Lymphoid Enhancer Factor Research Articles

VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Activation of activin/Smad2 and 3 signaling pathway and the potential involvement of endothelial‑mesenchymal transition in the valvular damage due to rheumatic heart disease

Rheumatic heart disease (RHD) is an autoimmune disease caused by rheumatic fever following group A hemolytic streptococcal infection and primarily affects the mitral valve. RHD is currently a major global health problem. However, the exact pathological mechanisms associated with RHD-induced cardiac valve damage remain to be elucidated. The endothelial-mesenchymal transition (EndMT) serves a key role in a number of diseases with an important role in cardiac fibrosis and the activin/Smad2 and 3 signaling pathway is involved in regulating the EndMT. Nevertheless, there are no studies to date, to the best of the authors' knowledge, investigating the association between RHD and EndMT. Thus, the aim of the current study was to investigate the potential role of EndMT in cardiac valve damage and assess whether activin/Smad2 and 3 signaling was activated during RHD-induced valvular injury in a rat model of RHD induced by inactivated Group A streptococci and complete Freund's adjuvant. Inflammation and fibrosis were assessed by hematoxylin and eosin and Sirius red staining. Serum cytokine and rheumatoid factor levels were measured using ELISA kits. Expression levels of activin/Smad2 and 3 signaling pathway-related factors [activin A, Smad2, Smad3, phosphorylated (p-)Smad2 and p-Smad3], EndMT-related factors [lymphoid enhancer factor-1 (LEF-1), Snail1, TWIST, zinc finger E-box-binding homeobox (ZEB)1, ZEB2, α smooth muscle actin (α-SMA) and type I collagen α 1 (COL1A1)], apoptosis-related markers (BAX and cleaved caspase-3) and valvular inflammation markers (NF-κB and p-NF-κB) were detected using reverse transcription-quantitative PCR and western blot analyses. Compared with the control group, the degree of valvular inflammation and fibrosis, serum levels of IL-6, IL-17, TNF-α and expression of apoptosis-related markers (BAX and cleaved caspase-3) and valvular inflammation marker (p-NF-κB), activin/Smad2 and 3 signaling pathway-related factors (activin A, p-Smad2 and p-Smad3), EndMT-related factors (LEF-1, Snail1, TWIST, ZEB 1, ZEB2, α-SMA and COL1A1) were significantly increased in the RHD group. These results suggested that the activin/Smad2 and 3 signaling pathway was activated during the development of valvular damage caused by RHD and that the EndMT is involved in RHD-induced cardiac valve damage.

S1440 A Case Series of Five Solid Pseudopapillary Neoplasm of Pancreas: Comparison of Four Putative Immunohistochemical Stains With Four Conventional Immunohistochemical Stains

INTRODUCTION: Solid pseudopapillary neoplasm (SPN), a rare exocrine tumor, accounts for 0.2-2.7% of primary pancreatic tumors occurs mostly in young females. The histologic origin is debatable and thus, it is important to identify the most sensitive and specific biomarkers. Through this case series, we are addressing the comparative analysis of four commonly used immunostains with 4 new putative immunohistochemical markers. CASE DESCRIPTION/METHODS: We performed an electronic search of pathology records for a 25 years-period (1994-2019) for SPN. Formalin-fixed, paraffin-embedded tissue blocks were retrieved and used for staining for four commonly used (CD-10, Beta- Catenin, Progesterone receptor, Alpha-1 antitrypsin) and four new (LEF-1, TFE-3, SOX-11 and AMACR) immunohistochemical markers.: In our case-series, female-to-male ratio is 4:1, with a mean age of 37.6 years. The tumor size ranged from 2.3 to 14 cm. Only one case showed aggressive features of necrosis, perineural invasion and lymphovascular invasion. None of the cases showed lymph node or distant metastasis. The results from the immunohistochemical staining are noted in Table 1. DISCUSSION: In our case series, lymphoid enhancer factor-1 (LEF-1) emerged out as the most sensitive new putative marker with 100% diffuse nuclear positivity among the other new markers. To the best of our knowledge, these novel results are noted for the first time in our case series. This significant finding is in congruence with the emerging molecular diagnostic finding that the long isoform of LEF-1 expresses b-catenin binding domain and functions as a transcriptional enhancer factor. It also emphasizes the involvement of Wnt/β-catenin signaling pathway in the histologic origin of this tumor. Further studies are needed to evaluate its specificity especially against SPN mimickers such as Acinar-cell carcinoma and pancreatic neuroendocrine tumors.Figure 1.: Figure 1: A. Solid pseudopapillary neoplasm H&E section at 200x. B. SPN TFE-3 stain at 200x C. SPN Alpha 1 antitrypsin 200x D. SPN AMACR stain at 400x E. SPN Beta Catenin at 400x F. SPN CD10 at 400x G. SPN LEF-1 stain at 400x H. SPN SOX-11 stain at 400x.Table 1.: Staining pattern and comparison of four conventional stains with 4 putative stains in 5 cases of Solid Pseudopapillary neoplasm of pancreas.

Protective effect of Matricaria chamomilla extract against 1,2-dimethylhydrazine-induced colorectal cancer in mice.

Background Colorectal cancer (CRC) is a major public health problem, with almost 1.8 million newly diagnosed cases and about 881,000 deaths annually. Chamomile (Matricaria chamomilla) is a well-documented medicinal herb that possesses anti-inflammatory and anti-carcinogenic properties. This study aimed to unravel the effect of aqueous chamomile extract against 1,2-dimethylhydrazine(DMH)-induced CRC in mice. Methods Male Balb/c mice received a weekly intraperitoneal injection of DMH (20 mg/kg body weight) for 12 weeks. Chamomile extract (150 mg/kg body weight/5 days/week p.o.) was administered at the initiation and post-initiation stages of carcinogenesis. Polyps count, histopathological analysis, real-time polymerase chain reaction (RT-PCR) analysis of Wnt signaling genes, ELISA of cyclooxygenase-2 (COX-2), and enzyme assay for inducible nitric oxide synthase (iNOS) were performed. Results Chamomile extract modulated the Wnt pathway in colonic tissues, where it significantly downregulated Wnt5a, β-catenin, T cell factor (Tcf4), lymphoid enhancer factor 1 (Lef1), c-Myc and Cyclin D1 expression levels, while it upregulated adenomatous polyposis coli (APC) and glycogen synthase kinase (GSK3β) expression levels. This extract significantly reduced COX-2 levels and iNOS activities. Polyps count and histopathological analysis provided supportive evidence for the biochemical and molecular analyses. Conclusions Chamomile can act as a potent dietary chemopreventive agent against DMH-induced CRC.

Screen and Function Study of the Key Regulative Odontogenic Genes in Mice

To screen the key odontogenic genes in mice and verify the odontogenic inducing effect on amniotic epithelial cells (WISH). The spatially and temporally different expression of bone morphogenetic proteins 4 (BMP4), fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH), lymphoid enhancer factor 1 (LEF1) proteins and their genes expression in the early odontogenesis stage (embryo day 10.5 (E10.5)、E11.5、E14.5) in fetal mice were detected by immunohistochemistry staining and quantitative real-time PCR (RT-qPCR). According to the results, we screened the probable key odontogenic genes. Then adding osteogenic inducing solution to induce non-odontogenic epithelium cells, WISH. After 3 weeks culture of non-odontogenic epithelial WISH for osteogenic induction, the epithelial-mesenchymal transformation cap ability was evaluated by using Alizarin (ALZ) red staining and RT-qPCR on the alkaline phosphatase ( ALP) mRNA expression level. Using germ layer recombination experiment to observe and verify whether the screened genes can induce non-odontogenic epithelium cells acquire odontogenesis ability. The recombined tissue grafts containing key genes were transplanted beneath the renal capsule of mice. The results of immunohistochemistry staining and RT-qPCR showed that on E10.5 BMP4 protein and gene were differently expressed in the first and second branchial arch epithelium, which synchronized the odontogenic capability transferring from epithelium to mesenchyme from E10.5-E14.5. Though the expression of FGF8 protein and gene existed such difference in the first and second branchial arch epithelium, there was no synchronization in transfer. The expression of LEF1 and SHH proteins and genes had neither difference nor synchronization. So far, we considered the BMP4 was the probable key odontogenic gene. Through 3 weeks' osteogenic induction, ALZ red stained positively and calcium nodules were observed in WISH, and the expression level of ALP mRNA increased. In the germ layer recombination experiment, exogenous BMP4 protein enabled the second branchial arch mesenchyme forming tooth-like structures after recombined with the second branchial arch epithelium or WISH. The proteins and genes of BMP4, FGF8, SHH and LEF1 are spatially and temporally differently expressed in the early tooth development stage in mice. The protein and gene of BMP4 are differently expressed between the first and second branchial arch epithelium and enables the non-odontogenic epithelium acquiring odontogenic ability. BMP4 is the possible key odontogenic gene.

MircoRNA-143-3p regulating ARL6 is involved in the cadmium-induced inhibition of osteogenic differentiation in human bone marrow mesenchymal stem cells

Cadmium, which is extensively distributed in the environment, accumulates in organisms through the trophic chain. Although cadmium can cause bone injury, its role in osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) remains unclear. The present study investigated the effect of cadmium chloride (CdCl2) on osteogenesis of hBMSCs and the underlying mechanism. CdCl2 dose-dependently reduced the viability of hBMSCs. Concentrations of CdCl2 (2.5 and 5.0 μM) increased miR-143-3p levels; decreased levels of adenosine diphosphate-ribosylation factor-like protein 6 (ARL6); inhibited Wnt family member 3A (Wnt3a), β-catenin, lymphoid enhancer factor (LEF1), and T-cell factor 1 (TCF1); and suppressed osteogenesis of hBMSCs. Inhibition of miR-143-3p or overexpression of ARL6 with lentivirus blocked these CdCl2-induced changes. Luciferase reporter assays confirmed that miR-143-3p binds to the 3’-UTR regions of ARL6 mRNA. Reduced-expression of miR-143-3p enhanced the CdCl2-induced suppression of the osteogenesis of hBMSCs and inhibition of the Wnt/β-catenin pathway, effects that were reversed by down-regulated expression of ARL6. Thus, miR-143-3p targets ARL6 to down-regulate the Wnt/β-catenin pathway, which is involved in the suppression of osteogenic differentiation of hBMSCs. The results provide new directions for clinical treatment of bone diseases resulting from cadmium toxicity.

Hymenialdisine: A Marine Natural Product That Acts on Both Osteoblasts and Osteoclasts and Prevents Estrogen-Dependent Bone Loss in Mice.

Excessive osteoclast (OC) activity together with relatively weak osteoblast (OB) function are strongly connected to osteolytic diseases, including osteoporosis, tumor-induced osteolysis, and inflammatory bone erosion. Very few natural products or compounds have been shown to exert therapeutic effects on both OCs and OBs, limiting the potential development of natural compounds for clinical application. Hymenialdisine (HMD) is a marine sponge-derived natural inhibitor of protein kinases with previously reported anti-osteoarthritis and anti-cancer properties. However, the roles of HMD in OCs, OBs, and osteoporosis have not yet been well established. Here, we found that HMD not only suppressed osteoclastogenesis but also promoted OB differentiation. HMD exerted dose-dependent inhibitory effects on RANKL-induced OC formation, bone resorption, and OC-specific gene expression. These strong inhibitory effects were achieved by blocking the NF-κB and MAPK signaling pathways, and NFATc1 expression. In addition, HMD potentially stimulated OB differentiation by activating alkaline phosphatase (ALP) and enhancing OB matrix mineralization. We found that HMD can activate the glycogen synthase kinase 3β (GSK-3β)/β-catenin/T-cell factor (TCF)/lymphoid enhancer factor (LEF) signaling pathway to upregulate Runx-2 expression, the main transcription factor in this pathway. Increased expression of Runx-2 was also correlated with expression of the OB-specific genes Col1a1 and osteocalcin (Ocn). Furthermore, we also evaluated the therapeutic potential of HMD in a female C57BL/6j mouse model of ovariectomy (OVX)-induced systematic bone loss. HMD showed a remarkable ability to prevent decreases in bone volume (BV/TV) and trabecular thickness (Tb.Th). In summary, HMD exerts notable effects in inhibiting OC-related osteolysis and enhancing OB-induced ossification, suggesting the potential application of HMD in osteoporosis treatment. © 2020 American Society for Bone and Mineral Research.

Lef‐1 is Required for Proper Cell Cycle Progression in Airway Basal Cells

The surface airway epithelium (SAE) is a pseudostratified layer of cells found within the mammalian airway, and serves as a barrier between the environment and the rest of the body. Its function is crucial for proper respiration and innate immunity. At homeostasis the SAE has very low cellular turnover, yet has the capacity to regenerate itself after injury or infection at an astonishingly fast rate. This is due primarily to the population of resident stem cells found within the SAE called basal cells. Though much work has been done to identify the molecular mechanisms that govern basal cell proliferation, migration, and differentiation, many questions still remain.The transcription factor Lymphoid Enhancer Binding Factor 1 (Lef‐1) is a Wnt effector protein expressed in stem cell populations in various epithelial tissues, including the developing lung. Our lab has shown that overexpression of Lef‐1 in primary airway stem cell cultures leads to increased proliferation and migration. Additionally, Lef‐1 is required for the development of airway submucosal glands (SMGs), epithelial structures that are continuous with the SAE. However, it is not known if Lef‐1 plays a role in basal cell function. Therefore, we sought to determine the effects of Lef‐1 deletion in basal cells. Though its expression in basal cells is almost undetectable on a population‐wide scale, deletion of Lef‐1 from primary mouse basal cells severely decreases their ability to proliferate in culture, and skews them more toward a secretory club cell fate. Lef‐1 deletion leads to a loss in DNA damage response (DDR) gene expression, including Tp73, Rad51, and Chek1. Lef‐1 knockout basal cells tend to stall in the cell cycle at the G1/S transition. Together these studies indicate an important role for Lef‐1 in basal cell replication and proliferation.Support or Funding InformationUniversity of Iowa Molecular Medicine ProgramUniversity of Iowa Department of Anatomy & Cell Biology Funding: 2R01 DK047967

Effect of electroacupuncture on neurological function and Wnt signaling pathway in ischemic brain tissue of cerebral infarction rats

To explore the mechanism of electroacupuncture (EA) underlying improvement of cerebral infarction (CI) by investigating its influence on expression of cerebral Wnt7a, lymphoid enhancer factor-1 (LEF1), glycogen synthase kinase 3β(GSK-3β) and Dickkopf-1(DKK1) mRNA and proteins in CI rats. A total of 280 male Wistar rats were randomly divided into blank control (n＝10), sham-operation, model and EA groups，and 90 rats of the last 3 groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups with 10 rats in each subgroup. The CI model was established by occlusion of the middle cerebral artery (MCAO). The sham-operation group received the same surgical operation but without thread embolus insertion. EA (2 Hz/15 Hz, 2 mA) was applied to "Shuigou" (GV26) for 20 min, once a day for 1, 3, 7 and 12 d, respectively. The neurological deficit was evaluated by using Neurological Severity Scores (NSS). The expression levels of Wnt7a，LEF1, GSK-3β and DKK1 mRNAs and proteins in the right ischemic brain tissues were detected by Quantative real-time PCR and Western blot, respectively. After MCAO, the NSS score was significantly increased in the model and EA groups relevant to the blank control and sham-operation groups (P<0.01) and gradually decreased with the prolongation of ischemia time. After EA, the NSS scores were notably decreased on day 3, 7 and 12 in the EA group compared with the model group (P<0.05, P<0.01). After modeling, the expression levels of Wnt7a and LEF1 mRNAs from 3 h to 12 d, Wnt7a and LEF1 proteins from 6 h to 12 d were considerably increased (P<0.01, P<0.05), while those of GSK-3β mRNA at 9, 12 and 24 h, GSK-3β protein at 24 h and 3 d, and DKK1 mRNA at 24 h and 3 d and DKK1 protein at 3 d were obviously decreased in the model group relevant to the sham-operation group (P<0.05, P<0.01). After the intervention, the expression levels of Wnt7a mRNA at 12 h to 3 d, Wnt7α protein from 24 h to 12 d, LEF1 mRNA from 24 h to 12 d, and LEF1 protein from 3 d to 12 d were further apparently up-regulated (P<0.01, P<0.05), while those of GSK-3β mRNA at 9 h, 3，7 and 12 d, and GSK-3β protein at 12 h, 7 d and 12 d, and DKK1 mRNA at 12 h, 24 h and 3 d, and DKK1 protein at 24 h to 12 d were obviously down-regulated in the EA group relevant to the model group (P<0.01, P<0.05). No significant difference was found between the blank and sham-operation groups in the NSS scores and expression levels of Wnt7a, LEF1, GSK-3β and DKK1 mRNAs and proteins at all the time points (P>0.05). EA of GV26 can significantly improve the neurological deficit symptoms in MCAO rats, which may be associated with its effects in up-regulating the expression of Wnt7a and LEF1 mRNAs and proteins, and in down-regulating the expression of GSK-3β and DKK1 mRNAs and proteins.

PCDHGA9 represses epithelial-mesenchymal transition and metastatic potential in gastric cancer cells by reducing \u03b2-catenin transcriptional activity

Gastric cancer (GC) has a high mortality rate, and metastasis is the main reason for treatment failure. It is important to study the mechanism of tumour invasion and metastasis based on the regulation of key genes. In a previous study comparing the expression differences between GES-1 and SGC-7901 cells, PCDHGA9 was selected for further research. In vitro and in vivo experiments showed that PCDHGA9 inhibited invasion and metastasis. A cluster analysis suggested that PCDHGA9 inhibited epithelial-mesenchymal transition (EMT) through the Wnt/β-catenin and TGF-β pathways. Laser confocal techniques and western blotting revealed that PCDHGA9 inhibited the nuclear translocation of β-catenin, regulated T cell factor (TCF)/ /lymphoid enhancer factor (LEF) transcriptional activity, directly impacted the signal transmission of the TGF-β/Smad2/3 pathway, strengthened the adhesion complex, weakened the effects of TGF-β, and blocked the activation of the Wnt pathway. In addition, PCDHGA9 expression was regulated by methylation, which was closely related to poor clinical prognosis. The aim of this study was to elucidate the molecular mechanism by which PCDHGA9 inhibits EMT and metastasis in GC to provide a new theoretical basis for identifying GC metastasis and a new target for improving the outcome of metastatic GC.

Prognostic Impact of Lymphoid Enhancer Factor 1 Expression and Serum Galectin.3 in Egyptian AML Patients

Background Deregulation of the Wnt signaling pathway had a role in haematological malignancies. Previous studies reported that lymphoid enhancer factor 1 (LEF1) expression and serum Galectin-3 level could affect clinical parameters and outcome in acute myeloid leukemia patients, but as far as we know, no study has addressed their combined effect on AML patients. Aim We studied the expression of LEF1 by real-time qPCR and measured serum level of Gal.3 by ELISA technique in peripheral blood of 69 AML patients and correlated it with different clinicopathological criteria of patients, response, PFS and OS. Results We found high expression (LEF1high) was associated with better OS (p = 0.02) and EFS (p = 0.019) compared to LEF1low, low serum Gal.3 level had better OS (p = 0.014) and EFS (p = 0.02) compared to high serum Gal.3 level. LEF1high less likely to carry a FLT3-ITD (p = 0.047) compared to LEF1low patient, also LEF1high characterized by favorable risk (p = 0.02) than LEF1low patients. While patients with higher Gal-3 levels characterized by poor risk (p = 0.02) than lower Gal.3 lels, also more likely to carry a FLT3-ITD with borderline significance (p = 0.054). Combined LEF1high/Gal.3 low patients had lower baseline blast percentages (p = 0.02), favorable risk (p = 0.01), less likely to carry FLT3-ITD (p = 0.02), higher CR rate (p = 0.055), shorter time to CR (0.001) than other groups. Among high Gal.3 level group, LEF1highexpression improved OS and EFS (20 and 15 months respectively) vs LEF1low expression (13 and 8 months respectively). Conclusion We conclude that high LEF1 expression was a favorable prognostic marker which can define AML patient risk and outcome independent from assessing the serum galectin.3 level.

The osteogenic differentiation of human adipose-derived stem cells is regulated through the let-7i-3p/LEF1/\u03b2-catenin axis under cyclic strain

BackgroundThe Wnt/β-catenin pathway is involved in the osteogenic differentiation of human adipose-derived stem cells (hASCs) under cyclic strain. Very little is known about the role of microRNAs in these events.MethodsCells were obtained using enzyme digestion methods, and proliferation was detected using Cell Counting Kit 8. Cell cycles and immunophenotypes were detected by flow cytometry. The multilineage potential of hASCs was induced by induction media. Cyclic strain was applied to hASCs (0.5 Hz, 2 h/day, 6 days) to induce osteogenic differentiation and miRNA changes. Bioinformatic and dual-luciferase analyses confirmed lymphoid enhancer factor 1 (LEF1) as a potential target of let-7i-3p. The effect of let-7i-3p on LEF1 in hASCs transfected with a let-7i-3p mimic and inhibitor was analyzed by immunofluorescence. hASCs were transfected with a let-7i-3p mimic, inhibitor, or small interfering RNA (siRNA) against LEF1 and β-catenin. Quantitative real-time PCR (qPCR) and western blotting were performed to examine the osteogenic markers and Wnt/β-catenin pathway at the mRNA and protein levels, respectively. Immunofluorescence and western blotting were performed to confirm the activation of the Wnt/β-catenin pathway.ResultsFlow cytometry showed that 82.12% ± 5.83% of the cells were in G1 phase and 17.88% ± 2.59% of the cells were in S/G2 phase; hASCs were positive for CD29, CD90, and CD105. hASCs could have the potential for osteogenic, chondrogenic, and adipogenic differentiation. MicroRNA screening via microarray showed that let-7i-3p expression was decreased under cyclic strain. Bioinformatic and dual-luciferase analyses confirmed that LEF1 in the Wnt/β-catenin pathway was the target of let-7i-3p. Under cyclic strain, the osteogenic differentiation of hASCs was promoted by overexpression of LEF1and β-catenin and inhibited by overexpression of let-7i-3p. hASCs were transfected with let-7i-3p mimics and inhibitor. Gain- or loss-of-function analyses of let-7i-3p showed that the osteogenic differentiation of hASCs was promoted by decreased let-7i-3p expression and inhibited by increased let-7i-3p expression. Furthermore, high LEF1 expression inactivated the Wnt/β-catenin pathway in let-7i-3p-enhanced hASCs. In contrast, let-7i-3p inhibition activated the Wnt/β-catenin pathway.ConclusionsLet-7i-3p, acting as a negative regulator of the Wnt/β-catenin pathway by targeting LEF1, inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro.

CircMYO10 promotes osteosarcoma progression by regulating miR-370-3p/RUVBL1 axis to enhance the transcriptional activity of \u03b2-catenin/LEF1 complex via effects on chromatin remodeling

BackgroundCircMYO10 is a circular RNA generated by back-splicing of gene MYO10 and is upregulated in osteosarcoma cell lines, but its functional role in osteosarcoma is still unknown. This study aimed to clarify the mechanism of circMYO10 in osteosarcoma.MethodsCircMYO10 expression in 10 paired osteosarcoma and chondroma tissues was assessed by quantitative reverse transcription polymerase chain reaction (PCR). The function of circMYO10/miR-370-3p/RUVBL1 axis was assessed regarding two key characteristics: proliferation and endothelial–mesenchymal transition (EMT). Bioinformatics analysis, western blotting, real-time PCR, fluorescence in situ hybridization, immunoprecipitation, RNA pull-down assays, luciferase reporter assays, chromatin immunoprecipitation, and rescue experiments were used to evaluate the mechanism. Stably transfected MG63 cells were injected via tail vein or subcutaneously into nude mice to assess the role of circMYO10 in vivo.ResultsCircMYO10 was significantly upregulated, while miR-370-3p was downregulated, in osteosarcoma cell lines and human osteosarcoma samples. Silencing circMYO10 inhibited cell proliferation and EMT in vivo and in vitro. Mechanistic investigations revealed that miR-370-3p targets RUVBL1 directly, and inhibits the interaction between RUVBL1 and β-catenin/LEF1 complex while circMYO10 showed a contrary effect via the inhibition of miR-370-3p. RUVBL1 was found to be complexed with chromatin remodeling and histone-modifying factor TIP60, and lymphoid enhancer factor-1 (LEF1) to promote histone H4K16 acetylation (H4K16Ac) in the vicinity of the promoter region of gene C-myc. Chromatin immunoprecipitation methods showed that miR-370-3p sponge promotes H4K16Ac in the indicated region, which is partially abrogated by RUVBL1 small hairpin RNA (shRNA) while circMYO10 showed a contrary result via the inhibition of miR-370-3p. Either miR-370-3p sponge or ShRUVBL1 attenuated circMYO10-induced phenotypes in osteosarcoma cell lines. MiR-370-3p inhibition abrogated the inhibition of proliferation, EMT of osteosarcoma cells in vitro and in vivo seen upon circMYO10 suppression via Wnt/β-catenin signaling.ConclusionsCircMYO10 promotes osteosarcoma progression by regulating miR-370-3p/RUVBL1 axis to promote chromatin remodeling and thus enhances the transcriptional activity of β-catenin/LEF1 complex, which indicates that circMYO10 may be a potential therapeutic target for osteosarcoma treatment.

Guhong Injection promotes fracture healing by activating Wnt/beta-catenin signaling pathway in vivo and in vitro

Guhong Injection (GHI), composed of aceglutamide and safflower aqueous extract, has been applied to the clinical treatment of orthopedic diseases, but the relevant mechanism by which GHI exerts effects on bone remodeling has not been reported. In the present study, we investigated the effects of various concentrations of GHI (2.5, 5 and 10 ml/kg) in accelerating rat tibia healing progress by observing haematoxylin and eosin (HE) stained sections, detecting the activity of bone metabolism biochemical markers such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), osteocalcin (OC) and C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-1) in rat serum, as well as measuring the expressions of collagen I (COL-1) and collagen II (COL-2) in rat tibia. Also, we investigated the effects of different concentrations of GHI (30, 60 and 90 μl/ml) on the proliferation and differentiation of osteoblasts (OBs) through proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and type I collagen (COL-1). At the same time, the expression of important factors of Wnt/beta-catenin signaling pathway including Wnt-3a, beta-catenin, disheveled-1 (Dvl-1), glycogen synthase kinases-3beta (GSK-3beta), lymphoid enhancing factor-1 (LEF-1) and axis inhibition protein-2 (Axin-2) after GHI intervention was detected by quantitative real-time PCR (q-PCR), immunohistochemistry and Western blotting. In vivo, rats of tibia fracture model treated with intraperitoneal injection (ip) of GHI had more mature fibroblasts, as well as shorter period formation of new bone. The levels of BMP-2, TGF-beta and OC in rat serum were significantly up-regulated, while the level of CTX was down-regulated. After 4 weeks of drug treatment, the level of COL-1 in the rat tibia increased, but there was no significant change in the level of COL-2. In vitro, after drug intervention, the number of OBs increased significantly, the activities of PCNA, ALP and COL-1 were enhanced. Treatment with GHI increased the mRNA and protein expression of Wnt-3a, beta-catenin, Dvl-1 and LEF-1, and decreased the expression of mRNA of Axin-2 and GSK-3beta. All results demonstrate that GHI accelerates the proliferation of OBs and shortens the recovery time of bone structure, and the Wnt/beta-catenin signaling pathway is involved in the regulation process.

Abstract 134: Dopamine D 2 Receptor Modulates Wnt Expression and Control of Cell Proliferation

Injury to the kidney triggers a cell proliferation response that can be adaptive, leading to repair of the tubular epithelium, or maladaptive, leading to fibrosis and chronic kidney disease. The Wnt pathway is activated in response to kidney injury and contributes to the fibrotic response. Although transient activation of the pathway is needed for repair of injured tissue and increase in cell proliferation, the sustained activation may promote fibrosis. The mechanisms regulating the expression of a key Wnt ligand, Wnt3a, or the modulation of its activity are unknown. We tested the hypothesis that dopamine D 2 receptor (D2R) signaling modulates β-catenin and T-cell factor (TCF)/lymphoid enhancer factor (LEF)-mediated transcriptional activity by effects on Wnt3a expression. In mice and human renal proximal tubule cells (RPTCs) D2R downregulation (siRNA, 20 nM, 72h) or antagonism (sulpiride, 1 μM, 6h), decreased β-catenin phosphorylation and increased TCF/LEF reporter activity (50-70%, P&lt;0.05) and Wnt3a expression. Conversely D2R agonism (quinpirole, 1 μM, 24h) increased β-catenin phosphorylation (20%, P&lt;0.05), and decreased TCF/LEF reporter activity (35%; P&lt;0.05) and Wnt3a expression (45%, P&lt;0.05). Human RPTCs carrying D2R variants (SNPs) resulting in decreased D2R expression have increased expressions of Wnt3a (50%, p&lt;0.05) and Ki-67, a cell proliferation marker, that are decreased by transient overexpression of the D2R. Furthermore, selective renal D2R silencing or renal ischemia/reperfusion in mice increases renal expression of Wnt3a and Ki-67, which are both are reversed by selective D2R overexpression in the kidney. Wnt3a and Ki-67 colocalize in the nucleus of proximal tubules cells of mice subjected to ischemia/reperfusion indicating the involvement of the Wnt/β-catenin pathway in injury-induced cell proliferation. Conversely, Wnt3a downregulation in RPTCs carrying D2R variants not only decreased Ki-67 but also decreased cell apoptosis (Tunel reaction). Our results suggest that D2R is a transcriptional modulator of canonical Wnt signal transduction with broad implications for health and development of new therapeutics.

LncRNA-H19 inhibits apoptosis of acute myeloid leukemia cells via targeting miR-29a-3p.

To explore the influences of long non-coding ribonucleic acid (lncRNA)-H19 on the proliferation and apoptosis of acute myeloid leukemia (AML) cells via the Wnt signaling pathway. Blood samples were collected from 40 AML patients. The AML cells were cultured. Cell counting kit-8 (CCK-8) was used to detect cell proliferation and flow cytometry was applied to analyze cell cycle and determine the apoptosis rate. Moreover, the action target of lncRNA-H19 was detected through a dual-luciferase reporter assay and Western blotting was performed to detect the change in protein level. The expression of lncRNA-H19 in AML patients was markedly higher than that in normal controls and compared with human embryonic kidney (HEK)-293T cells, AML cell Kasumi-1 exhibited an increased lncRNA-H19 expression. LncRNA-H19 could promote cell proliferation, but suppress cell apoptosis. It is bound to micro RNA (miR)-29a-3p in a targeted manner. and the expression level of miR-29a-3p in AML patients was prominently lower than that in normal controls. After miR-29a-3p was inhibited, the expression of intranuclear β-catenin was significantly increased and the Wnt/β-catenin pathway critical molecules T-cell factor (TCF) and lymphoid enhancer factor 1 (LEF1) were evidently up-regulated after the down-regulation of miR-29a-3p. LncRNA-H19 targets miR-29a-3p to promote the proliferation of AML cells, but inhibit the apoptosis through the Wnt/ β-catenin signaling pathway.

Opposing actions of renal tubular- and myeloid-derived porcupine in obstruction-induced kidney fibrosis

Wnt/β-catenin signaling is essential in the pathogenesis of renal fibrosis. We previously reported inhibition of the Wnt O-acyl transferase porcupine, required for Wnt secretion, dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Here, we investigated the tissue-specific contributions of porcupine to renal fibrosis and inflammation in ureteral obstruction using mice with porcupine deletion restricted to the kidney tubular epithelium or infiltrating myeloid cells. Obstruction of the ureter induced the renal mRNA expression of porcupine and downstream targets, β-catenin, T-cell factor, and lymphoid enhancer factor in wild type mice. Renal tubular specific deficiency of porcupine reduced the expression of collagen I and other fibrosis markers in the obstructed kidney. Moreover, kidneys from obstructed mice with tubule-specific porcupine deficiency had reduced macrophage accumulation with attenuated expression of myeloid cytokine and chemokine mRNA. In co-culture with activated macrophages, renal tubular cells from tubular-specific porcupine knockout mice had blunted induction of fibrosis mediators compared with wild type renal tubular cells. In contrast, macrophages from macrophage-specific porcupine deficient mice in co-culture with wild type renal tubular cells had markedly enhanced expression of pro-fibrotic cytokines compared to wild type macrophages. Consequently, porcupine deletion specifically within macrophages augmented renal scar formation following ureteral obstruction. Thus, our experiments suggest a benefit of interrupting Wnt secretion specifically within the kidney epithelium while preserving Wnt O-acylation in infiltrating myeloid cells during renal fibrogenesis.

Wnt/β-catenin signaling in brain development and mental disorders: keeping TCF7L2 in mind.

Canonical Wnt signaling, which is transduced by β‐catenin and lymphoid enhancer factor 1/T cell‐specific transcription factors (LEF1/TCFs), regulates many aspects of metazoan development and tissue renewal. Although much evidence has associated canonical Wnt/β‐catenin signaling with mood disorders, the mechanistic links are still unknown. Many components of the canonical Wnt pathway are involved in cellular processes that are unrelated to classical canonical Wnt signaling, thus further blurring the picture. The present review critically evaluates the involvement of classical Wnt/β‐catenin signaling in developmental processes that putatively underlie the pathology of mental illnesses. Particular attention is given to the roles of LEF1/TCFs, which have been discussed surprisingly rarely in this context. Highlighting recent discoveries, we propose that alterations in the activity of LEF1/TCFs, and particularly of transcription factor 7‐like 2 (TCF7L2), result in defects previously associated with neuropsychiatric disorders, including imbalances in neurogenesis and oligodendrogenesis, the functional disruption of thalamocortical circuitry and dysfunction of the habenula.

Opposing roles of TCF7/LEF1 and TCF7L2 in cyclin D2 and Bmp4 expression and cardiomyocyte cell cycle control during late heart development

Bone morphogenetic protein (BMP) and Wnt pathways regulate cell proliferation and differentiation, but how these two pathways interact and mediate their nuclear actions in the heart, especially during late cardiac development, remains poorly defined. T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors, including Lef1, Tcf7, Tcf7l1, and Tcf7l2, are important nuclear mediators of canonical Wnt/β-catenin signaling throughout cardiac development. We reveal that these TCF/LEF family members direct heart maturation through distinct temporal and spatial control. TCF7 and LEF1 decrease while TCF7L1 and TCF7L2 remain relatively stable during heart development. LEF1 is mainly expressed in mesenchymal cells in valvular regions. TCF7 and TCF7L1 are detected in the nucleus of mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. Tcf7l2 is the primary TCF/LEF family member in cardiomyocytes and undergoes alternative splicing during heart development. A TCF7L2 intensity gradient opposite to that of β-catenin and cardiomyocyte proliferative activity is present in fetal hearts. Wnt activation by cardiac deletion of APC, a negative Wnt regulator, dramatically increases Cyclin D2 and Bmp4 expression. BMP signal transducing transcription factors, the mothers against decapentaplegic homologs (SMADs) are increasingly phosphorylated upon Wnt activation. LEF1/TCF7 displaces TCF7L2 and cooperates with pSMAD 1/5/8 in the regulatory elements of Cyclin D2 and Bmp4 promoters to promote β-catenin recruitment and transcriptional activation. Finally, we demonstrate that TCF7L2 is a transcriptional suppressor of Cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments.

Apoptotic protease activating factor-1 negatively regulates Wnt signaling in hepatocellular carcinoma.

The current study aims to evaluate the mechanism of apoptotic protease activating factor-1 (Apaf-1) in hepatocellular carcinoma (HCC) cells by verifying the regulation of the wnt/beta-catenin signaling pathway via Apal-1. Our data showed that transfection with Ad-Apaf-1 could inhibit the activity of a lymphoid enhancer factor (LEF) luciferase plasmid activated by β-catenin. Overexpressing Apaf-1 could suppress the β-catenin-induced LEF luciferase activity in a dose-dependent manner. Western blot assays demonstrated that the overexpression of Apaf1 significantly suppressed the expression of Wnt/β-catenin signaling-related proteins. Further study demonstrated that Apaf-1 suppressed HepG2 cell migration, invasion, and viability. Knocking down the expression of Apaf-1 activated the wnt/β-catenin pathway in HepG2 cells. In contrast, silencing β-catenin decreased the activation of wnt/β-catenin, even in the presence of si-Apaf-1. Cell cycle distribution analysis demonstrated a decrease in the number of cells in the G0/G1 phase in the Apaf-1 silencing group. In contrast, knocking down the expression of β-catenin increased the number of cells in the G0/G1 phase, even in the presence of si-Apaf-1. In summary, the Apaf-1-mediated suppression of HepG2 cell malignancy is achieved by inhibiting the wnt/β-catenin pathway.