Nkd1 Functions as a Passive Antagonist of Wnt Signaling

Wnt signaling is involved in many aspects of development and in the homeostasis of stem cells. Its importance is underscored by the fact that misregulation of Wnt signaling has been implicated in numerous diseases, especially colorectal cancer. However, how Wnt signaling regulates itself is not well understood. There are several Wnt negative feedback regulators, which are active antagonists of Wnt signaling, but one feedback regulator, Nkd1, has reduced activity compared to other antagonists, yet is still a negative feedback regulator. Here we describe our efforts to understand the role of Nkd1 using Wnt signaling compromised zebrafish mutant lines. In several of these lines, Nkd1 function was not any more active than it was in wild type embryos. However, we found that Nkd1’s ability to antagonize canonical Wnt/β-catenin signaling was enhanced in the Wnt/Planar Cell Polarity mutants silberblick (slb/wnt11) and trilobite (tri/vangl2). While slb and tri mutants do not display alterations in canonical Wnt signaling, we found that they are hypersensitive to it. Overexpression of the canonical Wnt/β-catenin ligand Wnt8a in slb or tri mutants resulted in dorsalized embryos, with tri mutants being much more sensitive to Wnt8a than slb mutants. Furthermore, the hyperdorsalization caused by Wnt8a in tri could be rescued by Nkd1. These results suggest that Nkd1 functions as a passive antagonist of Wnt signaling, functioning only when homeostatic levels of Wnt signaling have been breached or when Wnt signaling becomes destabilized.     

Role of Raf-kinase inhibitor protein in colorectal cancer and its regulation by hydroxycamptothecine

Background

Recently accumulated evidence suggests that Raf kinase inhibitor protein (RKIP) participates in regulation of many signaling pathways and plays an important role in tumorigenesis and tumor metastasis. However, studies investigating the role of RKIP in colorectal cancer have not been reported. The aim of this study was to investigate the role of RKIP on colorectal cancer cell differentiation, progression and its correlation with chemosensitivity.

Results

Immunohistochemical analysis revealed that RKIP expression was higher in non-neoplastic colorectal tissue (NCRCT) and colorectal cancer tissue (CRCT) than that in metastatic lymph node tissue (MLNT) (P <0.05). P-ERK protein expression was higher in MLNT and CRCT than that in NCRCT (P = 0.02). Immunocytochemical analysis further revealed that RKIP expression was higher in the well differentiated cell line SW1116 as compared to that in the poorly differentiated cell line LoVo. Matrigel invasive assay demonstrated that the inhibition of RKIP by short hairpin RNA (shRNA) 271 transfection significantly increased the number of migrated cells (90.67 ± 4.04 vs. 37.33 ± 2.51, P <0.05), whereas over-expression of RKIP by PEBP-1 plasmid transfection significantly suppressed the number of migrated cells (79.24 ± 5.18 vs. 154.33 ± 7.25, P <0.05). Meanwhile, down-regulation of RKIP induced an increase in the cell survival rate by inhibiting apoptosis induced by hydroxycamptothecine.

Conclusions

RKIP was also found to be associated with cell differentiation, with a higher activity in well differentiated colorectal cancer cells than in poorly differentiated ones. The upregulated expression of RKIP in colorectal cancer cells inhibited cell invasion and metastasis, while downregulation of RKIP reduced chemosensitivity by inhibiting apoptosis induced by HCPT.     

Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-β Signaling

Human Dachshund homologue 1 (DACH1) is a major component of the Retinal Determination Gene Network. Loss of DACH1 expression was found in breast, prostate, lung, endometrial, colorectal and hepatocellular carcinoma. To explore the expression, regulation and function of DACH1 in human esophageal cancer, 11 esophageal cancer cell lines, 10 cases of normal esophageal mucosa, 51 cases of different grades of dysplasia and 104 cases of primary esophageal squamous cancer were employed. Methylation specific PCR, immunohistochemistry, western blot, flow cytometry, small interfering RNAs, colony formation techniques and xenograft mice model were used. We found that DACH1 expression was regulated by promoter region hypermethylation in esophageal cancer cell lines. 18.8% (6 of 32) of grade 1, 42.1% (8 of 19) of grade 2 and grade 3 dysplasia (ED2,3), and 61.5% (64 of 104) of esophageal cancer were methylated, but no methylation was found in 10 cases of normal esophageal mucosa. The methylation was increased in progression tendency during esophageal carcinogenesis (P<0.01). DACH1 methylation was associated with poor differentiation (P<0.05) and late tumor stage (P<0.05). Restoration of DACH1 expression inhibited cell growth and activated TGF-β signaling in KYSE150 and KYSE510 cells. DACH1 suppressed human esophageal cancer cell tumor growth in xenograft mice. In conclusion, DACH1 is frequently methylated in human esophageal cancer and methylation of DACH1 is involved in the early stage of esophageal carcinogenesis. DACH1 expression is regulated by promoter region hypermethylation. DACH1 suppresses esophageal cancer growth by activating TGF-β signaling.     

Kinetics of changes in gene and microRNA expression related with muscle inflammation and protein degradation following LPS-challenge in weaned piglets

To test the dynamic changes of the expression of genes and microRNA in the gastrocnemius muscle after LPS challenge, 36 piglets were assigned to a control group (slaughtered 0 h after saline injection) and LPS groups (slaughtered at 1 h, 2 h, 4 h, 8 h, and 12 h after LPS treatment, respectively). After LPS treatment, the mRNA expression of IL-1β, IL-6, and TNF-α reached maximal levels at 1 h, 2 h, and 1 h, respectively (P < 0.05), and mRNA expression of TLR4, NODs, muscle-specific ring finger 1, and muscle atrophy F-box peaked at 12 h (P < 0.05). Moreover, the expression of miR-122, miR-135a, and miR-370 reduced at 1 h, 1 h, and 2 h, respectively (P < 0.05), and miR-34a, miR-224, miR-132, and miR-145 reached maximum expression levels at 1 h, 1 h, 2 h, and 4 h, respectively (P < 0.05). These results suggested that mRNA expression of pro-inflammatory cytokines was elevated in the early stage, mRNA expression of genes related to TLR4 and NODs signaling pathways and protein degradation increased in the later phase, and the expression of microRNA related to muscle inflammation and protein degradation changed in the early stage after LPS injection.     

MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene

Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.     

Thrombospondin-1 expression and modulation of Wnt and hippo signaling pathways during the early phase of Trypanosoma cruzi infection of heart endothelial cells

The protozoan parasite, Trypanosoma cruzi, causes severe morbidity and mortality in afflicted individuals. Approximately 30% of T. cruzi infected individuals present with cardiac pathology. The invasive forms of the parasite are carried in the vascular system to infect other cells of the body. During transportation, the molecular mechanisms by which the parasite signals and interact with host endothelial cells (EC) especially heart endothelium is currently unknown. The parasite increases host thrombospondin-1 (TSP1) expression and activates the Wnt/β-catenin and hippo signaling pathways during the early phase of infection. The links between TSP1 and activation of the signaling pathways and their impact on parasite infectivity during the early phase of infection remain unknown. To elucidate the significance of TSP1 function in YAP/β-catenin colocalization and how they impact parasite infectivity during the early phase of infection, we challenged mouse heart endothelial cells (MHEC) from wild type (WT) and TSP1 knockout mice with T. cruzi and evaluated Wnt signaling, YAP/β-catenin crosstalk, and how they affect parasite infection. We found that in the absence of TSP1, the parasite induced the expression of Wnt-5a to a maximum at 2 h (1.73±0.13), P< 0.001 and enhanced the level of phosphorylated glycogen synthase kinase 3β at the same time point (2.99±0.24), P<0.001. In WT MHEC, the levels of Wnt-5a were toned down and the level of p-GSK-3β was lowest at 2 h (0.47±0.06), P< 0.01 compared to uninfected control. This was accompanied by a continuous significant increase in the nuclear colocalization of β-catenin/YAP in TSP1 KO MHEC with a maximum Pearson correlation coefficient of (0.67±0.02), P< 0.05 at 6 h. In WT MHEC, the nuclear colocalization of β-catenin/YAP remained steady and showed a reduction at 6 h (0.29±0.007), P< 0.05. These results indicate that TSP1 plays an important role in regulating β-catenin/YAP colocalization during the early phase of T. cruzi infection. Importantly, dysregulation of this crosstalk by pre-incubation of WT MHEC with a β-catenin inhibitor, endo-IWR 1, dramatically reduced the level of infection of WT MHEC. Parasite infectivity of inhibitor treated WT MHEC was similar to the level of infection of TSP1 KO MHEC. These results indicate that the β-catenin pathway induced by the parasite and regulated by TSP1 during the early phase of T. cruzi infection is an important potential therapeutic target, which can be explored for the prophylactic prevention of T. cruzi infection.     

Wnt ligand–dependent activation of the negative feedback regulator Nkd1

Misregulation of Wnt signaling is at the root of many diseases, most notably colorectal cancer, and although we understand the activation of the pathway, we have a very poor understanding of the circumstances under which Wnt signaling turns itself off. There are numerous negative feedback regulators of Wnt signaling, but two stand out as constitutive and obligate Wnt-induced regulators: Axin2 and Nkd1. Whereas Axin2 behaves similarly to Axin in the destruction complex, Nkd1 is more enigmatic. Here we use zebrafish blastula cells that are responsive Wnt signaling to demonstrate that Nkd1 activity is specifically dependent on Wnt ligand activation of the receptor. Furthermore, our results support the hypothesis that Nkd1 is recruited to the Wnt signalosome with Dvl2, where it becomes activated to move into the cytoplasm to interact with β-catenin, inhibiting its nuclear accumulation. Comparison of these results with Nkd function in Drosophila generates a unified and conserved model for the role of this negative feedback regulator in the modulation of Wnt signaling.     

The Influence of the CHIEF Pathway on Colorectal Cancer-Specific Mortality

Many components of the CHIEF (Convergence of Hormones, Inflammation, and Energy Related Factors) pathway could influence survival given their involvement in cell growth, apoptosis, angiogenesis, and tumor invasion stimulation. We used ARTP (Adaptive Rank Truncation Product) to test if genes in the pathway were associated with colorectal cancer-specific mortality. Colon cancer (n = 1555) and rectal cancer (n = 754) cases were followed over five years. Age, center, stage at diagnosis, and tumor molecular phenotype were considered when calculating ARTP p values. A polygenic risk score was used to summarize the magnitude of risk associated with this pathway. The JAK/STAT/SOC was significant for colon cancer survival (P_ARTP = 0.035). Fifteen genes (DUSP2, INFGR1, IL6, IRF2, JAK2, MAP3K10, MMP1, NFkB1A, NOS2A, PIK3CA, SEPX1, SMAD3, TLR2, TYK2, and VDR) were associated with colon cancer mortality (P_ARTP <0.05); JAK2 (P_ARTP  = 0.0086), PIK3CA (P_ARTP = 0.0098), and SMAD3 (P_ARTP = 0.0059) had the strongest associations. Over 40 SNPs were significantly associated with survival within the 15 significant genes (P_ARTP<0.05). SMAD3 had the strongest association with survival (HR_GG 2.46 95% CI 1.44,4.21 P_Ttrnd = 0.0002). Seven genes (IL2RA, IL8RA, IL8RB, IRF2, RAF1, RUNX3, and SEPX1) were significantly associated with rectal cancer (P_ARTP<0.05). The HR for colorectal cancer-specific mortality among colon cancer cases in the upper at-risk alleles group was 11.81 (95% CI 7.07, 19. 74) and was 10.99 (95% CI 5.30, 22.78) for rectal cancer. These results suggest that several genes in the CHIEF pathway are important for colorectal cancer survival; the risk associated with the pathway merits validation in other studies.     

Expression Characteristics of β-Catenin in Scallop Chlamys farreri Gonads and Its Role as a Potential Upstream Gene of Dax1 through Canonical Wnt Signalling Pathway Regulating the Spermatogenesis

β-catenin is a key signaling molecule in the canonical Wnt pathway, which is involved in animal development. However, little information has been reported for β-catenin in bivalves. In the present study, we cloned a homolog of β-catenin from the scallop Chlamys farreri and determined its expression characteristics. The full-length cDNA of β-catenin was 3,353 bp, including a 2,511 bp open reading frame that encoded a predicted 836 amino acid protein. Level of the β-catenin mRNA increased significantly (P<0.05) with C. farreri gonadal development and presented a sexually dimorphic expression pattern in the gonads, which was significantly high in ovaries detected by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis revealed that the β-catenin was mainly located in germ cells of the gonads, with obvious positive immune signals in the oogonia and oocytes of ovaries as well as in the spermatogonia and spermatocytes of testes, implying β-catenin might be involved in the gametogenesis of C. farreri. Furthermore, when 0.1 µg/mL and 0.2 µg/mL DKK-1 (an inhibitor of the canonical Wnt pathway) were added in vitro to culture medium containing testis cells of C. farreri, the expression of β-catenin decreased significantly detected by qRT-PCR (P<0.05), suggesting the canonical Wnt signal pathway exists in the scallop testis. Similarly, when 50 µM and 100 µM quercetin (an inhibitor of β-catenin) were added in vitro to the culture system, Dax1 expression was significantly down-regulated compared with controls (P<0.05), implying the β-catenin is an upstream gene of Dax1 and is involved in the regulation of C. farreri spermatogenesis.     

Metabolic balance in colorectal cancer is maintained by optimal Wnt signaling levels

Wnt pathways are important for the modulation of tissue homeostasis, and their deregulation is linked to cancer development. Canonical Wnt signaling is hyperactivated in many human colorectal cancers due to genetic alterations of the negative Wnt regulator APC. However, the expression levels of Wnt‐dependent targets vary between tumors, and the mechanisms of carcinogenesis concomitant with this Wnt signaling dosage have not been understood. In this study, we integrate whole‐genome CRISPR/Cas9 screens with large‐scale multi‐omic data to delineate functional subtypes of cancer. We engineer APC loss‐of‐function mutations and thereby hyperactivate Wnt signaling in cells with low endogenous Wnt activity and find that the resulting engineered cells have an unfavorable metabolic equilibrium compared with cells which naturally acquired Wnt hyperactivation. We show that the dosage level of oncogenic Wnt hyperactivation impacts the metabolic equilibrium and the mitochondrial phenotype of a given cell type in a context‐dependent manner. These findings illustrate the impact of context‐dependent genetic interactions on cellular phenotypes of a central cancer driver mutation and expand our understanding of quantitative modulation of oncogenic signaling in tumorigenesis.     

Effects of Ovarian Hormones and Oral Contraceptive Pills on Cardiac Vagal Withdrawal at the Onset of Dynamic Exercise

The purpose of this study was to investigate the effects of the ovarian hormones and the use of oral contraceptive pills (OCP) on cardiac vagal withdrawal at the onset of dynamic exercise. Thirty physically active women aged 19–32 years were divided into two groups: OCP users (n = 17) and non-OCP users (n = 13). Participants were studied randomly at three different phases of the menstrual cycle: early follicular (day 3.6 ± 1.2; range 1–5), ovulatory (day 14.3 ± 0.8; range 13–16) and midluteal (day 21.3 ± 0.8; range 20–24), according to endogenous (in non-OCP users) or exogenous (in OCP users) estradiol and progesterone variations. The cardiac vagal withdrawal was represented by the cardiac vagal index (CVI), which was obtained by the 4-s exercise test. Additionally, resting heart rate, systolic (SBP) and diastolic blood pressure (DBP) were obtained. The CVI was not significantly different between the three phases of the menstrual cycle in either the non-OCP users (early follicular: 1.58 ± 0.1; ovulatory: 1.56 ± 0.1; midluteal: 1.58 ± 0.1, P > 0.05) or OCP users (early follicular: 1.47 ± 0.1; ovulatory: 1.49 ± 0.1; midluteal: 1.47 ± 0.1, P > 0.05) (mean ± SEM). Resting cardiovascular responses were not affected by hormonal phase or OCP use, except that the SBP was higher in the OCP users than non-OCP users in all phases of the cycle (P < 0.05). In summary, our results demonstrate that cardiac vagal withdrawal at the onset of dynamic exercise was not impacted by the menstrual cycle or OCP use in physically active women.     

Role of Toll-Like Receptor 4 in Colorectal Carcinogenesis: A Meta-Analysis

Objective

This meta-analysis was performed to evaluate the role of toll-like receptor 4 (TLR-4) in colorectal carcinogenesis.

Methods

The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases were searched from inception through November 1st, 2013 without language restrictions. Odds ratios (ORs) or standardized mean differences (SMD) with their 95% confidence intervals (CI) were calculated.

Results

Fourteen case-control studies met the inclusion criteria for this meta-analysis. A total of 1,209 colorectal cancer (CRC) cases and 1,218 healthy controls were involved in this meta-analysis. Two common polymorphisms (299 A>G and 399 C>T) in the TLR-4 gene, TLR-4 mRNA and protein expression were assessed. Our meta-analysis results revealed that the TLR-4 399 C>T polymorphism might increase the risk of CRC (allele model: OR = 1.77, 95%CI = 1.32∼2.36, P<0.001; dominant model: OR = 1.83, 95%CI = 1.32∼2.52, P<0.001; respectively). However, we found no correlation between the TLR-4 299 A>G polymorphism and CRC risk (all P>0.05). A subgroup analysis by ethnicity suggested that TLR-4 genetic polymorphisms were associated with an increased risk of CRC among Asians (allele model: OR = 1.50, 95%CI = 1.19∼1.88, P = 0.001; dominant model: OR = 1.49, 95%CI = 1.16∼1.92, P = 0.002; respectively), but not among Caucasians and Africans (all P>0.05). Furthermore, our results showed that TLR-4 mRNA and protein levels in CRC patients were higher than those in healthy controls (TLR-4 mRNA: SMD  = 2.51, 95%CI  = 0.98∼4.05, P = 0.001; TLR-4 protein: OR  = 4.75, 95%CI  = 1.16∼19.36, P = 0.030; respectively).

Conclusion

Our findings provide empirical evidence that TLR-4 may play an important role in colorectal carcinogenesis. Thus, TLR-4 is a promising potential biomarker for the early diagnosis of CRC.     

Wnt/β-catenin signaling mediates the abnormal osteogenic and adipogenic capabilities of bone marrow mesenchymal stem cells from chronic graft-versus-host disease patients

Chronic graft-versus-host disease (cGVHD) is the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cells (MSCs) in bone marrow (BM) remain unclear in the pathophysiology of cGVHD. In this study, we analyzed BM-MSCs from 66 patients after allo-HSCT, including 33 with active cGVHD and 33 without cGVHD. BM-MSCs showed similar morphology, frequency, phenotype, and proliferation in patients with or without cGVHD. MSCs from the active cGVHD group showed a decreased apoptosis rate (P < 0.01). Osteogenic capacity was increased while adipogenic capacity was decreased in the active cGVHD MSCs compared with no-cGVHD MSCs. The expressions of osteogenic gene RUNX2 and COL1A1 were higher (P < 0.001) while adipogenic gene PPAR-γ and FABP4 were lower (P < 0.001) in the active cGVHD MSCs than no-cGVHD MSCs. These changes were associated with the severity of cGVHD (P < 0.0001; r = 0.534, r = 0.476, r = −0.796, and r = −0.747, respectively in RUNX2, COL1A1, PPAR-γ, and FABP4). The expression of Wnt/β-catenin pathway ligand Wnt3a was increased in cGVHD-MSCs. The dysfunction of cGVHD-MSCs could be reversed by Dickkopf related protein 1(DKK1) to inhibit the binding of Wnt3a. In summary, the differentiation of BM-MSCs was abnormal in active cGVHD, and its underlying mechanism is the upregulated of Wnt3a through Wnt/β-catenin signaling pathway of MSCs.Subject terms: Cell signalling, Mesenchymal stem cells     

Effect of Non-Alcoholic Fatty Liver Disease on Estimated Glomerular Filtration Rate Could Be Dependent on Age

There is a gap between the association of non-alcoholic fatty liver disease (NAFLD) and renal function in an apparently healthy population. This study aims to assess whether NAFLD is associated with estimated glomerular filtration rate (eGFR) levels and to understand early changes of eGFR in NAFLD. A cross-sectional study was performed among apparently healthy persons who underwent general health screening including laboratory assessments and hepatic ultrasonography from January 2013 to December 2013 at the First Affiliated Hospital of Zhejiang University, College of Medicine, China. This study included 1,193 subjects with a mean age of 48 years. Prevalence of NAFLD was 31.3%. Mean eGFR was significantly lower in NAFLD than in controls (107 ± 19 mL/min/1.73 m² vs. 113 ± 23 mL/min/1.73 m², P<0.001). Correlation analysis between eGFR and NAFLD related risk factors revealed an inverse correlation between eGFR levels and some NAFLD risk factors (all P<0.01). All subjects were classified into five phases according to age. Average eGFR levels of NAFLD were lower than controls in three phases for subjects with ≤ 50 years of age (all P<0.05), while there were no significant differences on average eGFR levels between NAFLD and controls in two phases for subjects with >50 years of age (Both P>0.05). The eGFR level is significantly associated with NAFLD and its risk factors in an apparently healthy population. Effects of NAFLD on eGFR could be dependent on age.