Wnt5a Protein Levels Research Articles

Human epicardial adipose tissue-secreted miR-92a-3p regulates myocardial redox state via paracrine signalling: implications for cardiovascular clinical outcomes

Abstract Background Visceral obesity is directly linked to increased cardiovascular risk, including heart failure. Purpose We explored the ability of human epicardial adipose tissue (EAT)-derived microRNAs (miRNAs) to regulate the myocardial redox state and clinical outcomes. Methods The 5 study arms included 466 patients undergoing cardiac surgery to perform: 1) the discovery phase in which we screened for 351 miRNAs expressed and released from human EAT; 2) correlation analyses between EAT microRNAs and myocardial expression of their targets or myocardial superoxide production in paired EAT/atrial biopsies; 3) genome-wide association screening for miR-92a-3p expression in EAT to test causality/directionality; 4) ex vivo experiments to investigate the underlying mechanisms that were also studied in vitro and in vivo; 5) an 8-year follow-up study to test the prognostic value of the discovered miRNA. Superoxide (O2.-) generation was measured by lucigenin chemiluminescence with NADPH 100μM stimulation as indicator of NADPH-oxidases activity. Vas2870 400 μM (a specific pan-NADPH oxidase inhibitor) was used to obtain the Vas2870-inhibitable O2.- signal which constitutes a more specific index of NADPH oxidase activity. Activation of Rac1, a key NADPH-oxidases subunit, was evaluated by a commercially available kit. Differentiated H9c2 cells were used as an in vitro model of cardiomyocytes. Doxycycline-inducible Wnt5a-overexpressing mice were used for in vivo experiments. Results The EAT secretome profiling on study 1 patients led us to identify 3 microRNAs both expressed and released by EAT whose levels in EAT correlated with oxidative stress in human myocardium. Among these miRNAs only miR-92a-3p reduced NADPH-oxidase-derived superoxide (O2.-) in cardiomyocytes (A). A genetic screening identified 7 SNPs that were associated with high miR-92a-3p levels in EAT (EAT-miR-92a-3p SNPs) and were related to lower myocardial superoxide production (B). miR-92a-3p decreased both activation of Rac1 (not shown) and Wnt5a protein levels in vitro (C). Patients with high EAT miR-92a-3p levels had lower WNT5A levels in the myocardium (not shown). Ex vivo, in vivo and in vitro experiments showed opposite effects of Wnt5a on Rac1-mediated NADPH-oxidases activity (D and not shown). Finally, we found an association of miR-92a-3p levels in EAT with lower relative risk of adverse cardiovascular events (E). Conclusions EAT-derived miRNAs exert paracrine effects on the human heart. Indeed miR-92a-3p suppresses the Wnt5a/Rac1/NADPH oxidase axis and improves myocardial redox state. EAT-derived miR-92a-3p is related with improved clinical outcomes and is a rational therapeutic target for the prevention and treatment of obesity-related heart disease.

Wnt5a-Ror2 signaling mediates root resorption

This study aimed to investigate the role of wingless-type MMTV integration site family member 5a (Wnt5a)-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling in root resorption. The messenger RNA (mRNA) expression of Wnt5a, Ror2, and RANKL in periodontal ligament cells (PDLCs) under compression force (CF) with or without Ror2 small interfering RNA (siRNA) were measured by quantitative reverse transcription-polymerase chain reaction, and these proteins released into culture supernatants were measured using enzyme-linked immunosorbent assay. Then these PDLC-conditioned media under CF with or without Ror2 siRNA were used to culture osteoclast precursors to detect osteoclastogenesis effects via tartrate-resistant acid phosphatase staining. In invivo studies, the odontoclast number and the root resorption volume under excessive CF with or without Ror2 siRNA were investigated by tartrate-resistant acid phosphatase immunohistochemical staining and microcomputed tomography. The protein levels for Wnt5a, Ror2, and receptor activator of nuclear factor-kappa B ligand (RANKL) in the periodontal ligament tissues were also detected using immunohistochemical staining. Finally, the odontoclast number, root resorption volume, and the mRNA and protein expressions were compared between immature and mature teeth. The mRNA production and protein release level of Wnt5a, Ror2, and RANKL increased after CF, whereas they were significantly downregulated with Ror2 siRNA. The osteoclast number increased treating with culture medium from PDLC applying CF, but the increase was inhibited after adding Ror2 siRNA. In the animal model, the odontoclast number and root resorption volume significantly increased in the CF group but decreased in the CF with the Ror2 siRNA group. The protein levels of Wnt5a, Ror2, and RANKL in periodontal ligament were upregulated under excessive CF, and the pathway was inhibited with Ror2 siRNA. In the immature tooth group, the odontoclast number, root resorption volume, and the mRNA and protein expressions of Wnt5a-Ror2 signaling were reduced. Wnt5a-Ror2 signaling in PDLCs enhanced by excessive CF could promote RANKL release and induce precursor differentiation, partly leading to increased odontoclast activity and ultimate root resorption. The less resorption of the immature tooth may be due to odontoclastogenesis inhibition by decreased expression of Wnt5a-Ror2 signaling.

Wnt 5a mediated inflammatory injury of renal tubular epithelial cells dependent on calcium signaling pathway in Trichloroethylene sensitized mice

Patients with trichloroethene-induced Trichloroethylene hypersensitivity syndrome (THS) often present kidney injury. However, the role of Wnt 5a/Ca2+ pathway in renal tubular injury in Trichloroethylene (TCE) sensitized mice remains unclear. This study aimed to investigate how Wnt 5a/Ca2+ pathway induced renal tubular epithelial cell injury in TCE sensitized mice. A total of 84 female BALB/c Specific Pathogen Free mice aged 6–8 weeks were used to establish TCE sensitized mouse models. Renal histology and serum levels of α1-MG and β2-MG were used to assess the renal injury. The renal protein levels of Wnt 5a, ROR2, FZD5, PLC, p-CaMKII, IκB α, p-IκB α, NF-κB(p65), TNF α, IL 6 and IL 1β were measured. The levels of serum α1-MG and β2-MG and TNF α, IL 6 and IL 1β levels in the kidney tissue were significantly increased in TCE sensitized positive group. However, Box5 pretreatment inhibited the expression of PLC, p-CaMKII, p65 and attenuated the injury of renal tubular epithelial cells and suppressed the upregulated expression of the above cytokines. In addition, KN93 also reduced nuclear translocation of p65 and renal injury as well as the elevated cytokines by inhibiting CaMKII. These data identify Wnt 5a binding to ROR2 and FZD5, p65 nuclear translocation, and inflammatory cytokine release as a novel mechanism for renal tubular epithelial cells injury by sensitization with TCE. Box5 or KN93 pretreatment can block the expression of inflammatory cytokines and reduce the injury of renal tubular epithelial cells.

A Novel WNT5A-Mimicking Peptide Affects Leukemia Cell Survival in the Bone Marrow Microenvironment

Background: The crosstalk between hematopoietic cells and bone marrow (BM) microenvironment in hematological malignancies is related to disease initiation, maintenance and relapse. BM niche sustains a protective response against currently available treatments that have shown unwanted adverse effects and high levels of toxicity for patients. WNT5a is a glycoprotein secreted by mesenchymal stromal cells (MSC) that activates the WNT non-canonical pathway in hematopoietic cells, modulating important biological processes related to neoplasia development.Aims: To investigate WNT5a mRNA expression, protein levels and methylation pattern in Myelodysplastic Syndrome (MDS) and de novo Acute Myeloid Leukemia (AML) patients and their impact on clinical outcomes, and to analyze effects of Foxy-5 (WNTResearch), a new WNT5a-mimicking compound.Methods: WNT5a mRNA expression was analyzed in mononuclear cells (MC) from 371 AML patients (212 male, median age 61 years [range: 2-87]) (Ohsu, Nature 2018). BMMSC and BM biopsies from 5 healthy donors (HD), 6 MDS and 13 AML patients at diagnosis were submitted to analysis of WNT5a mRNA expression and methylation after Azacytidine (1μM) treatment by real-time PCR, and WNT5a protein levels by immunohistochemistry and immunofluorescence (IF). A panel of myeloid leukemia cell lines (U937, HL60, THP1, KG1a, K562) were treated with Foxy-5 (1 to 100μM) for 72h in monoculture, coculture and 3D-coculture (with MSC) and evaluated for ROS production (DCFDA dye), cell proliferation (Ki-67 stain), autophagy (acridine orange dye), chemotaxis (Transwell), actin polymerization (phalloidin stain), cell cycle (PI/RNAse stain), cell viability (MTT assay), apoptosis (Annexin-V stain) and protein expression (Western blot, WB). MC and CD34+ cells from HD were submitted to cytotoxic assays. Statistical analyzes were performed using ANOVA or Mann-Whitney tests, as appropriate.Results: WNT5a gene expression was reduced in MC from AML patients with adverse cytogenetic risk compared to favorable and intermediate cytogenetic risk (fold-decrease [FD]: 42.9; 18.8, respectively) (P<.05) and in BMMSC from AML compared to HD (FD: 53.3) (P<.05). Accordingly, WNT5a gene expression in MDS and AML BMMSC treated with Azacytidine was restored (fold-increase [FI]: 3.99; 1.50, respectively). WNT5a protein expression were diminished in BMMSC from MDS and AML patients compared to HD onto a 3D-coculture (IF)(P<.05) and immunohistochemically detected in all BM hematopoietic lineages. Foxy-5 reduced ROS production in U937 (FD of mean fluorescence intensity [MFI]: 48.2; 46.6), HL60 (FD: 47.1; 115.0), KG1a (FD: 34.9; 20.7) and K562 (FD: 19.0; 24.3) at 100μM in monoculture and coculture, respectively (P<.05). Foxy-5 also significantly decreased proliferation in U937 (FD: 41.0), HL60 (FD: 18.0), THP1 (FD: 36.0) and K562 (FD: 68.0) at 100μM (P<.05), confirmed by a 3D-coculture containing these cell lines and MSC. Foxy-5 reduced monocyte differentiation and inhibited CD11b expression in U937 (FD: 16.4) and THP1 (FD: 14.4). Cell cycle progression was blocked in sub G0/G1 phase in all cell lines (P<.05) after Foxy-5 treatment, probably mediated by the reduction of cyclin D1 protein levels, as verified by WB. Further, Foxy-5 reduced AKT1/2/3 and ERK1/2 phosphorylation levels, possibly by beta-catenin inhibition, with disruption of actin polymerization (U937 (FD: 65.3), HL60 (FD: 35.9), THP1 (FD: 58.5), K562 (FD: 15.0)) at 100μM (P<.05) and consequent impairment of CXCL12-induced chemotaxis (U937 (FD: 27.9), HL60 (FD: 42.5), THP1 (FD: 82.4), K562 (FD: 45.1)) at 100μM (P<.05). In coculture, cell autophagy was reduced in U937 (FD: 27.8), HL60 (FD: 35.9), KG1a (FD: 16.4) and K562 (FD: 35.8) when treated with Foxy-5 at 100μM (P<.05). Finally, Foxy-5 treatment did not affect cytotoxicity in MC and CD34+ cells from HD.Conclusion: WNT5a downregulation in MDS and AML patients occurs probably by methylation and contributes to poor prognosis. Foxy-5, by restoring WNT5a levels, could represent a strategy to counterbalance several oncogenic processes present in leukemia by reducing ROS production and, consequently, inhibiting cell growth and differentiation, downregulating PI3K and MAPK pathways, disrupting actin polymerization and decreasing autophagy. Thus, Foxy-5 treatment may be an important approach to impair leukemia growth and maintenance and arises as a promising therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Sfrp5/Wnt5a and leptin/adiponectin levels in the serum and the periarterial adipose tissue of patients with peripheral arterial occlusive disease

BackgroundLeptin, adiponectin, secreted frizzled-related protein 5 (Sfrp5) and wingless-type family member 5a (Wnt5a) are novel adipokines that are involved in insulin sensitivity and atherosclerosis. The aim of the present study was to investigate the serum and periarterial adipose tissue leptin/adiponectin and Sfrp5/Wnt5a levels in patients with peripheral arterial occlusive disease (PAOD). MethodsA total of 75 patients with PAOD and 39 control subjects were recruited. The serum concentrations of leptin, adiponectin, Sfrp5 and Wnt5a were measured by ELISAs, and the leptin, adiponectin, Sfrp5 and Wnt5a levels in the periarterial adipose tissue were observed by western blotting. ResultsThe serum Sfrp5 levels were significantly lower in the patients with PAOD than in the control subjects (p < 0.001) and Wnt5a levels were higher in the patients with PAOD (p < 0.001). The serum leptin levels were significantly higher in the patients with PAOD than in the control subjects (p < 0.001), and adiponectin levels were significantly lower in the patients with PAOD (p < 0.001). The serum Sfrp5 levels were associated with ABI (rs = 0.274; p = 0.018), Wnt5a (rs = −0.409; p < 0.001), adiponectin (rs = 0.244; p = 0.035) and Leptin/Adiponetin ratio (rs = −0.244; p = 0.037). The adiponectin and Sfrp5 protein levels were decreased in the periarterial adipose tissue of patients with PAOD compared with control subjects. The leptin and Wnt5a protein levels were increased in the periarterial adipose tissue of patients with PAOD compared with control subjects. ConclusionWe demonstrated that the adiponectin and Sfrp5 levels in the serum and periarterial adipose tissue were significantly lower in the patients with PAOD than in the control subjects. The leptin and Wnt5a levels in the serum and periarterial adipose tissue were significantly higher in the patients with PAOD than in the control subjects.

PRDX1 stimulates non-small-cell lung carcinoma to proliferate via the Wnt/β-Catenin signaling.

Previous studies have shown that PRDX1 is upregulated in some types of malignant tumors. The role of PRDX1 in non-small-cancer lung carcinoma (NSCLC) remains unclear. This study aims to identify the role of PRDX1 in influencing in-vitro biological functions of NSCLC and the molecular mechanism. We collected 50 cases of fresh NSCLC and adjacent non-tumoral tissues for detecting differential expressions of PRDX1 by quantitative real-time polymerase chain reaction (qRT-PCR). Survival time of NSCLC patients, defined as the period from the operation to the latest follow-up or death due to recurrence or metastasis, was recorded for assessing the relationship between PRDX1 and prognosis in NSCLC. Using lentivirus transfection, PRDX1 level was downregulated in NSCLC cells. Subsequently, proliferative and apoptotic abilities, and expression levels of vital genes in the Wnt/β-Catenin signaling were examined. Finally, the significance of activated Wnt/β-Catenin signaling during PRDX1-regulated NSCLC proliferation was explored. Using GEPIA database and NSCLC tissues we collected, PRDX1 was detected to be upregulated in NSCLC samples than controls. PRDX1 level was related to tumor staging and prognosis in NSCLC. Knockdown of PRDX1 attenuated proliferative ability and stimulated apoptosis in NSCLC. Protein levels of Wnt5A was downregulated in H1299 and SPC-A1 cells with PRDX1 knockdown. Overexpression of β-Catenin enhanced proliferative ability and inhibited apoptosis in NSCLC cells with PRDX1 knockdown. PRDX1 is upregulated in NSCLC samples, and linked to tumor staging and prognosis. It stimulates NSCLC to proliferate by activating the Wnt/β-Catenin signaling.

Gastrin-releasing peptide induces fibrotic response in MRC5s and proliferation in A549s

Idiopathic pulmonary fibrosis (IPF) is a complex lung disease, whose build-up scar tissue is induced by several molecules. Gastrin-releasing peptide (GRP) is released from pulmonary neuroendocrine cells, alveolar macrophages, and some nerve endings in the lung. A possible role of GRP in IPF is unclear. We aimed to investigate the fibrotic response to GRP, at the cellular level in MRC5 and A549 cell lines. The proliferative and fibrotic effects of GRP on these cells were evaluated by using BrdU, immunoblotting, immunofluorescence and qRT-PCR for molecules associated with myofibroblast differentiation, TGF-β and Wnt signalling. All doses of GRP increased the amount of BrdU incorporation in A549 cells. In contrast, the amount of BrdU increased in MRC5 cells in the first 24 h, though progressively decreased by 72 h. GRP did not stimulate epithelial-mesenchymal transition in A549 cells, rather, it stimulated the differentiation of MRC5 cells into myofibroblasts. Furthermore, GRP induced gene and protein expressions of p-Smad2/3 and Smad4, and reduced the levels of Smad7 in MRC5 cells. In addition, GRP decreased Wnt5a protein levels and stimulated β-catenin activation by increasing Wnt4, Wnt7a and β-catenin protein levels. GRP caused myofibroblast differentiation by inducing TGF-βand Wnt pathways via paracrine and autocrine signalling in MRC5 cells. In conclusion, GRP may lead to pulmonary fibrosis due to its proliferative and fibrotic effects on lung fibroblasts. The abrogation of GRP-mediated signal activation might be considered as a treatment modality for fibrotic lung diseases.BiWGXPLUnt4V7uQDSjX_9sVideo .Graphical abstract

159 Downregulation of serum exosome miR-1305 contribute to the development of psoriasis through non-canonical Wnt signaling pathway

Background: The extensive involvement of microRNAs (miRNAs) in the pathogenesis of psoriasis is well documented. However, whether the serum exosome derived miRNAs play a role in psoriasis remains unclear. Objectives: To explore the role of serum exosome microRNA-1305 (miR-1305) in psoriasis. Methods: Exosomes were isolated from serum by differential ultracentrifugation. The morphology was identified by transmission electron microscope (TEM). Observation of serum exosomes uptake by keratinocytes though confocal fluorescence microscopy. MiRNA microarray was performed in serum exosomes from 4 patients with psoriasis and 4 controls. qRT-PCR were used to identify the differential expression miRNAs. Using bioinformatic predicted the signaling pathways related to mir-1305. Western blot was used to investigate the protein levels of Wnt5a and its downstream effectors. Normal human epidermal keratinocytes (NHEKs) and HaCat cells were transfected with miR-1305 mimic/Ctrl (50 nM) or miR-1305 inhibitor/Ctrl (100 nM); or siWnt5a/ctrl (60 nM) with Lipofectamine 2000. Results: The diameter of serum exosomes were around 100 nm. Serum exosomes could be uptake by keratinocytes after co-culture for 12h. Using miRNA microarray, we found 16 differentially expressed miRNAs in serum exosomes; 14 (87.5%) were downregulated and 2 (12.5%) were upregulated (fold change>2, P<0.05). Among these differentially expressed miRNAs, miR-1305 was down-regulated in serum exosomes from psoriasis patients (fold change=0.20, P<0.01, n=4). Through the methods of bioinformatics analysis, qRT-PCR and Western blot, we found that miR-1305 was closely related to non-canonical WNT signaling pathway (P<0.01), and miR-1305 regulated the expression of Wnt5a in Normal human epidermal keratinocytes (NHEKs) and HaCat cells. Conclusions: MiR-1305 was downregulated in serum exosomes from psoriasis patients. Downregulation of serum exosomes miR-1305 was involved in the pathogenesis of psoriasis through regulating non-canonical WNT pathways. However, much work remains to be done in the future to test our point of view.

LncRNA MALAT1 Inhibits Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Down-Regulating WNT5A

ncRNA involves in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). WNT5A participates in the growth and development of osteogenic differentiation. This study aims to investigate whether lncRNA MALAT1 regulates BMSCs osteogenesis through WNT5A. qRT-PCR was done to detect the lncRNA MALAT1 level and osteogenic markers in osteoporosis patients and control groups. The above markers and WNT5A protein levels were detected by Western blot. The degree of osteogenic differentiation was detected by ALP activity assay and ALP staining. The differentiation ability of BMSCs after lncRNA MALAT1 overexpression was detected by ARS staining. The binding site of lncRNA MALAT1 to WNT5A was determined by dual luciferase reporter assay. lncRNA MALAT1 expression was higher in patients with osteoporosis, and decreased significantly with increased osteogenic induction. Overexpression of lncRNA MALAT1 in BMSCS reduced WNT5A level, while interference with lncRNA MALAT1 increased WNT5A levels. In cells with overexpression of lncRNA MALAT1, transfection of si-WNT5A can significantly downregulate the RUNX2, OSX, ALP, OCN, OPN, and COL1A1, thereby inhibiting osteogenic differentiation, interfering with the regulation of WNT signaling pathway and regulating BMSCs osteogenic differentiation. lncRNA MALAT1 and WNT5A can regulate BMSCs osteogenesis, thus accelerating the progression of osteoporosis.

Sclerostin and WNT-5a gingival protein levels in chronic periodontitis and health.

Wnt signaling pathways regulate osteoblast differentiation and bone formation and are associated with inflammatory responses driven by innate and adaptive immunity via the NF-κB pathway. The aim of this study was to compare the levels of sclerostin (SOST), WNT-5a, and TNF-α between chronic periodontitis and periodontally healthy sites and determine their value as diagnostic markers of chronic periodontitis. In a cross-sectional assessment 25 chronic periodontitis cases and 25 periodontally healthy controls were selected upon clinical and radiographic periodontal evaluation. Gingival crevicular fluid (GCF) was collected cross-sectionally from diseased and healthy sites in periodontitis patients and from healthy sites in each control subject. In a subgroup analysis, ten patients with generalized moderate and severe chronic periodontitis and ten generalized periodontally healthy individuals were included. The protein levels of SOST, WNT-5a, and TNF-α in GCF were measured by sandwich ELISA. The Shapiro-Wilk test was utilized to assess the normality of the distribution and non-parametric comparisons were performed. The protein levels of SOST were significantly higher in the generalized moderate and severe chronic periodontitis subgroup when compared to the generalized healthy (P=0.002), while the WNT-5a and TNF-α GCF total amounts were similar (P>0.05). Diseased sites in the periodontitis patients exhibited significantly higher total protein levels of WNT-5a than in healthy sites (P=0.017), whereas no differences were detected for SOST and TNF-α (P>0.05). The total protein levels of SOST, WNT-5a, and TNF-α in GCF were similar in periodontitis and non-periodontitis patients (P>0.05). Sclerostin and WNT-5a gingival protein levels demonstrated a high diagnostic value for generalized moderate and severe chronic periodontitis, while a low accuracy was detected for localized chronic periodontitis.

Interleukin-1 beta promotes neuronal differentiation through the Wnt5a/RhoA/JNK pathway in cortical neural precursor cells

Pro-inflammatory cytokine interleukin-1 beta (IL-1β) is a key mediator of inflammation and stress in the central nervous system (CNS), and is highly expressed in the developing brain. In this study, we investigated the possible role of IL-1β in neuronal differentiation of cortical neural precursor cells (NPCs). We showed that stimulation with IL-1β increased expression levels of neurotrophin-3 (NT3) and neurogenin 1 (Ngn1) and promoted neurite outgrowth. We also found that IL-1β increased mRNA and protein levels of Wnt5a. Knockdown of Wnt5a by transfection with Wnt5a siRNA inhibited IL-1β-induced neuronal differentiation. Moreover, IL-1β-induced Wnt5a expression was regulated by nuclear factor kappa B (NF-κB) activation, which is involved in IL-1β-mediated neuronal differentiation. To examine the role of Wnt5a in neuronal differentiation of NPCs, we exogenously added Wnt5a. We found that exogenous Wnt5a promotes neuronal differentiation, and activates the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway. In addition, Wnt5a-induced neuronal differentiation was blocked by RhoA siRNA, as well as by a specific Rho-kinase inhibitor (Y27632) or a SAPK/JNK inhibitor (SP600125). Furthermore, treatment with RhoA siRNA, Y27632, or SP600125 suppressed the IL-1β-induced neuronal differentiation. Therefore, these results suggest that the sequential Wnt5a/RhoA/ROCK/JNK pathway is involved in IL-1β-induced neuronal differentiation of NPCs.

Cross talk between androgen and Wnt signaling potentially contributes to age-related skeletal muscle atrophy in rats.

We sought to determine whether age-related gastrocnemius muscle mass loss was associated with parallel decrements in androgen receptor (AR) or select Wnt signaling markers. To test this hypothesis, serum-free and total testosterone (TEST) and gastrocnemius AR and Wnt signaling markers were analyzed in male Fischer 344 rats that were 3, 6, 12, 18, and 24 mo (mo) old ( n = 9 per group). Free and total TEST was greatest in 6 mo rats, and AR protein and Wnt5 protein levels linearly declined with aging. There were associations between Wnt5 protein levels and relative gastrocnemius mass ( r = 0.395, P = 0.007) as well as AR and Wnt5 protein levels (r = 0.670, P < 0.001). We next tested the hypothesis that Wnt5 affects muscle fiber size by treating C2C12-derived myotubes with lower (75 ng/ml) and higher (150 ng/ml) concentrations of recombinant Wnt5a protein. Both treatments increased myotube size ( P < 0.05) suggesting this ligand may affect muscle fiber size in vivo. We next tested if Wnt5a protein levels were androgen-modulated by examining 10-mo-old male Fischer 344 rats ( n = 10-11 per group) that were orchiectomized and treated with testosterone-enanthate (TEST-E); trenbolone enanthate (TREN), a nonaromatizable synthetic testosterone analogue; or a vehicle (ORX only) for 4 wk. Interestingly, TEST-E and TREN treatments increased Wnt5a protein in the androgen-sensitive levator ani/bulbocavernosus muscle compared with ORX only ( P < 0.05). To summarize, aromatizable and nonaromatizable androgens increase Wnt5a protein expression in skeletal muscle, age-related decrements in muscle AR may contribute Wnt5a protein decrements, and our in vitro data imply this mechanism may contribute to age-related muscle loss. NEW & NOTEWORTHY Results from this study demonstrate androgen and Wnt5 protein expression decrease with aging, and this may be a mechanism involved with age-related muscle loss.

Wnt5a mediates chronic post-thoracotomy pain by regulating non-canonical pathways, nerve regeneration, and inflammation in rats

As well-characterized ligands involved in neurogenesis, Wnts are emerging as promising targets in pain pathogenesis. Our previous pilot study showed that intrathecal inhibition of Wnt5a, but not Wnts, relieves chronic post-thoracotomy pain (CPTP) in rats. In the present study, we aimed to further explore the regulatory mechanism of Wnt5a in CPTP development. Increased protein levels of Wnt5a, transmembrane receptor Ror2, and activated non-canonical Wnt pathway members were found in the thoracic dorsal root ganglions from postoperative day (POD) 7 to POD 21. However, the levels of canonical Wnt pathway members showed no change by reverse transcriptase-PCR. In addition, elevated nerve regeneration, activated pro-inflammatory factors, and glial cells were detected from POD 7 to POD 21. Furthermore, intrathecal Wnt5a blockade during the early phase (POD 0 to POD 9) significantly increased the pain threshold, and intervention in the late phase (POD 14 to POD 16) alleviated pain; however, the analgesic response was not as effective as that in the early phase. Additionally, early but not late Wnt5a blockade significantly reversed CPTP-induced activation of the non-canonical Wnt pathways, nerve regeneration, and inflammation. In contrast, a Wnt5a agonist decreased the pain threshold in both naïve and painless rats. These results suggest that Wnt5a promotes the development of CPTP by activating non-canonical Wnt pathways, nerve regeneration, and inflammation. Therapeutic intervention by targeting Wnt5a may represent an effective strategy for preventing and treating CPTP.

The secreted protein WNT5A regulates condylar chondrocyte proliferation, hypertrophy and migration

ObjectiveOur previous study showed that WNT5A, a member of the noncanonical WNT pathway, is involved in interleukin-1beta induced matrix metalloproteinase expression in temporomandibular joint (TMJ) condylar chondrocytes. The purpose of this study is to further explore the roles of WNT5A in cartilage biology of the TMJ. MethodsAn early TMJ osteoarthritis-like rat model was constructed by a mechanical method (steady mouth-opening). The gene and protein levels of WNT5A during the condylar cartilage changes were measured. Effects of WNT5A on chondrocyte proliferation, hypertrophy and migration were analyzed after WNT5A gain or loss of function in vitro. A c-Jun N-terminal kinase (JNK) inhibitor SP600125 was used to evaluate the involvement of JNK pathway in these effects of WNT5A. The expression and transcription activity of cell cycle regulators c-MYC and Cyclin D1 were examined to determine the mechanism behind WNT5A regulation of chondrocyte proliferation. ResultsWNT5A was significantly upregulated in the condylar cartilage of rats in the early TMJ osteoarthritis-like model. Activating WNT5A facilitated condylar chondrocyte proliferation, hypertrophy and migration. Conversely, inhibiting WNT5A activity in chondrocytes decreased their proliferation, hypertrophy and migration. Blockage of the JNK pathway by its inhibitor, SP600125, impaired these effects of WNT5A on chondrocytes. WNT5A regulated both the expression and transcriptional activity of c-MYC and Cyclin D1 in chondrocytes, both of which were upregulated in condylar cartilage of the rat early TMJ osteoarthritis. ConclusionWNT5A regulates condylar chondrocyte proliferation, hypertrophy and migration. These findings provide new insights into the role of WNT5A signaling in TMJ cartilage biology and its potential in future therapy for TMJ degenerative diseases.

Effects of bone marrow mesenchymal stem cells on the mineralization of calcified vascular smooth muscle cells

Objective To study the effects of bone marrow mesenchymal stem cells(MSC)on the mineralization of calcified vascular smooth muscle cells(CSMC). Methods Osteogenic differentiation of vascular smooth muscle cells(SMC)was induced by osteogenic medium for 14 days.Then von Kossa staining was performed to observe the mineral deposits in SMC.The transwell system was used to establish the indirect co-culture environment.And hence, CSMC was indirectly co-cultured without or with MSC at a ratio of 1∶1 in the down plates or up plates.The cells were divided into 4 groups.Normal group included the normal SMC cultured with the normal medium.Calcification inducers group included the normal SMC cultured with the osteogenic medium.Calcified cells group included CSMC cultured with the normal medium.MSC intervention group included MSC and CSMC cultured with the normal medium.14 days later, SMC from 4 groups was harvested.Alkaline phosphatase(AKP)activity was detected to evaluate the severity of calcification.Moreover, the protein levels of Wnt5a and β-catenin were measured by Western blot analysis. Results Compared with normal group, the calcification inducers group showed that AKP activity, Wnt5a and β-catenin protein levels were notably increased.Additionally, AKP activity and the protein levels of Wnt5a, β-catenin were decreased in calcified cells group as compared with calcification inducers group.Furthermore, AKP activity and the protein levels of Wnt5a, β-catenin were significantly reduced(P<0.01)in MSC intervention group versus calcified cells group Conclusions When MSC is indirectly co-cultured with CSMC, MSC can relieve the vascular calcification, which might be mediated by blocking the Wnt5a/β-catenin signaling through immunomodulatory and paracrine. Key words: Mesenchymal stem cells; Muscle, Smooth, Vasculan; Calcification; The Wnt5a/β-catenin signaling

Wnt5a signaling antagonizes Leishmania donovani infection.

Abstract Leishmania donovani, the causative agent for visceral leishmaniasis creates a niche within host immune cells to sustain itself. In light of the fact that Wnt5a utilizes a Rac1–NFκB circuitry for sustaining immune homeostasis in macrophages and alters actin assembly (Naskar.et.al 2014, Witze et.al 2008) we deciphered whether there is any influence of Wnt5a signaling on L. donovani infection. Upon infection of macrophages with L.donovani, we noted significant decrease in Wnt5a protein levels. Infection with both SAG sensitive and SAG resistant parasite strains yielded similar results. Interestingly, upon treatment of macrophages with recombinant Wnt5a prior to infection separately with SAG senstive and SAG resistant L.donovani, there was significant decrease in the intracellular load of both strains of parasite, as judged by microscopic analysis and expression of Leishmania specific genes (Amastin and G6PDH). Depleting Wnt5a from the macrophages by si-RNA in-vitro or by use of Inhibitor of Wnt Production (IWP2) in-vivo, on the other hand, led to an increase in parasite load. Using specific inhibitors of the Wnt5a signaling intermediates Rac1 and NFκB, we demonstrated that Wnt5a induced reduction in parasite load is due to Wnt5a dependent Rac1 activity but not NFκB signalling. In light of the known involvement of Rac1 in actin organization, the altered actin assembly of Wnt5a pre-treated macrophages, as evident from flow cytometric analysis corroborated the observed Rac1 mediated inhibition of L.donovani infection. Taken together, our results indicate that (i) Wnt5a signaling is detrimental to L. donovani infection and (ii) the parasite evades host immune response at least partly through obliteration of Wnt5a signaling.

High levels of WNT-5A in human glioma correlate with increased presence of tumor-associated microglia/monocytes

Malignant gliomas are among the most severe types of cancer, and the most common primary brain tumors. Treatment options are limited and the prognosis is poor. WNT-5A, a member of the WNT family of lipoglycoproteins, plays a role in oncogenesis and tumor progression in various cancers, whereas the role of WNT-5A in glioma remains obscure. Based on the role of WNT-5A as an oncogene, its potential to regulate microglia cells and the glioma-promoting capacities of microglia cells, we hypothesize that WNT-5A has a role in regulation of immune functions in glioma. We investigated WNT-5A expression by in silico analysis of the cancer genome atlas (TCGA) transcript profiling of human glioblastoma samples and immunohistochemistry experiments of human glioma tissue microarrays (TMA). Our results reveal higher WNT-5A protein levels and mRNA expression in a subgroup of gliomas (WNT-5Ahigh) compared to non-malignant control brain tissue. Furthermore, we show a significant correlation between WNT-5A in the tumor and presence of major histocompatibility complex Class II-positive microglia/monocytes. Our data pinpoint a positive correlation between WNT-5A and a proinflammatory signature in glioma. We identify increased presence of microglia/monocytes as an important aspect in the inflammatory transformation suggesting a novel role for WNT-5A in human glioma.

Abstract 4476: Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer

Abstract Background and Aims Wnt5a protein levels are upregulated in prostate cancer (PCa) compared to benign tissue and patients with high Wnt5a protein levels have a better outcome after radical prostatectomy compared to patients with low Wnt5a levels (Syed Khaja AS, 2011; 2012). Thus, the reconstitution of the Wnt5a signaling pathway could be a promising therapeutic approach in PCa, as already shown in breast cancer (Säfholm A, 2008). Constitutively active STAT3 (pSTAT3) has been correlated to PCa progression and disruption of its signaling pathway could represent a promising strategy for the treatment of patients with advanced PCa (Hellsten R, 2008). We have analyzed the expression of Wnt5a and pSTAT3 in a cohort of patients with metastatic Castration Resistant Prostate Cancer (mCRPC). Using in vitro and in vivo techniques we have explored the effect of the Wnt5a-mimicking peptide FOXY5 for the reconstitution of the Wnt5a pathway in PCa, and we have studied the effect of the STAT3 inhibitor Galiellalactone (GL) on PCa cell viability, apoptosis, invasion, tumor growth and metastases. Methods Wnt5a and pSTAT3 protein expression was analyzed by IHC on a TMA construct with duplicate cores of malignant tissues from 38 patients with mCRPC. Statistical and correlation analyses were performed using the SPSS version 21. Cell viability was determined by MTT assay; apoptotic cells were visualized with M30 CytoDeath antibody 24h after treatment with FOXY5 or GL. Cell invasion was analyzed using matrigel pre-coated cell culture inserts in the presence or absence of FOXY5 or GL. For the animal study, NMRI nude mice were injected orthotopically with 1×106 DU145-Luc cells, and treated IP with GL every day or with FOXY5 every second day. Primary tumor growth and metastatic spread were evaluated weekly by in vivo luminescence using the IVIS Lumina II system. 9 weeks after injection animals were sacrificed and organs were analyzed for the presence of metastases. Results IHC analyses of TMA showed high pSTAT3 and low Wnt5a expression in CRPC patients, with a small but significant correlation (r = 0.4646; p = 0.0038). Our results show that GL inhibits viability and induces apoptosis of DU145-Luc cells in vitro, and that it reduces tumor growth and metastatic spread to lymph nodes in vivo. FOXY5 treatment has no effects on cell viability or on primary tumor growth, but it significantly reduces cell invasion in vitro and metastatic spread to lymph nodes in vivo. Conclusions Our results confirm that the reconstitution of the Wnt5a pathway and the inhibition of the STAT3 pathway are a promising therapeutic approach for the treatment of mCRPC. We found that GL and FOXY5 are good candidates for the treatment of mCRPC, as they inhibit tumor growth and metastatic spread to lymph nodes. Since these two compounds act through different mechanisms, our future studies will explore the possibility of a combination of GL and FOXY5 for the treatment of PCa. Citation Format: Giacomo Canesin, Susan Evans-Axelsson, Rebecka Hellsten, Nicholas Don-Doncow, Tommy Andersson, Anders Bjartell. Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2015-4476

EGF-reduced Wnt5a transcription induces epithelial-mesenchymal transition via Arf6-ERK signaling in gastric cancer cells.

Wnt5a, a ligand for activating the non-canonical Wnt signaling pathway, is commonly associated with Epithelial-to-mesenchymal transition (EMT) in cancer cell metastasis. Here, we show that downregulation of Wnt5a mRNA and protein by EGF is necessary for EGF-induced EMT in gastric cancer SGC-7901 cells. To further explore the mechanisms, we investigated the effect of EGF signaling on Wnt5a expression. EGF increased Arf6 and ERK activity, while blockade of Arf6 activation repressed ERK activity, up-regulated Wnt5a expression and repressed EMT in response to EGF. We also demonstrate that EGF inactivated Wnt5a transcription by direct recruitment of ERK to the Wnt5a promoter. On the other hand, inhibition of ERK phosphorylation resulted in decreased movement of ERK from the cytoplasm to the nucleus, following rescued Wnt5a mRNA and protein expression and favored an epithelial phenotype of SGC-7901 cells. In addition, we notice that kinase-dead, nuclear-localised ERK has inhibitory effect on Wnt5a transcription. Analysis of gastric cancer specimens revealed an inverse correlation between P-ERK and Wnt5a protein levels and an association between Wnt5a expression and better prognosis. These findings indicate that Wnt5a is a potential suppressor of EMT and identify a novel Arf6/ERK signaling pathway for EGF-regulated Wnt5a expression at transcriptional level of gastric cancer cells.

RETRACTED: IL-1β-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway

We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3 mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells.