OBJECTIVETo observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells (HSCs) and their the expression of the transforming growth factor beta (TGFb) and /Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.

METHODSSynchronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a (50, 100, 200, 250 and 300 ng/mL). Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a, and the Effects on the protein expression of TGFb/Smad signaling factors was assessed by western blot detection (gray-value analysis) of alpha-smooth muscle actin (a-SMA), a-SMA, TGFb1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling.

RESULTSIn general, HSC The proliferation of hepatic stellate cells increased after the addition of in response to Wnt3a stimulation for 24 h, reaching its peak at the maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration (63.00+/-2.30%), and it increased dramatically compared with those in which was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mLl group (P less than 0.05), but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P more than 0.05).; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of a-SMA, TGFb1, and Smad3 increased with the addition of Wnt3a and the extension of time . For all three, The maximal amount of increased protein expression all reached to the was maximal produced by stimulation when hepatic stellate cells were treated by with 300 ng/mLl Wnt3a for 48 h hours,and the rations of(normalized gray- values:s of a-SMA, 1.0860+/-0.0101; TGFb1, 1.0346+/-0.0118; Smad3, to GAPDH were 1.0860+/-0.0101, 1.0346+/-0.0118, 1.0306+/-0.0122)respectively. However in contrast, the Wnt3a stimulation led to concentration- and time-dependent decreases in Smad7 expression varied inversely, with to them with the minimal ration of it to GAPDH the maximal decrease occurring with 300 ng/mL Wnt3a for 48 h (0.7736+/-0.0139) after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, (P less than 0.05).There were positive correlations between a-SMA expression and was found to be positively correlated to TGFb1, Smad3 (r=0.968, P less than 0.05) and; Smad3 (r=0.997, P less than 0.01), but a-SMA and Smad7 had negatively correlated to Smad7 ion(r=0.960, P less than 0.05).

CONCLUSIONWnt3a can increase the stimulates proliferation as well as and activation of rat the hepatic stellate cells HSCs , and upregulate modifies the expression of TGFb/Smad signaling factors, of the hepatic stellate cells, and which may promote the hepatic fibrosis.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; metabolism ; Wnt3A Protein ; pharmacology

Wnt3a, one of Wnt family members, plays key roles in regulating pleiotropic cellular functions, including self-renewal, proliferation, differentiation, and motility. Accumulating evidence has suggested that Wnt3a promotes or suppresses tumor progression via the canonical Wnt signaling pathway depending on cancer type. In addition, the roles of Wnt3a signaling can be inhibited by multiple proteins or chemicals. Herein, we summarize the latest findings on Wnt3a as an important therapeutic target in cancer.
Cell Division ; physiology ; Gene Expression Regulation, Neoplastic ; physiology ; Humans ; Neoplasm Proteins ; metabolism ; physiology ; Neoplasms ; genetics ; metabolism ; pathology ; Tumor Cells, Cultured ; Wnt Signaling Pathway ; physiology ; Wnt3A Protein ; metabolism ; physiology

To explore the effects and molecular mechanisms of triptolide（TP）on improving podocyte epithelial-mesenchymal transition（EMT）induced by high dose of D-glucose（HG）, the immortalized podocytes of mice were divided into the normal group（N）, the high dose of D-glucose group（HG）, the low dose of TP group（L-TP）, the high dose of TP group（H-TP）and the mannitol group（MNT）, and treated by the different measures respectively. More specifically, the podocytes in each group were separately treated by D-glucose（DG, 5 mmol·L⁻¹）or HG（25 mmol·L⁻¹）or HG（25 mmol·L⁻¹）+ TP（3 μg·L⁻¹）or HG（25 mmol·L⁻¹）+ TP（10 μg·L⁻¹）or DG（5 mmol·L⁻¹）+ MNT（24.5 mmol·L⁻¹）. After the intervention for 24, 48 and 72 hours, firstly, the activation of podocyte proliferation was investigated. Secondly, the protein expression levels of the epithelial markers in podocytes such as nephrin and podocin, the mesenchymal markers such as desmin and collagen Ⅰ and the EMT-related mediators such as snail were detected respectively. Finally, the protein expression levels of Wnt3α and β-catenin as the key signaling molecules in Wnt3α/β-catenin pathway were examined severally. The results indicated that, HG could cause the low protein expression levels of nephrin and podocin and the high protein expression levels of desmin, collagen Ⅰ and snail in podocytes, and inducing podocyte EMT. On the other hand, HG could cause the high protein expression levels of Wnt3α and β-catenin in podocytes, and activating Wnt3α/β-catenin signaling pathway. In addition, L-TP had no effect on the activation of podocyte proliferation, the co-treatment of L-TP and HG could significantly recover the protein expression levels of nephrin and podocin, inhibit the protein expression levels of desmin, collagen I and snail in podocytes, thus, further improving podocyte EMT. And that, the co-treatment of L-TP and HG could obviously decrease the high protein expression levels of Wnt3α and β-catenin induced by HG in podocytes, and inhibit Wnt3α/β-catenin signaling pathway activation. On the whole, HG can induce podocyte EMT by activating Wnt3α/β-catenin signaling pathway; L-TP can ameliorate podocyte EMT through inhibiting Wnt3α/β-catenin signaling pathway activation, which may be one of the effects and molecular mechanisms .
Animals ; Cells, Cultured ; Diterpenes ; pharmacology ; Epithelial-Mesenchymal Transition ; Epoxy Compounds ; pharmacology ; Glucose ; Mice ; Phenanthrenes ; pharmacology ; Podocytes ; drug effects ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; beta Catenin ; metabolism

The aim of this paper was to investigate the effects of curcumin on the proliferation,migration,invasion and apoptosis of human gastric cancer cells and to explore the potential mechanisms. SGC7901,MKN45 and NCI N87 cells lines were cultured under different concentrations of curcumin( 2. 5,5,10,20,40,80 and 160 μmol·L~(-1)) at different time points( 12,24,48 and 72 h),and the effect of curcumin on cell proliferation was detected by CCK-8 assay. The migration and invasiveness of cells were determined by wound healing and Transwell assays,the apoptosis rate was assessed by flow cytometry,the expression of N-cadherin,E-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6,Bcl-2 and Bax were detected by Western blot,and the enzymatic activity of caspase-3,caspase-8 and caspase-9 was evaluated via caspase kit. RESULTS:: indicated that the proliferation of MKN45 cells was significantly inhibited by curcumin in a dose-and time-dependent manner( IC50= 21. 93 μmol·L~(-1)). Moreover,curcumin could inhibit the migration and invasion of MKN45 cells,downregulate the expression of N-cadherin,snail1,Wnt3 a,p-β-catenin,p-LRP6 and Bcl-2,and upregulate the expression of E-cadherin and Bax,it could increase the activity of caspase-3,caspase-8,caspase-9 and induce apoptosis as well. The potential mechanism is through inhibiting the Wnt3 a/β-catenin/EMT pathway,regulating Bcl-2 signaling and caspase pathway,which might provide new potential strategies for gastric cancer treatment.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Curcumin ; pharmacology ; Humans ; Stomach Neoplasms ; drug therapy ; pathology ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; beta Catenin ; metabolism

As a member of the hox gene family, hoxB4 gene encodes a class of DNA-dependent homeobox domain nucleoprotein, which is a specific transcription factor, playing an important role in regulating the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Therefore, it is important to understand the mechanisms involved in regulating expression of hoxB4 in the HSC. Previous studies have suggested that some hoxB4 upstream regulatory factors, such as USF-1 (upstream activating factor -1), USF-2 (upstream activating factor -2) and NF-Y complex, as well as hematopoietic cytokines, such as platelet growth factor (TPO) and Wnt3a protein, play important regulatory roles in the expression of hoxB4 in hematopoietic stem cells. In this review the structure and biological characteristics of hoxB4, mechanisms involved in regulating expression of hoxB4 in the HSC are summarized.
CCAAT-Binding Factor ; metabolism ; Gene Expression Regulation ; Genes, Homeobox ; genetics ; physiology ; Hematopoietic Stem Cells ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; physiology ; Humans ; Transcription Factors ; genetics ; metabolism ; physiology ; Upstream Stimulatory Factors ; metabolism ; Wnt Proteins ; metabolism ; Wnt3 Protein ; Wnt3A Protein

OBJECTIVETo study the role of abnormally activated Wnt/β-catenin signal pathway in the pathogenesis of scleroderma (SD) and its association with the clinical classification of SD.

METHODSThe expression and distribution of Wnt 2, Wnt 3a, and β-catenin in the skin lesions of 45 SD patients, including 25 with systemic sclerosis (SSc) and 20 with localized scleroderma (LSc), were detected with SP immunohistochemistry, using 20 samples from healthy skin tissues as normal control.

RESULTSIn the dermis and epidermis of the SD skin lesions, Wnt 2 and Wnt 3a were located in the cytoplasm and cell nuclei, respectively; β-catenin was distributed in the nuclei of dermal fibroblast-like cells, glandular epithelium cells and infiltrating lymphocytes, and on the cell membrane in normal and a part of the affected epidermis. The skin lesions of SD patients showed obviously increased staining intensity of cytoplasmic Wnt 2, nuclear Wnt 3a and β-catenin, but markedly lowered cell membrane staining of β-catenin than normal skins (P<0.01). Both Wnt 2 and Wnt 3a were positively correlated with nuclear β-catenin deposition (r=0.663 and 0.654, P<0.01) and negatively with cell membrane β-catenin staining (r=-0.532 and -0.529, P<0.01). No significant difference was found in the staining intensities of the 3 proteins between SSc and LSc (P>0.05).

CONCLUSIONAbnormal activation of Wnt/β-catenin pathway occurs in the skin lesions of SD patients, which may play an important role in the pathogenesis of SD. SSc and LSc represent the two opposite ends of the SD spectrum rather than two separate diseases.

Adolescent ; Adult ; Case-Control Studies ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Scleroderma, Systemic ; metabolism ; pathology ; Signal Transduction ; Skin ; pathology ; Wnt Signaling Pathway ; Wnt2 Protein ; metabolism ; Wnt3A Protein ; metabolism ; Young Adult ; beta Catenin ; metabolism

OBJECTIVETo investigate regulation function of anodonta glucan HBP-A on chondrocytes through Wnt pathway in vitro.

METHODSRat chondrocytes were cultured and differentiated induced with IL-1beta (10 ng/ml) in vitro. Chondrocytes were divided into five groups:IL-13 group,IL-1beta + IWP-2 (5 microM,Wnt pathway inhibitor) group, IL-1beta + HBP-A (0.3 mg/ml) group and IL-1beta + IWP-2 + HBP-A group. Wnt-3a, beta-catenin (24 h,48 h,72 h) and MMP-13(72 h) genes expression were detected by Rt-PCR, while beta-catenin, MMP-13, Sox-9 and coll-II (48 h) protein expression were measured by Western-blot.

RESULTSAfter induction of IL-1beta, gene expression of Wnt-3a, beta-catenin and MMP-13 were increased,so were the protein expression of beta-catenin and MMP-13. In contrast,protein expression of Sox-9 and Coll-II were declined. Following addition of HBP-A, Wnt-3a, beta-catenin and MMP-13 were shown as induction of IL-1beta, but protein expression of Sox-9 and Coll-II were upgraded. Combining HBP-A with IWP-2 led to the lowest level in Wnt-3a, beta-catenin gene and beta-catenin protein expression and highest expression of Sox-9 protein.

CONCLUSIONHBP-A could not only delay the differentiation of chondrocytes through downgrading the signal expression of Wnt/beta-catenin,but also adjust the expression of Wnt-3a, beta-catenin and Sox-9 when combinated with the Wnt inhibitor.

Animals ; Anodonta ; chemistry ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrocytes ; cytology ; drug effects ; metabolism ; Glucans ; pharmacology ; Interleukin-1beta ; metabolism ; Rats ; Wnt Signaling Pathway ; drug effects ; Wnt3A Protein ; genetics ; metabolism ; beta Catenin ; metabolism

BACKGROUNDBone morphogenetic protein 9 (BMP9) and Wnt/β-catenin signaling pathways are able to induce osteogenic differentiation of mesenchymal stem cells (MSCs), but the role of Wnt/β-catenin signaling pathway in BMP9-induced osteogenic differentiation is not well understood. Thus, our experiment was undertaken to investigate the interaction between BMP9 and Wnt/β-catenin pathway in inducing osteogenic differentiation of MSCs.

METHODSC3H10T1/2 cells were infected with recombinant adenovirus expressing BMP9, Wnt3a, and BMP9+Wnt3a. ALP, the early osteogenic marker, was detected by quantitative and staining assay. Later osteogenic marker, mineral calcium deposition, was determined by Alizarin Red S staining. The expression of osteopotin (OPN), osteocalcin (OC), and Runx2 was analyzed by Real time PCR and Western blotting. In vivo animal experiment was carried out to further confirm the role of Wnt3a in ectopic bone formation induced by BMP9.

RESULTSThe results showed that Wnt3a enhanced the ALP activity induced by BMP9 and increased the expressions of OC and OPN, with increase of mineral calcium deposition in vitro and ectopic bone formation in vivo. Furthermore, we also found that Wnt3a increased the level of Runx2, an important nuclear transcription factor of BMP9.

CONCLUSIONCanonical Wnt/β-catenin signal pathway may play an important role in BMP9-induced osteogenic differentiation of MSCs, and Runx2 may be a linkage between the two signal pathways.

Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Growth Differentiation Factor 2 ; genetics ; metabolism ; Humans ; Osteocalcin ; genetics ; metabolism ; Osteogenesis ; genetics ; physiology ; Wnt3A Protein ; genetics ; metabolism

This study was aimed to investigate the protective effect of Wit3a gene modification on mouse bone marrow mesenchymal stem cells against the injury induced by Ara-C. The gene-modified MSC steadily expressing Wnt3a were established by adenovirus system. The acute direct damage effects of different concentrations of Ara-C on the unmodified MSC and the gene-modified MSC were assessed by using an in vitro culture system, and the corresponding controls were set. The proliferation and apoptosis of MSC exposed to Ara-C were detected by cell count kit-8 (CCK-8) and flow cytometry. The expression of BCL-2 protein related with cell apoptosis was assayed by Western blot. The results indicated that as compared with unmodified MSC, Ara-C exhibited a less inhibitory effect on the proliferation of gene-modified MSC. There was obvious difference between unmodified MSC and gene-modified MSC (p < 0.05). The proliferation of gene-modified MSC began to recover at 72 hours after removal of Ara-C. However, unmodified MSC showed sustained suppression of proliferation after withdrawal of Ara-C. In apoptosis, the apoptosis rate of gene-modified MSC induced by Ara-C was significantly lower than those of unmodified MSC (p < 0.05). In addition, the expression levels of BCL-2 protein in gene-modified MSC were up-regulated compared with unmodified MSC (p < 0.05). It is concluded that Wnt3a gene modification can significantly mitigate the damage of mouse bone marrow MSC induced by Ara-C.
Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Cytarabine ; adverse effects ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Mice ; Organisms, Genetically Modified ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Wnt3A Protein ; genetics

OBJECTIVETo observe the effect of Wnt3a-transduced mouse bone marrow mesenchymal stem cells (MSC) on the proliferation of T lymphocytes.

METHODSMSC were isolated from C57BL/6 mouse bone marrow and expanded in vitro, then identified by flow cytometry and their differentiation capacity into osteocytes and adipocytes were determined. Recombinant plasmids containing Wnt3a gene, were transfected with lipofectamine into HEK293 cells by the AdEasy system. Viral particles were collected to infect MSC and adenovirus vector expressing GFP (Ad-GFP) was used as control. The expression of GFP in MSC was observed using fluorescence microscopy and the protein levels of Wnt3a and β-catenin were determined by Western blot. Wnt3a-transduced and Ad-GFP transduced MSC were separately cocultured with spleen lymphocytes stimulated by ConA, at the ratio of 1:100, 1:50 or 1:10 respectively. The proliferation rate of T lymphocytes was estimated by Cell Cout Kit-8 (CCK-8) and the level of cytokine by ELISA.

RESULTSFCM analysis showed that the MSC were highly positive for CD90.2, CD44 and negative for CD34, CD45, they could differentiate into osteoblasts and adipocytes after induction; The titer of recombinant adenoviruses was up to 1 × 10(10) pfu/ml. After infected with the adenoviruses, MSC had the strongest GFP expression at 72 h and the efficiency of infection was 50%-60%. The expressions of Wnt3a and β-catenin protein in the Wnt3a-transduced MSC were significantly increased. MSC could suppress the proliferation of T lymphocytes in a dose-dependent manner. When MSC cocultured with spleen lymphocytes at 1:10 ratio, T lymphocyte proliferation rate and the level of IFN-γ were (55.41 ± 1.75)% and (326.70 ± 14.41) pg/ml respectively in Ad-GFP transduced MSC group, while in Wnt3a-transduced MSC group, they were (37.27 ± 2.66)% and (218.80 ± 12.93) pg/ml respectively. There was no effect on the production of IL-2.

CONCLUSIONCompared to Ad-GFP transduced MSC, Wnt3a-transduced MSC exhibit a more potent inhibitory effect on the proliferation of T lymphocytes.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cell Proliferation ; Female ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; cytology ; Transduction, Genetic ; methods ; Wnt3A Protein ; genetics ; metabolism