of Wnt4 and Wnt5a and active β-catenin protein levels decreased from Day 1 to Day 4, but reached a peak on the fifth day of pregnancy. Fzd2 also reached its highest level on Day 5. Fzd6 expression showed a decreasing trend towards the day of implantation. Lack of seminal vesicle secretion decreased Wnt4 and Wnt5a expression on Days 1 and 5 and β-catenin levels on Day 5. There were almost no significant differences in expression levels of the Fzd2 and Fzd6 receptors between groups. There were positive and negative correlations, respectively, between implantation rates and embryo spacing and Wnt4, Wnt5a and active β-catenin in the control group, but such correlations were not observed in the SVX-mated mice.ConclusionsSignificant changes occurred in the expression of several Wnt signaling members and there was a significant association between Wnt signaling and embryo implantation. Seminal vesicle secretion affects Wnt signaling in mice and consequently also affects murine embryo implantation.

Spatio-temporal expression patterns of Wnt signaling pathway during the development of temporomandibular condylar cartilage

ObjectiveBoth alarmins S100A8/A9 and canonical Wnt signaling have been found to play active roles in the development of experimental osteoarthritis (OA). However, what activates canonical Wnt signaling remains unknown. This study was undertaken to investigate whether S100A8 induces canonical Wnt signaling and whether S100 proteins exert their effects via activation of Wnt signaling.MethodsExpression of the genes for S100A8/A9 and Wnt signaling pathway members was measured in an experimental OA model. Selected Wnt signaling pathway members were overexpressed, and levels of S100A8/A9 were measured. Activation of canonical Wnt signaling was determined after injection of S100A8 into naive joints and induction of collagenase‐induced OA in S100A9‐deficient mice. Expression of Wnt signaling pathway members was tested in macrophages and fibroblasts after S100A8 stimulation. Canonical Wnt signaling was inhibited in vivo to determine if the effects of S100A8 injections were dependent on Wnt signaling.ResultsThe alarmins S100A8/A9 and members of the Wnt signaling pathway showed coinciding expression in synovial tissue in an experimental OA model. Synovial overexpression of selected Wnt signaling pathway members did not result in increased expression of S100 proteins. In contrast, intraarticular injection of S100A8 increased canonical Wnt signaling, whereas canonical Wnt signaling was decreased after induction of experimental OA in S100A9‐deficient mice. S100A8 stimulation of macrophages, but not fibroblasts, resulted in increased expression of canonical Wnt signaling members. Overexpression of Dkk‐1 to inhibit canonical Wnt signaling decreased the induction of matrix metalloproteinase 3, interleukin‐6, and macrophage inflammatory protein 1α after injection of S100A8.ConclusionOur findings indicate that the alarmin S100A8 induces canonical Wnt signaling in macrophages and murine knee joints. The effects of S100A8 are partially dependent on activation of canonical Wnt signaling.

Wnt family members are best known for their roles in cell fate determination, differentiation, proliferation and apoptosis during embryonic development. Wnt signaling becomes effective during these cellular processes through the proper interaction between its ligands, receptors, effectors and inhibitors. Here we review Wnt signaling in terms of embryonic development to the blastocyst stage implantation with emphasis on endometrial changes that are critical for receptivity in the uterus. The relationship between Wnt signaling and implantation clearly reveals that, Wnt family members are critical for both early embryonic development and changing of the endometrium before implantation. Specific Wnt signaling pathway members are demonstrated to be critical for endometrial events such as decidualization and endometrial gland formation in addition to cyclic changes in the endometrium controlled by reproductive hormones. In conclusion, specific roles of Wnt members and associated factors for both uterine function and embryonic development should be further investigated with respect to the efficiency of human ARTs.

Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.

SAT0567 Wnt and WISP1 Expression in the Synovium Induces Production of Cartilage-Degrading Metalloproteinases by Synovial Cells

Activation of the wingless-type (Wnt) signaling pathway is common in various human cancers including colorectal cancer (CRC). Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt ligands and therefore inhibits the Wnt signaling pathway. In this study, we aimed to analyze the expression of two members of Wnt signaling (WIF-1 and Wnt5a) in Tunisian patients with sporadic CRC. WIF-1 was frequently methylated in tumor tissues (87.95 %) compared to normal mucosa (39.54 %) and correlated with distant metastasis and vascular invasion (P = 0.001 and 0.037, respectively). The unmethylated profile of the WIF-1 promoter conferred a benefit to patients in terms of overall survival (P log rank = 0.024). In addition, in the group of patients with methylated WIF-1 promoter, the overall survival rate was significantly prolonged for those with small tumor size (<5 cm) and absence of distant metastasis (P log rank = 0.007 and 0.036, respectively). Aberrant CpG methylation of the WIF-1 promoter leads to transcriptional silencing of this tumor suppressor gene in tumor tissues (P = 0.001). Furthermore, we showed that the level of Wnt5a mRNA was significantly lower in tumor compared to normal tissues (P = 0.031) and lower still in those showing more aggressive behavior (presence of lymph nodes and advanced TNM stage). Our finding supports that WIF-1 is frequently methylated and that Wnt5a acts as a tumor suppressor gene in CRC. Loss of WIF-1 and Wnt5a functions results in more aggressive behavior of the disease.

To explore the effect of Wnt signaling suppression on proliferation of non small cell lung cancer to gefitinib, and its related mechanisms. PC9 and PC9/AB2 cells of both gefitinib sensitive and resistant were treated with different concentrations of gefitinib, and the proliferation index was measured using CCK8 kit. The members of Wnt signaling pathway were detected by Western blot. Dual luciferase reportor gene assay (TOP Flash) was used to document the transcriptional level of β-catenin. β-catenin siRNA was transfected into PC9/AB2 cells to suppress the Wnt signaling transcription, followed by treatment with different concentrations of gefitinib. Western blot was then used to detect the expression of EGFR and its downstream signaling after inhibit the expression of β-catenin. Treating with different concentrations of gefitinib, the resistance of PC9/AB2 cells to gefitinib was significantly increased (P < 0.05). The members of Wnt signaling expressed at higher level in PC9/AB2 cells than in PC9 cells (t = 24.590, P = 0.000). TOP Flash examination showed that the endogenous transcriptional activity of Wnt signaling was higher in PC9/AB2 cell than that in PC9 cell (t = 4.983, P = 0.008). Compared with the negative control group, apoptotic rate and sensitivity to gefitinib significantly increased in interfered group (P < 0.05). The expression of p-ERK1/2 significantly decreased after Wnt signaling suppression, although other proteins showed no significant alterations. Suppressing the activity of Wnt signaling can partly reverse the celluar resistance to gefitinib in non small cell lung cancer.

OP0122 Synovial WNT and WISP1 Expression Induces Expression of Cartilage-Degrading Metalloproteinases in the Synovium

Noncanonical Wnt Signaling Maintains Hematopoietic Stem Cells in the Niche