The Low-density Lipoprotein Receptor-related Protein 6 Pathway in the Treatment of Intestinal Barrier Dysfunction Induced by Hypoxia and Intestinal Microbiota through the Wnt/ß-catenin Pathway.

Our study is to explore the key molecular of Low-density lipoprotein receptor-related protein 6 (LRP6) and the related Wnt/ß-catenin pathway regulated by LRP6 during the intestinal barrier dysfunction. Colorectal protein profile analysis showed that LRP6 expression was decreased in dextran sulfate sodium (DSS)-induced colitis mice, and mice received fecal bacteria transplantation from stroke patients. Mice with intestinal hypoxia and intestinal epithelial cells cultured in hypoxia showed decreased expression of LRP6. Overexpression of LPR6 or its N-terminus rescued the Wnt/ß-catenin signaling pathway which was inhibited by hypoxia and endoplasmic reticulum stress. In mice overexpressing of LRP6, the expression of ß-catenin and DKK1 increased, Bcl2 decreased, and Bax increased. Mice with LRP6 knockout showed an opposite trend, and the expression of Claudin2, Occludin and ZO-1 decreased. Two drugs, curcumin and auranofin could alleviate intestinal barrier damage in DSS-induced colitis mice by targeting LRP-6. Therefore, gut microbiota dysbiosis and hypoxia can inhibit the LRP6 and Wnt/ß-catenin pathway, and drugs targeting LRP6 can protect the intestinal barrier.

The effect of folic acid deficiency on Mest/Peg1 in neural tube defects.

OBJECTIVE: Neural tube defects (NTDs) are one of the most common and serious birth defects in human beings caused by genetic and environmental factors. Folate insufficiency is involved in the occurrence of NTDs and folic acid supplementation can prevent NTDs occurrence, however, the underlying mechanism remains poorly understood. METHODS: We established cell and animal models of folic acid deficiency to detect the methylation modification and expression levels of genes by MassARRAY and real-time PCR, respectively. Results and conclusion: In the present study, we found firstly that in human folic acid-insufficient NTDs, the methylation level of imprinted gene Mest/Peg1 was decreased. By using a folic acid-deficient cell model, we demonstrated that Mest/Peg1 methylation was descended. Meanwhile, the mRNA level of Mest/Peg1 was up-regulated via hypomethylation modification under low folic acid conditions. Consistent with the results in cell models, Mest/Peg1 expression was elevated through hypomethylation regulation in folate-deficient animal models. Furthermore, the up-regulation of Mest/Peg1 inhibited the expression of Lrp6 gene, a crucial component of Wnt pathway. Similar results with Lrp6 down-regulation of fetal brain were verified in animal models under folic acid-deficient condition. Taken together, our findings indicated folic acid increased the expression of Mest/Peg1 via hypomethylation modification, and then inhibited Lrp6 expression, which may ultimately impact on the development of nervous system through the inactivation of Wnt pathway.

Curcumin suppresses osteogenesis by inducing miR-126a-3p and subsequently suppressing the WNT/LRP6 pathway.

Curcumin, a natural phenolic biphenyl compound derived from the plant Curcuma longa, modulates multiple steps of carcinogenesis partly by affecting the expression of miRNAs. Interestingly, cancer development shares many of the same signalling pathways with bone formation. Reduced bone mass creates favourable conditions for tumor metastasis. However, the effects and mechanism of curcumin on bone formation and osteogenesis are relatively unknown and controversial. We demonstrated that curcumin inhibited osteogenesis of human adipose-derived mesenchymal stem cells (hADSCs) in a concentration-dependent manner. In hADSCs, curcumin modulates the expression of a series of miRNAs, including miR-126a-3p, during osteogenesis. Overexpression or inhibition of miR-126a-3p is required for the effect of curcumin on osteogenesis. Further investigation indicated that miR-126a-3p directly targets and inhibits LRP6 through binding to its 3'-UTR, and then blocks WNT activation. Our findings suggest that the use of curcumin as an anti-tumor agent may lead to decreased bone mass through the suppression of osteogenesis. Knowing whether the long-term or high doses use of curcumin will cause decreased bone mass and bone density, which might increase the potential threat of tumor metastasis, also requires a neutral assessment of the role of curcumin in both regulating bone formation and bone absorption.

A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

INTRODUCTION: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding. METHODS: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System. RESULTS: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a KD in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC50. DISCUSSION: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.

HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5- (trifluoromethoxy)phenyl) thiourea) suppress proliferation and migration of human cervical HeLa cells via inhibiting Wnt/ß-catenin signaling pathway.

BACKGROUND: Drug therapy plays an important role in the treatment of cervical cancer, which is one of the most common solid tumors in women. Therefore, it is important to seek more effective and less toxic therapies. PURPOSE: The aim of this study is to investigate the therapeutic potential of HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5-(trifluoromethoxy) phenyl)thiourea) (F5) for cervical cancer and explore the related mechanism. METHODS: By performing MTT assay, colony formation assay, flow cytometry, wound-healing assay, transwell assay, immunofluorescent staining and siRNA assay, we study the effect of F5 on human cervical HeLa cells. The mechanism of F5 was also investigated. RESULTS: We found that F5 significantly inhibited HeLa cell proliferation, led to accumulation of cells in the S phase, and induced apoptosis and inhibited migration. Mechanistically, F5 inhibited HeLa cell growth and migration through repressing the expression and nuclear translocation of ß-catenin, enhancing Axin expression, inhibiting the phosphorylation of LRP5/6 and GSK3ß, as well as downregulating the Wnt downstream targeted proteins. Knockdown of a checkpoint ß-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of ß-catenin, was used to treat HeLa cells and the results demonstrated that F5 inhibited proliferation and migration via the inhibition of the Wnt/ß-catenin pathway. CONCLUSION: Our findings demonstrated that F5 can target ß-catenin potentially and is useful in the treatment of cervical cancer.

Gigantol inhibits Wnt/ß-catenin signaling and exhibits anticancer activity in breast cancer cells.

BACKGROUND: Gigantol is a bibenzyl compound derived from several medicinal orchids. This biologically active compound has been shown to have promising therapeutic potential against cancer cells, but its mechanism of action remains unclear. METHODS: The inhibitory effect of gigantol on Wnt/ß-catenin signaling was evaluated with the SuperTOPFlash reporter system. The levels of phosphorylated low-density lipoprotein receptor related protein 6 (LRP6), total LRP6 and cytosolic ß-catenin were determined by Western blot analysis. The expression of Wnt target genes was analyzed using real-time PCR. Cell viability was measured with a MTT assay. The effect of gigantol on cell migration was examined using scratch wound-healing and transwell migration assays. RESULTS: Gigantol decreased the level of phosphorylated LRP6 and cytosolic ß-catenin in HEK293 cells. In breast cancer MDA-MB-231 and MDA-MB-468 cells, treatment with gigantol reduced the level of phosphorylated LRP6, total LRP6 and cytosolic ß-catenin in a dose-dependent manner, resulting in a decrease in the expression of Wnt target genes Axin2 and Survivin. We further demonstrated that gigantol suppressed the viability and migratory capacity of breast cancer cells. CONCLUSION: Gigantol is a novel inhibitor of the Wnt/ß-catenin pathway. It inhibits Wnt/ß-catenin signaling through downregulation of phosphorylated LRP6 and cytosolic ß-catenin in breast cancer cells.

Chrysin, Abundant in Morinda citrifolia Fruit Water-EtOAc Extracts, Combined with Apigenin Synergistically Induced Apoptosis and Inhibited Migration in Human Breast and Liver Cancer Cells.

The composition of Morinda citrifolia (M. citrifolia) was determined using high-performance liquid chromatography (HPLC), and the anticancer effects of M. citrifolia extract evaluated in HepG2, Huh7, and MDA-MB-231 cancer cells. M. citrifolia fruit extracts were obtained by using five different organic solvents, including hexane (Hex), methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and ethanol (EtOH). The water-EtOAc extracts from M. citrifolia fruits was found to have the highest anticancer activity. HPLC data revealed the predominance of chrysin in water-EtOAc extracts of M. citrifolia fruit. Furthermore, the combined effects of cotreatment with apigenin and chrysin on liver and breast cancer were investigated. Treatment with apigenin plus chrysin for 72-96 h reduced HepG2 and MDA-MB-231 cell viability and induced apoptosis through down-regulation of S-phase kinase-associated protein-2 (Skp2) and low-density lipoprotein receptor-related protein 6 (LRP6) expression. However, the combination treatment for 36 h synergistically decreased MDA-MB-231 cell motility but not cell viability through down-regulation of MMP2, MMP9, fibronectin, and snail in MDA-MB-231 cells. Additionally, chrysin combined with apigenin also suppressed tumor growth in human MDA-MB-231 breast cancer cells xenograft through down-regulation of ki-67 and Skp2 protein. The experimental results showed that chrysin combined with apigenin can reduce HepG2 and MDA-MB-231 proliferation and cell motility and induce apoptosis. It also offers opportunities for exploring new drug targets, and further investigations are underway in this regard.

Low-Density Lipoprotein Receptor-Related Protein 6 (LRP6) Is a Novel Nutritional Therapeutic Target for Hyperlipidemia, Non-Alcoholic Fatty Liver Disease, and Atherosclerosis.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a member of the low-density lipoprotein receptor family and has a unique structure, which facilitates its multiple functions as a co-receptor for Wnt/ß-catenin signaling and as a ligand receptor for endocytosis. The role LRP6 plays in metabolic regulation, specifically in the nutrient-sensing pathway, has recently garnered considerable interest. Patients carrying an LRP6 mutation exhibit elevated levels of LDL cholesterol, triglycerides, and fasting glucose, which cooperatively constitute the risk factors of metabolic syndrome and atherosclerosis. Since the discovery of this mutation, the general role of LRP6 in lipid homeostasis, glucose metabolism, and atherosclerosis has been thoroughly researched. These studies have demonstrated that LRP6 plays a role in LDL receptor-mediated LDL uptake. In addition, when the LRP6 mutant impaired Wnt-LRP6 signaling, hyperlipidemia, non-alcoholic fatty liver disease, and atherosclerosis developed. LRP6 regulates lipid homeostasis and body fat mass via the nutrient-sensing mechanistic target of the rapamycin (mTOR) pathway. Furthermore, the mutant LRP6 triggers atherosclerosis by activating platelet-derived growth factor (PDGF)-dependent vascular smooth muscle cell differentiation. This review highlights the exceptional opportunities to study the pathophysiologic contributions of LRP6 to metabolic syndrome and cardiovascular diseases, which implicate LRP6 as a latent regulator of lipid metabolism and a novel therapeutic target for nutritional intervention.

Reduced LRP6 expression and increase in the interaction of GSK3ß with p53 contribute to podocyte apoptosis in diabetes mellitus and are prevented by green tea.

In diabetes mellitus (DM), podocyte apoptosis leads to albuminuria and nephropathy progression. Low-density lipoprotein receptor-related protein 6 (LRP6) is WNT pathway receptor that is involved in podocyte death, adhesion and motility. Glycogen synthase kinase 3 (GSK3) interaction with p53 (GSK3-p53) promotes apoptosis in carcinoma cells. It is unknown if GSK3-p53 contributes to podocyte apoptosis in DM. In experimental DM, green tea (GT) reduces albuminuria by an unknown mechanism. In the present study, we assessed the role of the GSK3ß-p53 in podocyte apoptosis and the effects of GT on these abnormalities. In diabetic spontaneously hypertensive rats (SHRs), GT prevents podocyte's p-LRP6 expression reduction, increased GSK3ß-p53 and high p53 levels. In diabetic SHR rats, GT reduces podocyte apoptosis, foot process effacement and albuminuria. In immortalized mouse podocytes (iMPs), high glucose (HG), silencing RNA (siRNA) or blocking LRP6 (DKK-1) reduced p-LRP6 expression, leading to high GSK3ß-p53, p53 expression, apoptosis and increased albumin influx. GSK3ß blockade by BIO reduced GSK3ß-p53 and podocyte apoptosis. In iMPs under HG, GT reduced apoptosis and the albumin influx by blocking GSK3ß-p53 following the rise in p-LRP6 expression. These effects of GT were prevented by LRP6 siRNA or DKK-1. In conclusion, in DM, WNT inhibition, via LRP6, increases GSK3ß-p53 and podocyte apoptosis. Maneuvers that inactivate GSK3ß-p53, such as GT, may be renoprotective in DM.

Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/ß-catenin signaling--an in vitro and in vivo study.

We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is increased via osteogenic differentiation of bone marrow stromal cells (BMSCs), which is regulated by Wnt3a/ß-catenin signaling. Our in vitro data showed that HBO increased cell proliferation, Wnt3a production, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated BMSCs. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx2 were upregulated while those of GSK-3ß were downregulated after HBO treatment. The relative density ratio (phospho-protein/protein) of Akt and GSK-3ß was both up-regulated while that of ß-catenin was down-regulated after HBO treatment. We next investigated whether HBO affects the accumulation of ß-catenin. Our Western blot analysis showed increased levels of translocated ß-catenin that stimulated the expression of target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of osteogenic markers of BMSCs, such as alkaline phosphatase activity, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the bone morphogenetic protein (BMP2) and osterix production. We further demonstrated that HBO increased the expression of vacuolar-ATPases, which stimulated Wnt3a secretion from BMSCs. Finally, we showed that the beneficial effects of HBO on bone formation were related to Wnt3a/ß-catenin signaling in a rabbit model by histology, mechanical testing, and immunohistochemical assays. Accordingly, we concluded that HBO increased the osteogenic differentiation of BMSCs by regulating Wnt3a secretion and signaling.

CCN1 secretion induced by cigarette smoking extracts augments IL-8 release from bronchial epithelial cells.

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.