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

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

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

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

Once pulp necrosis or apical periodontitis occurs on immature teeth, the weak root and open root apex are challenging to clinicians. Berberine (BBR) is a potential medicine for bone disorders, therefore, we proposed to apply BBR in root canals to enhance root repair in immature teeth. An in vivo model of immature teeth with apical periodontitis was established in rats, and root canals were filled with BBR, calcium hydroxide or sterilized saline for 3 weeks. The shape of the roots was analyzed by micro-computed tomography and histological staining. In vitro, BBR was introduced into stem cells from apical papilla (SCAPs). Osteogenic differentiation of stem cells from apical papilla was investigated by alkaline phosphatase activity, mineralization ability, and gene expression of osteogenic makers. The signaling pathway, which regulated the osteogenesis of SCAPs was evaluated by quantitative real time PCR, Western blot analysis, and immunofluorescence. In rats treated with BBR, more tissue was formed, with longer roots, thicker root walls, and smaller apex diameters. In addition, we found that BBR promoted SCAPs osteogenesis in a time-dependent and concentration-dependent manner. BBR induced the expression of β-catenin and enhanced β-catenin entering into the nucleus, to up-regulate more runt-related nuclear factor 2 downstream. BBR enhanced root repair in immature teeth with apical periodontitis by activating the canonical Wnt/β-catenin pathway in SCAPs.
Animals ; Berberine ; pharmacology ; Cell Differentiation ; drug effects ; Dental Papilla ; Male ; Osteogenesis ; drug effects ; Periapical Periodontitis ; therapy ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; Wnt3A Protein ; genetics ; metabolism ; X-Ray Microtomography

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

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 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

Recent studies have shown that Er-Zhi-Wan (EZW), a traditional Chinese medicine consisting of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL), had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan (EZW) on the microarchitecture and the regulation of Wnt/β-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups: sham operation group (sham, n=10), ovariectomy (OVX) group (n=10), and OVX with EZW treatment group (EZW group, n=10). From one week after ovariectomy, EZW (100 mg/mL) or vehicle (distilled water) was fed (1 mL/100 g) once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchitecture, serum levels of E2, bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b (TRAP5b), as well as mandibular mRNA expression of Wnt/β-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), β-catenin and dickkopf homolog 1 (DKK1). The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Furthermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and β-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/β-catenin signaling pathway.
Acid Phosphatase ; blood ; Alkaline Phosphatase ; blood ; Alveolar Process ; drug effects ; metabolism ; Animals ; Body Weight ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; blood ; Female ; Gene Expression ; drug effects ; Intercellular Signaling Peptides and Proteins ; genetics ; Isoenzymes ; blood ; Low Density Lipoprotein Receptor-Related Protein-5 ; genetics ; Mandible ; drug effects ; metabolism ; Medicine, Chinese Traditional ; methods ; Organ Size ; drug effects ; Ovariectomy ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tartrate-Resistant Acid Phosphatase ; Time Factors ; Up-Regulation ; drug effects ; Uterus ; drug effects ; growth & development ; Wnt Signaling Pathway ; drug effects ; genetics ; Wnt3A Protein ; genetics ; beta Catenin ; genetics