Wnt3a Promotes the Vasculogenic Mimicry Formation of Colon Cancer via Wnt/β-Catenin Signaling

Our previous study provided evidence that non-canonical Wnt signaling is involved in regulating vasculogenic mimicry (VM) formation. However, the functions of canonical Wnt signaling in VM formation have not yet been explored. In this study, we found the presence of VM was related to colon cancer histological differentiation (p < 0.001), the clinical stage (p < 0.001), and presence of metastasis and recurrence (p < 0.001). VM-positive colon cancer samples showed increased Wnt3a expression (p < 0.001) and β-catenin nuclear expression (p < 0.001) compared with the VM-negative samples. In vitro, over-regulated Wnt3a expression in HT29 colon cancer cells promoted the capacity to form tube-like structures in the three-dimensional (3-D) culture together with increased expression of endothelial phenotype-associated proteins such as VEGFR2 and VE-cadherin. The mouse xenograft model showed that Wnt3a-overexpressing cells grew into larger tumor masses and formed more VM than the control cells. In addition, the Wnt/β-catenin signaling antagonist Dickkopf-1(Dkk1) can reverse the capacity to form tube-like structures and can decrease the expressions of VEGFR2 and VE-cadherin in Wnt3a-overexpressing cells. Taken together, our results suggest that Wnt/β-catenin signaling is involved in VM formation in colon cancer and might contribute to the development of more accurate treatment modalities aimed at VM.     

Impact of an Altered Wnt1/β-Catenin Expression on Clinicopathology and Prognosis in Clear Cell Renal Cell Carcinoma

In renal cell carcinoma (RCC), single members of the Wnt/β-catenin signaling cascade were recently identified to contribute to cancer progression. However, the role of Wnt1, one of the key ligands in β-catenin regulation, is currently unknown in RCC. Therefore, alterations of the Wnt1/β-catenin axis in clear cell RCC (ccRCC) were examined with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS). Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and β-catenin expression by immunohistochemistry in tissue microarrays. Expression scores, including intensity and percentage of stained cells, were compared between normal kidney and ccRCCs. Data was categorized according to mean expression scores and correlated to tumor and patients’ characteristics. Survival was analyzed according to the Kaplan-Meier and log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to explore the independent prognostic value of Wnt1 and β-catenin. In ccRCCs, high Wnt1 was associated with increased tumor diameter, stage and vascular invasion (p ≤ 0.02). High membranous β-catenin was associated with advanced stage, vascular invasion and tumor necrosis (p ≤ 0.01). Higher diameter, stage, node involvement, grade, vascular invasion and sarcomatoid differentiation (p ≤ 0.01) were found in patients with high cytoplasmic β-catenin. Patients with a high cytoplasmic β-catenin had a significantly reduced OS (hazard ratio (HR) 1.75) and CSS (HR 2.26), which was not independently associated with OS and CSS after adjustment in the multivariable model. Increased ccRCC aggressiveness was reflected by an altered Wnt1/β-catenin signaling. Cytoplasmic β-catenin was identified as the most promising candidate associated with unfavorable clinicopathology and impaired survival. Nevertheless, the shift of membranous β-catenin to the cytoplasm with a subsequently increased nuclear expression, as shown for other malignancies, could not be demonstrated to be present in ccRCC.     

A Caspase-Dependent Pathway Is Involved in Wnt/β-Catenin Signaling Promoted Apoptosis in Bacillus Calmette-Guerin Infected RAW264.7 Macrophages

Apoptosis of alveolar macrophages following Mycobacterium tuberculosis infection have been demonstrated to play a central role in the pathogenesis of tuberculosis. In the present study, we found that Wnt/β-catenin signaling possesses the potential to promote macrophage apoptosis in response to mycobacterial infection. In agreement with other findings, an activation Wnt/β-catenin signaling was observed in murine macrophage RAW264.7 cells upon Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection at a multiple-of-infection of 10, which was accompanied with up-regulation of pro-inflammatory cytokines TNF-α and IL-6 production. However, the BCG-induced TNF-α and IL-6 secretion could be significantly reduced when the cells were exposed to a canonical Wnt signaling ligand, Wnt3a. Importantly, the activation of Wnt/β-catenin signaling was able to further promote apoptosis in BCG-infected RAW264.7 cells in part by a mitochondria-dependent apoptosis pathway. Immunoblotting analysis further demonstrated that Wnt/β-catenin signaling-induced cell apoptosis partly through a caspase-dependent apoptosis mechanism by down-regulation of anti-apoptotic protein Mcl-1, and up-regulation of pro-apoptotic proteins Bax and cleaved-caspase-3, as well as enhancement of caspase-3 activity in BCG-infected RAW264.7 cells. These data may imply an underlying mechanism of alveolar macrophages in response to mycobacterial infection, by which the pathogen induces Wnt/β-catenin signaling activation, which in turn represses mycobacterium-trigged inflammatory responses and promotes mycobacteria-infected cell apoptosis.     

Wnt16 Signaling Is Required for IL-1β-Induced Matrix Metalloproteinase-13-Regulated Proliferation of Human Stem Cell-Derived Osteoblastic Cells

We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7⁺hSMSC)-derived osteoblast-like (α7⁺hSMSC-OB) cells, and found that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-13-regulated proliferation of these cells. These data suggest that MMP-13 plays a potentially unique physiological role in the regeneration of osteoblast-like cells. Here, we examined whether up-regulation of MMP-13 activity by IL-1β was mediated by Wingless/int1 (Wnt) signaling and increased the proliferation of osteoblast-like cells. IL-1β increased the mRNA and protein levels of Wnt16 and the Wnt receptor Lrp5/Fzd2. Exogenous Wnt16 was found to increase MMP-13 mRNA, protein and activity, and interestingly, the proliferation rate of these cells. Treatment with small interfering RNAs against Wnt16 and Lrp5 suppressed the IL-1β-induced increase in cell proliferation. We revealed that a unique signaling cascade IL-1β→Wnt16→Lrp5→MMP-13, was intimately involved in the proliferation of osteoblast-like cells, and suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation are regulated by Wnt16.     

Sulfonamide Inhibitors of β-Catenin Signaling as Anticancer Agents with Different Output on c-MYC

The Wnt/β-catenin pathway is often found deregulated in cancer. The aberrant accumulation of β-catenin in the cell nucleus results in the development of various malignancies. Specific drugs against this signaling pathway for clinical treatments have not been approved yet. Herein we report inhibitors of β-catenin signaling of potential therapeutic value as anticancer agents. Ethyl 4-((4-(trifluoromethyl)phenyl)sulfonamido)benzoate (compound 14 ) inhibits the effect on Wnt reporter with an IC₅₀ value of 7.0 μM, significantly reduces c-MYC levels, inhibits HCT116 colon cancer cell growth (IC₅₀ 20.2 μM), does not violate Lipinski and Veber rules, and shows predicted Caco-2 and MDCK cell permeability P_app>500 nm s⁻¹. Compound 14 seems to have potential for the development of new anticancer therapies.     

Subchronic Exposure to Arsenic Represses the TH/TRβ1-CaMK IV Signaling Pathway in Mouse Cerebellum

We previously reported that arsenic (As) impaired learning and memory by down-regulating calmodulin-dependent protein kinase IV (CaMK IV) in mouse cerebellum. It has been documented that the thyroid hormone receptor (TR)/retinoid X receptor (RXR) heterodimer and thyroid hormone (TH) may be involved in the regulation of CaMK IV. To investigate whether As affects the TR/RXR heterodimer and TH, we determined As concentration in serum and cerebellum, 3,5,3’-triiodothyronine (T3) and thyroxin (T4) levels in serum, and expression of CaMK IV, TR and RXR in cerebellum of mice exposed to As. Cognition function was examined by the step-down passive avoidance task and Morris water maze (MWM) tests. Morphology of the cerebellum was observed by Hematoxylin-Eosin staining under light microscope. Our results showed that the concentrations of As in the serum and cerebellum of mice both increased with increasing As-exposure level. A significant positive correlation was found between the two processes. Adeficit in learning and memory was found in the exposed mice. Abnormal morphologic changes of Purkinje cells were observed in cerebellum of the exposed mice. Moreover, the cerebellar expressions of CaMK IV protein and the TRβ gene, and TRβ1 protein were significantly lower in As-exposed mice than those in controls. Subchronic exposure to As appears to increase its level in serum and cerebella of mice, impairing learning and memory and down-regulating expression of TRβ1 as well as down-stream CaMK IV. It is also suggested that the increased As may be responsible for down-regulation of TRβ1 and CaMK IV in cerebellum and that the down-regulated TRβ1 may be involved in As-induced impairment of learning and memory via inhibiting CaMK IV and its down-stream pathway.     

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl₄ (2.0 mL/kg of 10% CCl₄ v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl₄- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.     

β-Conglycinin Reduces the Tight Junction Occludin and ZO-1 Expression in IPEC-J2

Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) (p < 0.001) and metabolic activity (p < 0.001) and a remarkable increase of alkaline phosphatase (AP) activity (p < 0.001) in a dose-dependent manner. The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).     

Molecular Signaling Involved in Oxysterol-Induced β1-Integrin Over-Expression in Human Macrophages

The hypercholesterolemia-atherosclerosis association is now established; hypercholesterolemia may induce vascular-cell activation, subsequently increasing expression of adhesion molecules, cytokines, chemokines, growth factors, and other key inflammatory molecules. Among inflammatory molecules expressed by vascular cells, integrins play a critical role in regulating macrophage activation and migration to the site of inflammation, by mediating cell-cell and cell-extracellular matrix interactions. The main lipid oxidation products present in oxidized LDL that may be responsible for inflammatory processes in atherogenesis, are cholesterol oxidation products, known as oxysterols. This study demonstrates the effect of an oxysterol mixture, compatible with that detectable in human hypercholesterolemic plasma, on the expression and synthesis of β₁-integrin in cells of the macrophage lineage. The molecular signaling whereby oxysterols induce β₁-integrin up-regulation is also comprehensively investigated. Over-expression of β₁-integrin depends on activation of classic and novel members of protein kinase C and extracellular signal-regulated kinases 1 and 2, as well as of the up-stream G-protein (Gq and G13), c-Src, and phospholipase C. In addition, the localization of β₁-integrin in advanced human carotid plaques is highlighted, marking its importance in atherosclerotic plaque progression.     

How Are Synapses Born? A Functional and Molecular View of the Role of the Wnt Signaling Pathway

Synaptic transmission is a dynamic process that requires precise regulation. Early in life, we must be able to forge appropriate connections (add and remove) to control our behavior. Neurons must recognize appropriate targets, and external soluble factors that activate specific signaling cascades provide the regulation needed to achieve this goal. Wnt signaling has been implicated in several forms of synaptic plasticity, including functional and structural changes associated with brain development. The analysis of synapses from an electrophysiological perspective allows us to characterize the functional role of cellular signaling pathways involved in brain development. The application of quantal theory to principles of developmental plasticity offers the possibility of dissecting the function of structural changes associated with the birth of new synapses as well as the maturation of immature silent synapses. Here, we focus on electrophysiological and molecular evidence that the Wnt signaling pathway regulates glutamatergic synaptic transmission, specifically N-methyl-d-aspartate receptors (NMDARs), to control the birth of new synapses. We also focus on the role of Wnts in the conversion of silent synapses into functional synapses.     

Expression of an Endo-β-1,4-glucanase Gene from Orpinomyces PC-2 in Pichia pastoris

The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.     

The Involvement of Mutual Inhibition of ERK and mTOR in PLCγ1-Mediated MMP-13 Expression in Human Osteoarthritis Chondrocytes

The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLCγ1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLCγ1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLCγ1, p-PLCγ1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLCγ1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLCγ1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLCγ1 on MMP-13 may be partly attributed to the inhibition of the PLCγ1/mTOR axis on the PLCγ1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLCγ1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.     

Flaxseed Oil Supplementation Improve Gene Expression Levels of PPAR-γ, LP(a), IL-1 and TNF-α in Type 2 Diabetic Patients with Coronary Heart Disease

This study was carried out to determine the effects of flaxseed oil administration on gene expression levels related to insulin, lipid and inflammation in overweight diabetic patients with coronary heart disease (CHD). This randomized double‐blind, placebo‐controlled trial was conducted among 60 diabetic patients with CHD. Subjects were randomly allocated into two groups to intake either 1000 mg n‐3 fatty acid from flaxseed oil containing 400 mg α‐Linolenic acid [ALA (18:3n‐3)] (n = 30) or placebo (n = 30) twice a day for 12 weeks. Gene expression related to insulin, lipid and inflammation were quantified in peripheral blood mononuclear cells (PBMC) of diabetic patients with CHD with RT‐PCR method. Results of RT‐PCR demonstrated that after the 12‐week intervention, compared with the placebo, flaxseed oil supplementation could up‐regulate gene expression of peroxisome proliferator‐activated receptor gamma (PPAR‐γ) (P = 0.02) in PBMC of diabetic patients with CHD. In addition, compared with the placebo, taking flaxseed oil supplements down‐regulated gene expression levels of lipoprotein(a) [LP(a)] (P = 0.001), interleukin‐1 (IL‐1) (P = 0.001) and tumor necrosis factor alpha (TNF‐α) (P = 0.02) in PBMC of diabetic patients with CHD. We did not observe any significant effect of flaxseed oil supplementation on gene expression levels of low‐density lipoprotein receptor (LDLR), IL‐8 and transforming growth factor beta (TGF‐β) in PBMC of diabetic patients with CHD. Overall, flaxseed oil supplementation for 12 weeks in diabetic patients with CHD significantly improved gene expression levels of PPAR‐γ, LP(a), IL‐1 and TNF‐α, but did not influence LDLR, IL‐8 and TGF‐β.     

Morphological and Functional Expression of Fibroblast on Poly(lactide-co-glycolide)/β-Tricalcium Phosphate/Nature Bone

The in-vitro biocompatibility of degradable and bioactive composites consisting of poly(lactide-co-glycolide) (PLGA), β-tricalcium phosphate (β-TCP), and nature bone (NB) was investigated by culturing fibroblasts on the PLGA/β-TCP/NB substrates, and the cell morphology, proliferation, alkaline phosphatase (ALP) activity, and von Kossa mineralization were evaluated. The results showed that the incorporation of β-TCP and NB benefited fibroblasts attachment and the fibroblasts cultured on the PLGA/β-TCP/NB composite substrates spread better as compared to those on the pure PLGA after culturing for different times. MTT assay showed that the fibroblasts cultured on the PLGA/β-TCP/NB extraction revealed a higher proliferation rate than those on the pure PLGA extraction. ALP and von Kossa assay indicated that β-TCP and NB could promote biomineralization in vitro. All of the results showed that the addition of β-TCP and NB into PLGA could stimulate fibroblasts to proliferate and differentiate.