β‐catenin regulates IRF3‐mediated innate immune signalling in colorectal cancer

Objective

β‐catenin is one of the most critical oncogenes associated with many kinds of human cancers, especially in the human CRC. Innate immunity recognizes tumour derived damage‐associated molecular patterns (DAMPs) and primes the anti‐tumour adaptive responses. While the function of β‐catenin in CRC tumourigenesis is well established, its impact on innate immune evasion is largely unknown. The aim of this study is to characterize the role of β‐catenin in inhibiting RIG‐I‐like receptor (RLR)‐mediated IFN‐β signalling in colorectal cancer.

Materials and Methods

Immunohistochemical staining and western blotting were conducted to study the expression of β‐catenin, IRF3 and phospho‐IRF3 (p‐IRF3) in CRC samples and cell lines. Plaque assay determining virus replication was performed to assess the regulation of β‐catenin on IFN‐β signalling. The inhibition of β‐catenin on RLR‐mediated IFN‐β signalling was further studied by real‐time analyses and reporter assays in the context of lentiviral‐mediated β‐catenin stably knocking down. Lastly, co‐immunoprecipitation and nuclear fractionation assay were conducted to monitor the interaction between β‐catenin and IRF3.

Results

We found that high expression of β‐catenin positively correlated with the expression of IRF3 in CRC cells. Overexpression of β‐catenin increased the viral replication. Conversely knocking down of β‐catenin inhibited viral replication. Furthermore, our data demonstrated that β‐catenin could inhibit the expression of IFN‐β and interferon‐stimulated gene 56 (ISG56). Mechanistically, we found that β‐catenin interacted with IRF3 and blocked its nuclear translocation.

Conclusion

Our study reveals an unprecedented role of β‐catenin in enabling innate immune evasion in CRC.

     

E‐cadherin can limit the transforming properties of activating β‐catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

HIF‐1α regulates EMT via the Snail and β‐catenin pathways in paraquat poisoning‐induced early pulmonary fibrosis

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.     

Hopeahainol A attenuates memory deficits by targeting β‐amyloid in APP/PS1 transgenic mice

Increasing evidence demonstrates that amyloid beta (Aβ) elicits mitochondrial dysfunction and oxidative stress, which contributes to the pathogenesis of Alzheimer's disease (AD). Identification of the molecules targeting Aβ is thus of particular significance in the treatment of AD. Hopeahainol A (HopA), a polyphenol with a novel skeleton obtained from Hopea hainanensis, is potentially acetylcholinesterase‐inhibitory and anti‐oxidative in H₂O₂‐treated PC12 cells. In this study, we reported that HopA might bind to Aβ_1–42 directly and inhibit the Aβ_1–42 aggregation using a combination of molecular dynamics simulation, binding assay, transmission electron microscopic analysis and staining technique. We also demonstrated that HopA decreased the interaction between Aβ_1–42 and Aβ‐binding alcohol dehydrogenase, which in turn reduced mitochondrial dysfunction and oxidative stress in vivo and in vitro. In addition, HopA was able to rescue the long‐term potentiation induction by protecting synaptic function and attenuate memory deficits in APP/PS1 mice. Our data suggest that HopA might be a promising drug for therapeutic intervention in AD.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

Are Bacteria the Major Producers of Extracellular Glycolytic Enzymes in Aquatic Environments?

In aquatic microbial ecology, it has been considered that most extracellular enzymes except phosphatases are of bacterial origin. We tested this paradigm by evaluating the relationship between bacterial cell number and the activity of three glycolytic enzymes from 17 fresh waters and also from a laboratory experiment. Our large sets of pooled data do not seem to support such a simple explanation, because we found only a weak correlation of bacterial number with activity of α‐glucosidase (r_s = 0.63), β‐glucosidase (r_s = 0.45), and β‐N‐acetylhexosaminidase (r_s = 0.44). We also tested relations of the enzymatic activities to potential sources of natural substrates: dissolved organic carbon (DOC) and phytoplankton (as chlorophyll a). Their correlations with the enzymatic activities tested were very weak or insignificant. On the other hand, we found evidence for distinct producers of extracellular enzymes by analysing enzyme kinetics. The kinetics usually did not follow the simple Michaelis‐Menten model but a more complex one, indicating a mixture of two enzymes with distinct affinity to a substrate. In combination with size fractionation, we could sometimes even distinguish three or more different enzymes. During diatom blooms, the diatom biomass tightly correlated with β‐N‐acetylhexosaminidase activity (>4 μm fraction). We also documented very tight relationships between activity of both glucosidases and dry weight of Daphnia longispina (r_s = 1.0 and 0.60 for α‐ and β‐glucosidases, respectively) in an alpine clear‐water lake. Our data and evidence from other studies indicate that extracellular glycosidic activities in aquatic ecosystems cannot generally be assigned only to bacteria. Also invertebrate animals and other eukaryotes (fungi, diatoms, protozoa etc.) should be considered as potentially very important enzyme producers. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

miR‐29a modulates tumor necrosis factor‐α‐induced osteogenic inhibition by targeting Wnt antagonists

We previously found that miR‐29a was significantly downregulated in Ankylosing spondylitis (AS) patients, a chronic inflammatory disease associated with bone metabolic disorder, however, the underlying mechanism remains unclear. In this study, we demonstrated that miR‐29a regulates tumor necrosis factor‐α (TNF‐α) mediated bone loss mainly by targeting DKK1 and GSK3β, thus activating the Wnt/β‐catenin pathway. Our findings may provide new insight into the pathogenesis of the bone metabolism disorder in inflammation environment and provide promising therapeutic target.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Proteomic analysis of β‐catenin activation in mouse liver by DIGE analysis identifies glucose metabolism as a new target of the Wnt pathway

The Wnt/β‐catenin signaling pathway has been increasingly implicated in liver development and physiology. Aberrant activation of this pathway is one of the major genetic events observed during the process of human HCC development. To gain insight into the mechanism underlying β‐catenin action in the liver, we conducted a quantitative differential proteomic analysis using 2‐D DIGE combined with MS, in mice with liver‐specific deletion of Apc resulting in acute activation of β‐catenin signaling (Apc^KOliv mice). We identified 94 protein spots showing differential expression between mutant Apc^KOliv and control mice, corresponding to 56 individual proteins. Most of the proteins identified were associated with metabolic pathways, such as ammonia and glucose metabolism. Our analysis showed an increase in lactate dehydrogenase activity together with a downregulation of two mitochondrial ATPase subunits (ATP5a1 and ATP5b). These observations indicate that β‐catenin signaling may induce a shift in the glucose metabolism from oxidative phosphorylation to glycolysis, known as the “Warburg effect”. Imaging with ¹⁸F‐fluoro‐2‐deoxy‐D ‐glucose‐positron emission tomography suggests that the specific metabolic reprogramming induced by β‐catenin in the liver does not imply the first step of glycolysis. This observation may explain why some HCCs are difficult to assess by fluoro‐2‐deoxy‐D ‐glucose‐positron emission tomography imaging.     

Zbed3 promotes proliferation and invasion of lung cancer partly through regulating the function of Axin‐Gsk3β complex

Our previous work showed that Zbed3 is overexpressed in nonsmall cell lung cancer and that down‐regulation of Zbed3 inhibited β‐catenin expression and cancer cell proliferation and invasiveness. Here, we investigated Zbed3's ability to promote lung cancer cell proliferation and invasion and the involvement of the Axin/TPC/glycogen synthase kinase 3β (Gsk‐3β) complex to the response. Coimmunoprecipitation assays showed that wild‐type Zbed3 bound to Axin but a Zbed3 mutant lacking the Axin binding site did not. In A549 and H1299 lung cancer cells, Zbed3 overexpression promoted cancer cell proliferation and invasiveness, as well as Wnt signalling and expression of downstream mediators, including β‐catenin, cyclin D1 and MMP7 (P < 0.05). In contrast, the Zbed3 mutant failed to enhance β‐catenin expression (P > 0.05), and its ability to promote cancer cell proliferation and invasiveness was much less than wild‐type Zbed3 (P < 0.05). The ability of Zbed3 to increase β‐catenin levels was abolished by Axin knockdown in A549 cells (P > 0.05). Similarly, treating the cells with a GSK‐3β inhibitor abolished Zbed3's ability to increase β‐catenin levels and Wnt signalling. These results indicate that Zbed3 enhances lung cancer cell proliferation and invasiveness at least in part by inhibiting Axin/adenomatous polyposis coli/GSK‐3β‐mediated negative regulation of β‐catenin levels.     

Bos indicus introgression into (peri‐)alpine cattle breeds – evidence from the analysis of bovine whey protein variants

The major bovine whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG), exhibit breed‐specific genetic variation. The aim of this study was to identify possible new protein variants and determine the distribution of variants across a variety of 18 taurine and indicine cattle breeds applying a DNA‐based sequencing approach. To this end, the open reading frames of the respective genes (LALBA and LGB) were sequenced in 476 animals. Within the LALBA gene, a previously unknown synonymous and a previously undesignated non‐synonymous nucleotide exchange were identified. Furthermore, two known α‐LA variants (A and B) and four known β‐LG variants (A, B, C and W) were determined. The occurrence of typical indicine variants in some taurine cattle breeds, such as Suisse Eringer, German Hinterwälder and Hungarian Grey Steppe, further supports the hypothesis of ancient Bos indicus introgression into (peri‐)alpine cattle breeds.     

Lithium induces follicular atresia in rat ovary through a GSK‐3β/β‐catenin dependent mechanism

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23‐day‐old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl‐treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17β‐estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)‐positive cells and conversely increased the number of TUNEL‐positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase‐3β, and unexpectedly decreased the expression of active (stabilized) β‐catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized β‐catenin and 17β‐estradiol synthesis. Mol. Reprod. Dev. 80: 286–296, 2013. © 2013 Wiley Periodicals, Inc.     

Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.