Extrinsic sialylation by extracellular ST6Gal-1 augmented BAFF-mediated activation of the non-canonical NF-kB, p38 MAPK, and PI3K/AKT pathways, and accelerated tyrosine phosphorylation after B cell receptor stimulation. in vivo, systemic ST6Gal-1 did not influence homing of B cells to the spleen but was critical for their long-term survival and systemic IgG levels. Circulatory ST6Gal-1 levels respond to inflammation, infection, and malignancy in mammals, including humans. In turn, we have shown previously that systemic ST6Gal-1 regulates inflammatory cell production by modifying bone marrow myeloid progenitors. Our data here point to an additional role of systemic ST6Gal-1 in guiding B cell development, which supports the concept that circulating ST6Gal-1 is a conveyor of systemic cues to guide the development of multiple branches of immune cells.Humoral immunity depends on intrinsic B cell developmental programs guided by systemic signals that convey physiologic needs. Aberrant cues or their improper interpretation can lead to immune insufficiency or a failure of tolerance and autoimmunity. The means by which such systemic signals are conveyed remain poorly understood. Hence, further insight is essential to understanding and treating autoimmune diseases and to the development of improved vaccines. ST6Gal-1 is a sialyltransferase that constructs the α2,6-sialyl linkage on cell surface and extracellular glycans. The requirement for functional ST6Gal-1 in the development of humoral immunity is well documented. Canonically, ST6Gal-1 resides within the intracellular ER-Golgi secretory apparatus and participates in cell-autonomous glycosylation. However, a significant pool of extracellular ST6Gal-1 exists in circulation. Here, we segregate the contributions of B cell intrinsic and extrinsic ST6Gal-1 to B cell development. We observed that B cell-intrinsic ST6Gal-1 is required for marginal zone B cell development, while B cell non-autonomous ST6Gal-1 modulates B cell development and survival at the early transitional stages of the marrow and spleen. Exposure to extracellular ST6Gal-1  The humoral immune system is central to the successful management of infectious insults, through resolving established infections and generating long-term protection against future exposures. Although humoral insufficiency puts the host at risk, dysregulation of normal B cell function and development underlies autoimmune conditions such as lupus and rheumatoid arthritis –3. The bH1 and TH2 stimuli  facility. The reference wild-type strain used was C57BL/6J from JAX. μMT mice (JAX) and μMT/ST6KO mice, generated by crossing single knockout strains, were used in adoptive transfer experiments. When specifically stated, the CD45.1 expressing strain, B6.SJL-Ptprca Pepcb/BoyJ, was used in order to distinguish donor cells from C57BL/6J, which expresses the CD45.2 allele of the Ptprc locus. For transplantations, mice received 6 Gy whole body gamma-radiation and were rescued with 4.0 × 106 whole bone marrow cells. Mice were euthanized after 6 weeks for analysis. For B cell migration assays, splenocytes from 6 day-old wild-type mice were stained in 5 uM CFSE before intravenous transfer to recipients. For B cell survival assays, CD3-/B220+ splenocytes were intravenously transferred into recipients. Unless otherwise indicated, mice between 7 and 10 weeks of age were used, and both sexes were equally represented. Roswell Park Institute of Animal Care and Use Committee (IACUC) approved maintenance of animals and all procedures used under protocol 1071M.The For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend. For magnetic cell separation, biotinylated anti-IgM (RMM-1), anti-B220 (RA3-6B2), anti-Gr1 (RB6-8C5), anti-CD23 (B3B4), and anti-CD3e antibody (145-2C11) were purchased from BD Pharmingen. For microscopy, anti-α-smooth muscle actin , anti-IgM-Cy3 (EMD Millipore), anti-IgD-FITC , anti-MARCO-FITC (Bio-rad), and anti-B220  were used. For western blot, anti-NFkB2, anti-p-p38 (T180/Y182), anti-pAkt (S473) from Cell Signaling Technology, anti-β-actin (Invitrogen), anti-BAFFR and anti-ST6Gal-1 (R&D Biosystems), and anti-pTyr (EMD Millipore) were used.low/IgMlow), IgM-hi (B220+/IgMhi), and bone marrow mature (BMM: B220hi/IgMlow−mid); splenic IgD-/CD21-(B220+/CD19+/IgD−/IgM+/CD21−), IgD+/CD21+ (B220+/CD19+/IgD+/IgM+/CD21+), marginal zone (MZ: B220+/CD19+/IgD−/IgM+/CD21+), follicular (FO: B220+/CD19+/IgD+/IgMlow/mid/CD21mid) B cells, and splenic plasma cells (SLPC: B220−/CD138+). Gating schemes are shown in Supplementary Figure The parameters for flow cytometry visualization of B lineage populations were as follows. Bone marrow immature  and RNA extracted according to manufacturer's instructions. 1.5 μg of RNA was converted to cDNA , then amplified by qPCR  with intron-spanning primers. Primer sequences are as follows: B2M: F- 5′-CTGACCGGCCTGTATGCTAT-3′; R- 5′-TTCCCGTTCTTCAGCATTTGGAT-3′, ST6GAL1: F- 5′-CTTGGCCTCCAGACCTAGTAAAGT-3′; R- 5′-TCCCTTTCTTCCACACGCAGATGA-3′. Expression for St6gal1 was normalized to B2-microglobulin.Frozen spleens were sectioned onto glass slides. Slides were acetone fixed, rehydrated in PBS, then blocked in 5% BSA solution, followed by staining with antibodies per manufacturer's guidelines. Fluorescence was visualized immediately using a Nikon Eclipse E600 microscope with EXFO X-cite 120 light source. Spot RT3 camera and Spot Software were used to capture images.5 cells/ml as indicated in presence of 10 μg/ml ST6Gal-1, 0.05 mM CMP-sialic acid, 20 ng/ml BAFF, 10 μg/ml anti-IgM antibody, 1000 U/ml IL-4, and function-grade anti-CD40 antibody (eBioscience HM40-3) for 18–20 h. Live cells were quantified by DAPI flow cytometry. Recombinant rat secretory ST6Gal-1 was a generous gift from Dr. Kelley Moremen of the University of Georgia.Bone marrow from wild-type mice was depleted for IgM and Gr-1, then enriched for B220 by MACS columns (Miltenyi Biotechnology) for immature B cells (96% purity). Where indicated, B220+ IgM-low cells were cultured in RPMI with 10% non-mitogenic FBS and penicillin/streptomycin for 40 h. For B cell receptor (BCR) stimulation, CD23+ (rather than IgM+) cells were negatively selected to obtain immature and transitional B cells (~80% purity). For cell activation experiments, B cells were extrinsically sialylated with 40 μg/ml ST6Gal-1 and 0.05 mM CMP-sialic acid (Sigma C-8271) in serum-free RPMI for 2 h, then stimulated with 200 ng/ml murine BAFF (R&D Biosystems) or 10 μg/ml function-grade anti-IgM F(ab')2 (Invitrogen 16-5092-85). To model negative selection, cells were cultured at 1 × 10For western blots, indicated cells were lysed in NP-40 lysis buffer with protease and phosphatase inhibitors and immediately snap-frozen. Lysates were separated on 10% SDS-PAGE gels, transferred to PVDF membranes, and probed with primary antibodies overnight and secondary antibodies for 1 h. Membranes were developed using Pierce ECL WB Substrate (Thermo Scientific) and imaged using ChemiDoc Touch (Bio-rad). Where indicated, band intensity was quantified with ImageLab software. For immunoprecipitation, B cell membrane proteins were isolated using MEM-PER Plus kit (Thermo Scientific), then incubated with blocked SNA-agarose beads (Vector Laboratories) overnight. Beads were extensively washed and immunoprecipitate eluted by boiling in denaturing and reducing conditions, before western blot analysis. Uncropped Western blot images are included in Supplementary Figure Detection of serum immunoglobulin G was achieved by ELISA (Bethyl Laboratories) according to manufacturer's protocols. Autoantigen-specific IgG was detected by direct ELISA against salmon sperm DNA, calf thymus histone, recombinant TPO (Cloud-Clone Corp.), or recombinant MPO (R&D Biosystems). Serum from the Ets-1 KO autoimmune mouse model was used as positive control . Calf tht test in Prism 7 software (Graph Pad). P < 0.05 is considered statistically significant.In all graphs, data is presented as mean ± SD of a single experiment. Differences between mean values were determined by ANOVA or Student's St6gal1-KO mice that have been backcrossed for 15 generations onto the C57BL/6J background. First, we examined how inactivation of ST6Gal-1, resulting in inability to α2,6-sialylate N-glycans, perturbs major B-lineage cell populations in the bone marrow and spleen. Bone marrow B220+ cells were subdivided into B220-low immature fraction, B220-high mature fraction, and an B220-variable/IgM-high fraction, defined elsewhere as transitional  animals. However, the St6gal1-KO marrow contains enlarged immature (IM) and diminished IgM-high populations  and s Figure  34). Ab. AbSt6gas Figure  36). By. BySt6gaSt6gal1-KO animals in overall organ size or total number of splenocytes. However, the St6gal1-KO spleen had reduced IgD+/CD21+ and marginal zone (MZ) B cells, and an expanded follicular (FO) B cell population  cells with MARCO (in green), a marker of marginal zone macrophages  is a pro-survival mitogenic receptor induced by B cell activating factor (BAFF)  , 40. Furr (BAFF) .Sambucus nigra agglutinin), only rarely agreed with endogenous ST6Gal-1 expression in stage-by-stage comparisons , IgM-high, and mature B cells (BMM) of the marrow, and the IgD-/CD21-, IgD+/CD21+, marginal zone (MZ), and follicular (FO) B splenocytes Figure . In the s Figure . IgD+/CDs Figure . Most poTaken together, the data indicate not only the need for ST6Gal-1 mediated sialylation during B lymphopoiesis, but also suggests that stage-specific regulation of endogenously expressed ST6Gal-1 cannot fully account for the cognate α2,6-sialyl ligands in B cell development.c region of circulating IgG  led to a diminished IgD-/CD21- compartment. This reduction, dependent on host expression of ST6Gal-1, was quantitatively significant in comparisons of male, but not female mice  mice that either express or lack ST6Gal-1. The donor splenocytes, from post-natal day 6 wild-type mice, lacked B cells beyond the IgM-high transitional stages  were obtained by magnetic cell sorting and cultured for 40 hrs with ST6Gal-1 or vehicle control. Control cultures increased CD23 (FcεRII) and B220 expression, and downregulated CD24, as expected during development  for 2 h, then stimulated with murine BAFF. rST6G exposure resulted in increased degradation of cytosolic p100, a mediator of non-canonical NF-kB signaling, and robust p38 phosphorylation. There was also a modest elevation of AKT phosphorylation by rST6G , prolactin, and Toll-like receptor 7 (TLR7) agonists, augment the development and survival of early transitional B cells –44. BAFFG Figure . Each ofG Figure –49. ST6GG Figure . TherefoG Figure . Furtherin vitro, and observed that the rST6G-treated B cells exhibited increased tyrosine phosphorylation as soon as 1 min after stimulation (Figure Tonic, low-level B cell receptor activation is obligatory for BAFFR signaling . Moreoven Figure .As targets of negative selection, early transitional B cells are central to the development of autoimmunity . Since wex vivo studies to show that contextual differences in the origin of ST6Gal-1 are a significant determinant of B cell development and function.The inability to generate α2,6-sialyl glycans due to deficiency of the sialyltransferase ST6Gal-1 results in a plethora of humoral defects, including an attenuated response to antigenic challenges, impaired B cell proliferation, and reduced serum IgM . Howevercis-interacting CD22 ligands. In contrast, antigen presentation by ST6Gal-1 expressing non-B cells can recruit CD22 and Siglec-10/G to the immunological synapse by trans interactions, suppressing BCR signaling and enforcing apoptosis (The humoral defects in ST6Gal-1 deficient animals are attributed to disrupted engagement and signaling of CD22, and potentially Siglec-10/G . Both CDpoptosis –58. Thispoptosis , safeguapoptosis .St6gal1-KO and wild-type mice. Our data show an absolute requirement for B cell-autonomous ST6Gal-1 in marginal zone B cell lineage development; St6gal1-KO precursors, regardless of recipient ST6Gal-1 status, were unable to re-establish splenic late transitional and marginal zone B cell compartments (see Figure St6gal1-KO recipients, a population subject to negative selection by antigenic engagement, highlighting a role for B cell non-autonomous ST6Gal-1 in B lymphopoiesis at the early transitional stage (see Figure St6gal1-KO spleens (see Figure The differential contributions of B cell-autonomous and non-B cell-autonomous ST6Gal-1 were examined using bone marrow chimeras between St6gal1-KO hematopoietic cells, incapable of autonomously synthesizing α2,6-sialyl structures, nevertheless gained considerable α2,6-sialic acid in the B lineage when transplanted into wild-type recipients (see Figure in vitro using recombinant ST6Gal-1. When added to isolated immature B220+ marrow cells, rST6G sialylated immature and transitional subsets, enhanced cell surface IgM and CD23 expression (see Figure B cell non-autonomous ST6Gal-1 can influence B cell development through the intrinsic generation of glycans on antigen-presenting cells or extrinsic remodeling of B cell surface glycans by the extracellular ST6Gal-1 pool. Extracellular ST6Gal-1, dynamically upregulated by the liver in response to inflammation, can systemically modulate glycan-dependent processes by generating α2,6-sialic acid on both central and peripheral immune cells , 25, 26.Transitional B cells represent a heterogeneous population of immature B cells in the process of acquiring the hallmarks of maturity. The molecular and cellular requirements enabling this bridging population to survive as it migrates into splenic follicles and attains functional maturity are useful biological chokepoints in the enforcement of self-tolerance. The mechanisms maintaining tolerance include cell death by negative selection, clonal deletion by receptor editing, and anergy . The minin vivo differences in the IgD-/CD21- population upon loss of host ST6Gal-1, consistent with reported sex-associated variation in serum BAFF and the emerging role of sex hormones in the regulation of BAFF expression (St6gal1-KO mice, as well as in B cells treated with rST6G ex vivo, also suggest a BAFF-dependent mechanism (Early transitional B cells, the physiologic targets of negative selection, are a labile population with the potential to harbor autoreactive clones. In addition to intrinsic developmental cues, niche-based extrinsic factors are already known to influence B cell development and negative selection . One majpression , 70, 71.pression , 73. Altechanism . Finallyechanism . Serum Bechanism –76. The echanism . BAFFR, echanism . In our Taken together, our data outline different roles for the ST6Gal-1 natively expressed in B-lineage cells, non-B lineage cells, and the pool of systemic ST6Gal-1. In view of the newer understanding of the biochemical feasibility and biological importance of the pool of extracellular ST6Gal-1, our observations define a role for circulatory ST6Gal-1 as a systemic extrinsic factor guiding the development and maintenance of humoral immunity, with therapeutic potential as a vaccine adjuvant.EI conceived, designed and performed experiments, and wrote manuscript. JL provided critical oversight, conceived and designed research, and wrote manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P = 0.024) and tumor number (P = 0.019). Genetic depletion of DKK1 impaired the proliferation, colony-forming ability, invasion, and tumor formation of HCC cells (HepG2 and HUH-7). Conversely, forced expression of DKK1 increased the proliferation, colony-forming ability, and invasion of HepG2 and HUH-7 cells in vitro and enhanced tumor formation in vivo. Subsequent investigation revealed that the DKK1-mediated proliferation and tumorigenicity of HepG2 and HUH-7 cells is dependent on the Wnt/β-catenin signaling pathway. These findings indicate that DKK1 plays an oncogenic role in HCC by activating the Wnt/β-catenin signaling pathway.Dysregulation of dickkopf-related protein 1 (DKK1) expression has been reported in a variety of human cancers. We previously reported that DKK1 was upregulated in hepatocellular carcinoma (HCC). However, the role of DKK1 in HCC remains unclear. This study aimed to investigate the clinical significance and biological functions of DKK1 in HCC. The expression of DKK1 was examined in cirrhotic and HCC tissues by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). DKK1 was silenced or overexpressed in HCC cell lines, and in vitro and in vivo studies were performed. Immunohistochemistry revealed that DKK1 was weakly expressed in cirrhotic tissues  but upregulated in HCC tissues . Significant upregulation of DKK1 was observed in 57.6%  of HCC tissues by qRT-PCR, and the expression of DKK1 was associated with tumor size ( Molecules that might be used as diagnostic/prognostic biomarkers or that inhibit HCC progression must be identified.3Hepatocellular carcinoma (HCC) is the most frequent cause of cancer-related death worldwide.6 Accumulating evidence supports the involvement of DKK1 in human cancers. However, the role of DKK1 in tumor biology remains unclear.7 The expression of DKK1 was reported to be downregulated in colorectal carcinoma and lung cancer cells.10 Accumulation of Musashi RNA-binding protein 1 (MRP-1) in HCC cells significantly promotes proliferation, tumor formation, and cell cycle progression via silencing of DKK1.11 In contrast, upregulation of DKK1 has been found in a variety of human cancers, such as cholangiocarcinoma and pancreatic, esophageal, breast and kidney cancers.17 In the liver, the expression of DKK1 is downregulated in human HCC, and upregulation of DKK1 in HCC was able to inhibit the invasion of HCC cells.18 However, Chen and his colleagues19 reported that DKK1 enhances HCC cell invasion via the MMP-7 signaling pathway. Overexpression of DKK1 is associated with a poor prognosis in human HCC.20 In addition, DKK1 can be used as a serum biomarker for the diagnosis of HCC.22 It has been reported that HCC develops with increased frequency in hepatitis B virus surface antigen (HBsAg) transgenic mice.23 We previously reported that DKK1 was upregulated in HBsAg mouse liver tissues and in human HCC.24 Ultimately, the specific role of DKK1 in HCC remains controversial. In this study, the expression of DKK1 was examined in cirrhotic and HCC tissues by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The biological function of DKK1 was investigated in HCC cell lines (HepG2 and HUH-7) using proliferation, invasion, cell cycle, colony formation, and xenograft tumor formation assays.Dickkopf-related protein 1 (DKK1) is a secreted protein that has been clearly identified as a direct inhibitor of Wnt/β-catenin signaling via the LRP5/6 coreceptor of Frizzled proteins.Patient characteristics, immunohistochemistry, cell line establishment and cell culture, quantitative RT-PCR, western blot analysis, cell proliferation analyses, cell colony analyses, cell cycle analyses, cell invasion analyses, and subcutaneous xenograft assays in nude mice were performed as described in the Supplementary Materials and Methods.Enzyme-linked immunosorbent assay (ELISA) was performed to investigate the expression of DKK1 in the supernatant of HCC cells with a Human DKK1 ELISA Kit . Briefly, a 96-well plate was precoated with anti-human DKK1 antibodies, and supernatant samples were diluted with sample dilution buffer and added to the wells. The plate was incubated for 90 min at 37 °C. After DKK1 was bound to the wells by the immobilized antibody, the liquid was removed from each well, and biotinylated anti-human DKK1 antibody was immediately added to the wells and incubated at 37 °C for 1 h. After washing three times, HRP-conjugated streptavidin was added after removal of unbound biotinylated antibody. The wells were washed again, and TMB substrate solution was added. Finally, the absorbance was measured at 450 nm by using an ELISA plate reader. According to the standard curve generated by serial dilution of recombinant human DKK1, the level of soluble DKK1 in each supernatant sample was calculated.NotI digestion and inserted into a DKK1 retroviral vector (pCMGV/DKK1). HepG2 and HUH-7 cells were infected with the pGHGV/shRNA-DKK1, pGHGV/shRNA-β-catenin, pCMGV/DKK1, or pCMGV vectors after 10 population doublings, as previously described.25 After puromycin selection, loss or gain of DKK1 or β-catenin were confirmed by qRT-PCR and western blot analysis, and stable cell lines were established.Lentiviral vectors  expressing shRNA targeting human DKK1 or β-catenin or a negative control shRNA . Primary antibodies specific for DKK1 and β-catenin in DSP were added simultaneously for 2 h at room temperature. After washing three times with PBS, secondary antibodies in DSP were added for 2 h at room temperature. Coverslips were washed again three times with PBS and were then mounted on slides with DAPI Slow Fade. Representative images were acquired on a Leica TCS SP5 confocal microscope with an oil immersion objective at ×60magnification. Representative images are shown as enhanced using ImageJ software. The primary antibodies and secondary antibodies are listed in Supplementary Table 4 cells/well in 48-well plates and transfected with pGL3-blast or pGL3-LEF/TCF  using LipofectamineTM 3000 according to the manufacturer’s protocol. Luciferase activity was assessed using a Dual-Luciferase Reporter Assay system  according to the manufacturer’s instructions. The primer sequences used for amplification of TCF/LEF are shown in Supplementary Table HepG2 and HUH-7 cells were plated at a density of 2 × 10t-test, and in the in vivo data using the Mann–Whitney U-test. In all cases, P values of <0.05 were considered statistically significant.All statistical analyses were performed using SPSS for Windows . All experiments with cultured cells were carried out independently at least three times in experimental triplicate. All data are expressed as the means ± standard deviations (SDs) unless otherwise indicated. We determined the significance of differences in the human HCC data using Fisher’s exact test, in the in vitro data using Student’s Immunohistochemistry revealed positive staining for the DKK1 protein in the cytoplasm of tumor cells. In general, DKK1 was weakly expressed  in 22 human cirrhotic tissues Fig. . In contP = 0.024) and tumor number (P = 0.019). However, DKK1 expression was not correlated with age (P = 0.116), sex (P = 0.142), capsular invasion (P = 0.292), portal vein tumor thrombi (P = 1.000), bile duct tumor thrombi (P = 0.424), lymphatic metastasis (P = 1.000), tumor stage (P = 0.238), histological grade (P = 0.531), serum alpha fetoprotein level (P = 1.000), serum CA19-9 level (P = 0.698), serum CA125 level (P = 0.707), or extrahepatic metastasis (P = 0.620).As shown in Table qRT-PCR and western blotting were performed to analyze the expression levels of DKK1 in DKK1-short hairpin RNA (shRNA) HCC cells (HepG2 and HUH-7). As shown in Fig. To investigate the effects of DKK1 on HCC cells, pCMGV/DKK1-derived retroviruses were used to transduce HepG2 and HUH-7 cells to establish HCC cell lines with stable forced expression of DKK1. The qRT-PCR and western blot analysis results revealed that DKK1 expression was effectively and functionally induced in HepG2 and HUH-7 cells compared with control cells Fig. . In addiBecause the in vitro studies suggested that DKK1 plays a regulatory role in HepG2 and HUH-7 cell proliferation and invasion, the biological significance of these results was further evaluated in an in vivo model of HCC. To this end, a xenograft tumor formation assay was performed to investigate the effect of DKK1. As shown in Fig. 7 Increased expression of DKK1 is associated with cytoplasmic/nuclear β-catenin accumulation in HCC patients.20 Therefore, we investigated whether DKK1 can activate the Wnt/β-catenin signaling pathway. Fluorescently labeled anti-DKK1 and anti-β-catenin antibodies were used for confocal microscopy analysis. Confocal microscopy revealed that forced expression of DKK1 in HepG2 and HUH-7 cells increased the nuclear expression of β-catenin  remains unclear. To investigate the role of β-catenin, β-catenin-shRNA lentiviral vectors were used to deplete β-catenin expression in DKK1High HepG2 and HUH-7 cells and establish stable β-cateninLowDKK1High HepG2 and HUH-7 cell lines  compared with DKK1-DKK1High HepG2 and HUH-7 cells (green arrow)  Fig.  in vivo.24 However, the specific role of DKK1 in HCC remains to be ascertained. It has been reported that DKK1 promotes HCC cell invasion and that increased expression of DKK1 is associated with poor prognosis in HCC patients.20 These data suggest that DKK1 may be involved in the progression of HCC. Herein, we demonstrated that DKK1 is upregulated in HCC tissues and is associated with larger tumor sizes and multiple tumors in patients with HCC. These findings indicate that DKK1 may participate in the progression of HCC. Additionally, depletion of DKK1 in HCC cells impaired cell proliferation, colony formation, cell cycle progression, and cell invasion abilities in vitro. In contrast, forced expression of DKK1 dramatically enhanced the proliferation, colony-forming ability, cell cycle progression, and invasion of HCC cells. Taken together, these data suggest that DKK1 plays a vital role in the proliferation and invasion of HCC cells in vitro.We previously reported that DKK1 was overexpressed in HCC.31 In addition, DKK1 has been shown to be activated during the progression of multiple tumors, including HCCs.7 Moreover, Yu et al.20 reported that increased expression of DKK1 is associated with cytoplasmic/nuclear β-catenin accumulation in HCC patients. It seems likely that DKK1 is involved in the regulation of Wnt/β-catenin in HCC cells. Here, we found that the expression levels of WNT1 and β-catenin were dramatically increased in HepG2 and HUH-7 cells with forced expression of DKK1. Knockdown of DKK1 in HCC cells by DKK1-shRNA dramatically impaired the protein expression of WNT1 and β-catenin. Moreover, deletion of DKK1 decreased TCF/LEF activity. Most importantly, the findings from confocal microscopy and western blot analysis also revealed that forced expression of DKK1 promotes β-catenin nuclear translocation in HepG2 and HUH-7 cells. Taken together, these data suggest that the Wnt/β-catenin signaling pathway is activated by DKK1 in HCC cells in vitro. WNT1 and β-catenin may be important downstream targets of DKK1.The function of DKK1 in HCC tumor formation, has not been examined to date. Here, the results of the in vivo assays provide the first evidence that implantation of HepG2 and HUH-7 cells with forced expression of DKK1 into nude mice results in enhanced tumor formation, indicating that DKK1 plays a significant role in the tumorigenicity of HCC cells in vivo. Collectively, these results indicate that DKK1 functions as a tumor oncogene in HCC. In the present study, the possible mechanism by which DKK1 mediates proliferation and tumor formation in HCC was investigated. WNT-induced signaling, which is frequently involved in cancer progression, can be suppressed by DKK1.32 It has been reported that β-catenin translocates from the cytoplasm to the nucleus and is essential for tumorigenesis.34 However, whether DKK1 is involved in the regulation of β-catenin in HCC cells is unclear. Thus, β-catenin-shRNA lentiviral vectors were used to deplete β-catenin expression in HepG2 and HUH-7 cells with forced expression of DKK1. We found that knockdown of β-catenin markedly inhibited the in vitro proliferation and in vivo tumorigenicity of HCC cells with forced expression of DKK1 compared with the corresponding control cells. These findings imply that DKK1 may enhance the proliferation and tumorigenicity of HCC cells through β-catenin.β-Catenin is the key functional protein in the Wnt/β-catenin signaling pathway.In conclusion, DKK1 contributes to HCC tumorigenesis by activating the Wnt/β-catenin signaling pathway, thus providing us with a potential therapeutic option for HCC in clinical practice.Supplementary date


Diabetic nephropathy (DN) is one of the principal complications of diabetes and podocyte injury plays an important role in the DN pathogenesis. Wnt/β-catenin signaling overactivation confers podocyte injury and promotes multiple types of renal disease. However, the underlying mechanism of Wnt/β-catenin signaling activation in DN progression has not been fully elucidated. Long noncoding RNA (lncRNA) is a large class of endogenous RNA molecules lacking functional code capacity and which participates in the pathogenesis of human disease, including DN.A diabetes model was constructed by intraperitoneal injection of Streptozotocin in rats. The MPC5 cells were used to create the in vitro model. Western blot and Quantitative reverse-transcriptase-PCR were used to examine the expression of protein and mRNA. The migrated capacity was analyzed by Transwell migration assay. The cell viability was detected by CCK8.In the present study, we revealed the association of lncRNA Maternally Expressed Gene 3 (MEG3) with aberrant activation of Wnt/β-catenin signaling and the role of MEG3/Wnt axis in podocyte injury. We found that high glucose (HG) treatment suppressed MEG3 expression in cultured podocytes, activated Wnt/β-catenin signaling and caused podocyte injury as indicated by the downregulation of podocyte-specific markers (podocin and synaptopodin) and the upregulation of snail1 and α-smooth muscle actin. Overexpression of MEG3 attenuated HG-induced podocyte injury by reducing Wnt/β-catenin activity, repressing cell migration, reactive oxygen species production and increasing the viability of podocytes. Furthermore, we provided evidences that restoration of Wnt/β-catenin signaling by specific agonist impeded the protective effect of MEG3 on podocyte injury. Current results demonstrated that MEG3/Wnt axis plays an important role in fostering podocyte injury and may serve as a potential therapeutic target for the treatment of DN.lncRNA MEG3 ameliorates podocyte injury in DN via inactivating Wnt/β-catenin signaling. Diabetic nephropathy (DN), the primary cause of end-stage kidney diseases, is a major complication of diabetes mellitus (DM) . PodocytIt is well-known that the Wnt/β-catenin signaling plays an important role in homeostasis, organogenesis and human diseases . In patiLong noncoding RNAs (lncRNAs) are a class of non-protein coding transcripts (more than 200 nt) which have been demonstrated widely as vital regulators in gene expression, cellular function and disease processes . MountinAll animal experiments were undertaken at Shanghai Jiaotong University School of Medicine and approved by the Animal Ethics Committee of Shanghai Jiaotong University School of Medicine (SYKX-2008-0050). Male Sprague Dawley rats (6–8 weeks of age) were purchased from the SLAC Laboratory Animal Co. Ltd.  and kept in a specific-pathogen-free environment. Diabetes were induced by intraperitoneal injection of STZ (70 mg/kg B.W. in a 0.1 M citrate buffer)  along with only citrate buffer in the control. The blood glucose level was then examined until it is above 16.7 mmol/L as hyperglycemia was determined.2. In order to carry out MEG3 overexpression, plasmid pcDNA3-MEG3 was constructed by introducing the full-length MEG3 into the pcDNA3.1 as our previously described , was cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FBS (Gibco). Cells were maintained in a humidified incubator at 37 °C with 5% COescribed . pcDNA3-The TurboFect in vivo transfection reagent  was used as an in vivo delivery vehicle for pcDNA3-MEG3 to prevent degradation and enhance transfection efficiency as described previously .The RNA expression level of lncRNA and mRNA was assessed using Quantitative reverse-transcriptase-PCR (qPCR). Total RNA of cells or tissues was extracted from using Trizol reagent . The high capacity cDNA Reverse Transcription Kit  was used for reverse transcription. The SYBR™ Green PCR Master Mix  was used for qPCR amplification on StepOne Plus real-time PCR system . All qPCRs were carried out in triplicate for each sample and β-actin was used as endogenous control for mRNAs and lncRNA.2 and allowed to migrate for 24 h. The cells that had migrated were fixed with 4% formaldehyde and stained with 0.1% crystal violet and captured with a microscope . The experiments were performed in triple set and results were obtained at four randomized visual fields.The migrated capacity of MPC5 cells was analyzed using Transwell migration assay. For transwell migration assay, the 24-well plate transwell system  were used. The filter in the upper chamber was incubated for 2 h at 37 °C. Then, MPC5 cells were plates  in the upper chamber with serum-free DMEM media and DMEM media with 10% FBS was added to the bottom chamber. Cells were incubated at 37 °C with 5% COThe protein expression of β-catenin, snail1 and synaptopodin in tissues was assayed using Immunohistochemistry (IHC). Renal tissues were fixed with 10% formalin and embedded in paraffin and the embedded samples were cut into 5 μm sections and mounted on slides. After antigen retrieval, the sections were incubated with primary antibodies for anti-β-catenin  or anti-snail1  or anti-synaptopodin  at 4 °C overnight. After washing three times with PBS, the sections were then incubated with the secondary antibody . β-catenin, snail1 and synaptopodin expression were visualized using DAB staining.The protein expression of β-catenin, snail1, a-SMA and podocin in cells or tissues was assayed using western blot analysis. Total protein of MPC5 cells or tissues was extracted in RIPA lysis buffer and then quantified by BCA assay . A total of 50 μg protein was separated by 10% SDS-PAGE and then electro-transferred to polyvinylidene difloride membrane. The membrane was then blocked with TBST containing 5% nonfat dry milk at 4 °C overnight. β-catenin , snail1 , α-smooth muscle actin (α-SMA) , and podocin  were used as the primary antibodies. After washing three times with TBST, the membrane was incubated with appropriate secondary antibodies . Protein bands were visualized using the ECL chemiluminescence system . β-actin was set as the internal control.3 cells/well) were plated into 96-well plates and cultured. A total of 20 µL of CCK-8 liquid was added at the indicated time. Following incubation for 2 h at 37 °C, the optical density was detected at 450 nm through a microplate reader (Bio-Rad Model 550).Cell Counting Kit-8  was carried out to determine the viability of MPC5 cells. In brief, MPC5 cells transfected with pcDNA3-MEG3 (3 × 10Intracellular reactive oxygen species (ROS) generation in MPC5 cells was assayed using ELISA kit  according to the manufacturer’s protocol. In brief, cells were plated in a 96-well plate and cultured with their respective treatments for 24 h. Culture medium was removed and plates were washed thrice at 24 h following culture. Fluorescence was measured by a Victor3 1420 Multilabel Counter .t-test, or one-way analysis of variance followed by the Scheffé test. A value of P < 0.05 was considered statistically significant.The SPSS 16.0 statistical analysis software  was used for data analysis. The results were presented as mean ± standard deviation from three or more independent experiments. The difference between two groups was compared using two-tailed student’s To investigate the possible role of MEG3 in HG-induced podocyte injury, we firstly assessed the level of MEG3 in renal tissues of DN patients and HG (30 mM)-treated podocytes using qPCR analysis. As shown in We then investigated the role of MEG3 in podocyte injury during HG treatment. pcDNA3-MEG3 was constructed and transfected into podocytes to overexpress MEG3. We then investigated the role of MEG3 in regulating renal function in diabetic rats. Rat models of DM were established by intraperitoneal injection of STZ and pcDNA3-MEG3 was administered to diabetic rats and the blood glucose level remained above 16.7 mmol/L after MEG3 overexpression in vivo. MEG3 inhibits podocyte injury and suppresses Wnt/β-catenin signaling activation in DN. Given the important role of Wnt/β-catenin signaling in the pathogenesis of podocyte injury, we thus speculated whether MEG3 protects against podocyte injury by inactivating Wnt/β-catenin. To prove it, podocytes were treated with HG in the presence or absence of MEG3 overexpression and the activation of Wnt/β-catenin signaling was assessed in vitro. The TOPFlash/FOPFlash luciferase reporter system was first used to directly assess the activation of β-catenin. The luciferase reporter map is listed in Finally, we investigated whether MEG3 protected against podocyte injury by inactivating Wnt/β-catenin signaling. As expected, western blot analysis showed that MEG3 upregulated the expression of podocin in the presence of HG, whereas Wnt activation induced by SKL2001 partially destroyed the protective effect of MEG3 . More imIn the current study, the roles of MEG3 in protecting against podocyte injury and potential molecular mechanism were investigated. Our data demonstrate that (i) MEG3 expression was decreased after HG treatment in podocytes, (ii) MEG3 overexpression attenuated HG-induced podocyte injury, (iii) MEG3 contributed to improve renal function in diabetic rats, (iv) MEG3 protected against podocyte injury by inactivating Wnt/β-catenin signaling. These data identified the important role and underlying mechanism of MEG3 in inhibiting podocyte injury in DN and may provide a novel opportunity to the therapy of DN.As a frequent microvascular complication of DM, specific clinical hallmarks of DN have been revealed including the recession of glomerular filtration rate and the progressive urinary albumin excretion, eventually developing end-stage kidney diseases . PodocytOur previous studies identified MEG3 as a tumor suppressor gene in bladder cancer . As withThe interplay between MEG3 and Wnt/β-catenin signaling affects the onset of podocyte injury and renal dysfunction. Most importantly, MEG3 overexpression can interrupt the activation of Wnt/β-catenin signaling and restore the renal dysfunction with protection from podocyte injury. Therefore, our finding provides a new insight on potential drug target to treat podocyte injury and DN.10.7717/peerj.8016/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8016/supp-2Supplemental Information 2Click here for additional data file.


IKKβ transcription, reduced MDM2 stability, and ultimately removed the inhibitory effect of MDM2 on GADD45α induction. In addition, DAPK1 functioned as an upstream protein kinase triggering p53/Ets-1-dependent IKKβ and MDM2 reduction and GADD45α accumulation, thus promoting apoptosis in HepG2 cells. Subsequent studies further revealed that the activation of the DAPK1/p53/Ets-1/IKKβ/MDM2/GADD45α cascade was a common signaling event in mediating apoptosis of diverse cancer cells induced by arsenite and other tumor therapeutic agents. Therefore, we conclude that data in the current study have revealed a novel role for IKKβ in negatively regulating GADD45α protein stability and the contribution of p53-dependent IKKβ reduction to mediating cancer cell apoptosis.Our previous studies revealed that GADD45α is a liable protein, which undergoes MDM2-dependent constitutive ubiquitination and degradation in resting HepG2 hepatoma cells. Arsenite exposure induces ribosomal stress responses mediated by the ribosomal protein S7, which can block MDM2 activity and result in GADD45α accumulation and cell apoptosis. In the present study, we found that one of the catalytic subunits of IκB kinase (IKK), IKKβ, exerted a novel IKKα- and NF-κB-independent function in stabilizing MDM2 and therefore contributed to ubiquitination-dependent degradation of GADD45α in resting HepG2 cells. Arsenite stimulation induced transactivation of p53, which formed a complex with its downstream target, Ets-1, and then synergistically repressed  GADD (growth arrest and DNA-damage inducible) 45α exert multiple functions in diverse cellular stress responses, including cell cycle arrest, cell senescence, apoptosis, DNA-damage repair and epigenetic modifications , 2. The p53 is a transcriptional factor that can regulate the transcription of various downstream target genes and therefore plays multiple roles in growth arrest, DNA repair, senescence, autophagy, apoptosis, metabolism, development, and other processes . p53 actIKKα and IKKβ, which are catalytic subunits of the I-κB kinase (IKK) complex, cooperatively mediate the activation of the transcriptional factor NF-κB and therefore play multiple roles under various conditions. Despite structural similarity, the mechanisms underlying the actions of IKKα and IKKβ in NF-κB activation are quite different , 27. MorIKKβ, which resulted in GADD45α accumulation and cell apoptosis under the exposure of tumor therapeutic agents, including arsenite.In our previous reports, we demonstrated that both IKKα and IKKβ have the ability to mediate stress responses through NF-κB-independent mechanisms. Moreover, some specificity occurs between IKKα and IKKβ, because their substrates are exclusively regulated by one kinase but not the other , 31–33. We previously revealed that GADD45α protein undergoes a constitutive degradation via the ubiquitin-proteasome pathway in resting HepG2 human hepatoma cells, which can be blocked by arsenite treatment, and then the accumulation of GADD45α contributes greatly to arsenite-induced cytotoxicity . In the In the following study, we observed that overexpression of IKKβ in HepG2 cells attenuated cell death incidence, while knockdown of IKKβ expression increased the percentage of apoptotic cells in response to arsenite stimulation Fig. . These rGADD45α mRNA transcription did not change under either IKKβ overexpression or depletion conditions  shows large differences with the corresponding sequences in IKKα Fig. . TherefoMDM2 mRNA transcription, while interrupting the expression of endogenous IKKβ, but not IKKα, remarkably reduced the ectopic MDM2 levels. Consistently, degradation of HA-MDM2 was efficiently rescued by overexpression of IKKβ , upon arsenite exposure. Additionally, a strong association of Ets-1 with the chromatin regions containing two Ets-1-REs was readily observed , but was totally interrupted by proximal p53-RE1 deletion (M3 and M4) within the IKKβ promoter. However, the IKKβ promoter mutant containing only p53-RE1 but neither of the Ets-1-REs (M5) did not respond to arsenite stimulation, while the mutated IKKβ promoter containing p53-RE1 and Ets-1-RE2 (M7) showed a significant reduction in the luciferase activity similar to the wild type, which response was totally lost when Ets-1-RE2 was replaced by Ets-1-RE1 (M6). Taken together, p53-RE1 and Ets-1-RE2 function as the DNA responsive elements in mediating the synergistic effect of p53 and Ets-1 on the suppression of IKKβ transcription.To further confirm the above results, a luciferase assay was performed by using the constructed luciferase reporters driven by the wild type, deletion mutant, or point mutant of the IKKβ, evidenced by the inhibition of Ets-1 induction and rescue of IKKβ reduction with the impairment of DAPK1 expression  that might be responsible for triggering p53-dependent IKKβ repression induced by arsenite, we found the significant upregulation of DAPK1 expression Fig.  and actiion Fig. . Moreoveion Fig. . Additioion Fig. . These dIn the following study, we found that compared with the strong activation of the DAPK1/p53/Ets-1/IKKβ/MDM2/GADD45α pathway in arsenite-treated HepG2 cells, no signals indicating the activation of any of the components in this pathway were observed in either of the two normal human diploid hepatic cells, HL7702 and LO2, which are insensitive to arsentie exposure, as shown in our previous study Fig.  [5]. ThiThen, we observed the activation of the DAPK1/p53/Ets-1/IKKβ/MDM2/GADD45α pathway in a variety of other arsenite-sensitive cancer cell lines, including HeLa Fig. , MCF7, HNext, we found that the DAPK1/p53/Ets-1/IKKβ/MDM2/GADD45α cascade was also active in HepG2 cells treated with other tumor therapeutic agents, such as VP-16 and 5-Fu Fig. . Most imThe studies of disclosing the signaling events responsible for the induced expression of GADD45α have attracted wide attention due to the critical role of GADD45α in many important cellular functions . The conMDM2 mRNA transcription  RRRCWWGYYY . Any subtle differences in the sequence or spacing of the p53-binding sites at different genes could result in changes in the DNA secondary structure or in the topology of the chromatin, thereby altering the affinity of p53 for the target promoters [IKKα [IKKβ . Therefore, the difference between the affinity of p53 for the different architecture of p53-REs within the IKKα and IKKβ promoter may play a critical role in determining the selective transcriptional repression of IKKβ. For another reason, p53/Ets-1 complex formation and the binding of Ets-1 to Ets-1-RE2 within the IKKβ promoter were also shown to be required for p53 to deliver the transcriptional repression effect . The IKKβ promoter sequence containing three putative p53-responsive elements (p53-RE1/2/3) and two putative Ets-1-responsive elements (Ets-1-RE/2) (WT), deletion mutant of p53-RE3 (M1), deletion mutant of p53-RE3 and p53-RE2 (M2), deletion mutant of all three p53-REs (M3), deletion mutant of all three p53-REs and the distal Ets-1-RE2 (M4), the mutant only containing the proximal p53-RE1 (M5), the mutant containing p53-RE1 and the proximal Ets-1-RE1 (M6) and the mutant containing p53-RE1 and the distal Ets-1-RE2 (M7) were respectively obtained and then inserted into the pGL3 basic vector to construct the different IKKβ promoter-driven luciferase reporter plasmids.In addition to the plasmid containing wild type human TMRNAi MAX (Invitrogen) according to the manufacturer’s instructions.HepG2, HeLa, MCF7, A549, Hep3B, HCT116, HL7702, and LO2 cells were maintained in DMEM supplement with 10% fetal bovine serum, 1% penicillin and streptomycin. Mycoplasma contamination were tested and excluded. Cells were transfected using LipofectAMINE 2000 or LipofectAMINEIKKβ promoter-driven luciferase reporter plasmids were transiently transfected into HepG2 cells in combination of the internal control reporter. The stable transfectants and the HepG2 cells transiently transfected with IKKβ promoter-driven luciferase reporter plasmids were exposed to asenite for 36 h, and then the luciferase activity was tested as previously described [p53-Luc and NF-κB-Luc were transfected into HepG2 cells and then the stable transfectants were established. Wild type and the mutated escribed . TriplicIKKα, IKKβ, p53, ETS-1, MDM2, DAPK1, the specific primers (can be obtained if required) were designed to amplify the target cDNAs.Trizol reagent was used to extract total RNA and then RT-PCR was performed as previously described . To analIKKβ promoter. The sequences of the primers can be available by requesting.ChIP assay was performed as described in our previous report . ChIP prHepG2 cells were transfected with various combinations of the expression plasmids encoding wild type MDM2, IKKβ or their mutants. Then reciprocal immunoprecipitations (IPs) were performed to detect the binding domains involved in MDM2/IKKβ interaction. HepG2 cells stimulated with arsenite and then the complex formation for the endogenous MDM2/IKKβ, p53/Ets-1, DAPK1/p53/Ets-1 were determined by immunoprecipitation. Cellular protein preparation and immunoblot assays were performed as described previously .Myc-Ub and FLAG-GADD45α were transfected into HepG2 cells in the absence or presence of the constructs encoding IKKβ or of the specific siRNAs targeting IKKα or IKKβ. The ubiquitination of GADD45α was detected as described in our previous report .To detect the subcellular distribution of MDM2 and IKKβ, HepG2 cells with or without arsenite exposure were fixed and then incubated with the primary antibodies against MDM2 or IKKβ and the FITC or PE-conjugated secondary antibodies. The signal was monitored using the confocal microscopy .Arsenite-induced apoptosis in HepG2 cells was determined according to the percentage of propidium iodide (PI)-positive cells as described in our previous reprots , 6.P < 0.05.To determine the effect of a single treatment within a group, Student’s t-test was used to test the significance of the data. To determine the effects of treatment × group interactions, factorial design (ANOVA) was employed to test the significance of the data. At least three independent experiments were performed. The results were presented as the mean ± SD. The level of significance was set at supplemented data


NRF3 gene expression in cancer cells is highly elusive. We herein describe that NRF3 upregulation is induced by the β-catenin/TCF4 complex in colon cancer cells. We first confirmed high NRF3 mRNA expression in human colon cancer specimens. The genome database indicated that the human NRF3 gene possesses a species-conserved WRE sequence (TCF/LEF consensus element), implying that the β-catenin/TCF complex activates NRF3 expression in colon cancer. Consistently, we observed that the β-catenin/TCF4 complex mediates NRF3 expression by binding directly to the WRE site. Furthermore, inducing NRF3 activates cell proliferation and the expression of the glucose transporter GLUT1. The existence of the β-catenin/TCF4-NRF3 axis was also validated in the intestine and organoids of Apc-deficient mice. Finally, the positive correlation between NRF3 and β-catenin target gene expression strongly supports our conclusion. Our findings clearly demonstrate that NRF3 induction in cancer cells is controlled by the Wnt/β-catenin pathway.Remarkable upregulation of the NRF2 (NFE2L2)-related transcription factor NRF3 (NFE2L3) in several cancer tissues and its correlation with poor prognosis strongly suggest the physiological function of NRF3 in tumors. Indeed, we had recently uncovered the function of NRF3, which promotes cancer cell proliferation by p53 degradation via the 20S proteasome. Nevertheless, the molecular mechanism underlying the induction of  The development of human cancer genome databases such as The Cancer Genome Atlas (TCGA) and Oncomine have led directly to the discovery of significant functional mutations in genes and pathways ,2. This Nrf3 knockout mice do not show obvious abnormalities [NRF3 gene induction in cancer cells is poorly understood.In this regard, we focused on the transcription factor NRF3 (NFE2L3), because it is highly expressed in several cancers, including colorectal adenocarcinoma, and is correlated with poor prognosis , belongsmalities ,9,10. Remalities   gene and the CTNNB1 gene encoding β-catenin is an initial event leading to the development of adenoma in most sporadic cases. These genetic alterations stabilize the β-catenin protein and promote its nuclear entry, leading to the activation of gene expression by T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family proteins binding to the WRE sequence . The TCF/LEF family consists of TCF1, TCF3, TCF4 and LEF1. The upregulation of β-catenin/TCF target genes, such as c-MYC, AXIN2 and LGR5, regulates cell proliferation and stemness in epithelial stem and progenitor cells [APC mutations gives rise to hyperplastic epithelium. Further carcinoma development requires an activating mutation in the K-ras gene and loss of the p53 gene.A large body of evidence has revealed that the accumulation of multiple gene mutations underlies the development of cancer . For insor cells ,22,23. TGene alterations dramatically change the metabolic status of cancer cells ,24,25. ONRF3 induction in cancer cells involve the β-catenin/TCF4 complex. We first identified a species-conserved WRE site that is recognized by the β-catenin/TCF4 complex in the NRF3 gene. The β-catenin/TCF4 complex mediates NRF3 expression in several cancer cells through binding to its WRE site. We also found that upregulating NRF3 augments cell proliferation and the expression of the GLUT1 gene. The biological significance of the β-catenin/Tcf4-Nrf3 axis was confirmed by deletion of the Apc gene in the mouse intestine and its derived organoids. Finally, we found a positive correlation between NRF3 and c-MYC or LGR5, target genes of the β-catenin/TCF4 complex. Altogether, this study reveals the β-catenin/TCF4-NRF3 axis as the molecular basis for NRF3 gene upregulation in cancer cells.We herein report that the molecular mechanisms of NRF3 mRNA expression was induced in several types of cancer, especially in colon and rectal carcinoma. These results strengthen our hypothesis that NRF3 is involved in colorectal carcinogenesis. To further examine the biological relevance of NRF3 in cancer, we first conducted an NRF3 expression analysis using a human cancer RNA dot blot . The bloNRF3 gene induction in colorectal cancer cells, we considered possible regulation by the β-catenin/TCF complex. Mutations in the Wnt-β-catenin pathway are crucial initial events in colon carcinogenesis [NRF3 gene and successfully found a species-conserved WRE sequence as the TCF recognition element in its first intron , H1299 , A172 (glioblastoma) and HeLa   A. All thy active . Moreovey active C. The daNRF3 knockdown reduced DLD-1 cell growth [NRF3 siRNA significantly reduced cell growth (GLUT1 (SLC2A1) gene  gene B. BecausRE sites C. The reeriments D. NRF3 spression . To exam results . We thusApcΔ716 mice develop intestinal tumors due to a null mutation in the Apc gene, thereby activating β-catenin [ApcΔ716 mice, we observed significant upregulation of Nrf3 expression and the β-catenin target gene c-Myc  by infecting Apcflox/flox intestine organoids (f/f) with Cre-expressing lentivirus  culture system because of their self-organizing structures ,33,34. Wntivirus B. Consisand Cd44 C. Importene Nqo1 D. Our fic-MYC and LGR5 are direct target genes of the β-catenin/TCF4 complex, the analysis showed a significant correlation among them. We thus conclude that NRF3 is induced by the β-catenin/TCF4 complex in human cancer cells.Finally, we searched the GEPIA database to confirm the physiological relevance of the association between the Wnt-β-catenin pathway and NRF3 . BecauseNRF3 mRNA expression, consequently leading to the upregulation of cell proliferation and GLUT1 gene expression in cancer cells  Under physiological conditions, NRF3 is sequestered in the endoplasmic reticulum (ER), thereby preventing its nuclear translocation and transcriptional activity [Nrf3 knockout mice do not show apparent abnormalities under physiological conditions [APC mutations dramatically change the cell metabolic status by inducing the c-MYC gene, some metabolites or activated signal transduction pathways may activate the nuclear translocation of NRF3. For a comprehensive understanding of the physiological function of NRF3 in cancer cells, the identification of intrinsic NRF3 activation and/or stress signals remains a crucial issue.It appears that β-catenin/TCF4 merely mediates the expression of NRF3 target genes by inducing activity . (2) Theactivity . (3) Theactivity  . Because GLUT3 mRNA is also induced in an NRF3-independent mechanism in cancer cells [GLUT1 gene expression on cancer cells.We found that the glucose transporter get gene . We needer cells ,25, GLUTiCRT14 was purchased from Sigma-Aldrich (SML0203) . The antibodies utilized were anti-β-catenin , anti-TCF4 , anti-α-Tubulin , and anti-histone H3 . Normal mouse IgG  and normal rabbit IgG  were used for chromatin immunoprecipitation analysis. A monoclonal NRF3 antibody (#9408) raised against human NRF3 (amino acids 364-415) was generated as described previously [TM) Array, which contains dots of polyA+ RNA normalized to ubiquitin from 76 different human tissues, was purchased from Clontech . A radiolabeled probe was prepared from the 500-bp HindIII fragment of the human clone SKhNrf3/1-1 [The Human Multiple Tissue Expression  supplemented with 10% FBS  and 1% penicillin/streptomycin . H1299 cells were cultured in RPMI1640  supplemented with 10% FBS and 1% penicillin/streptomycin. L-WRN cells  were cultured in DMEM  supplemented with 10% FBS. All cell lines were cultured in a humidified incubator at 5% COflox/floxApc  (580S flox/580S floxApc ) mice were kind gifts from Ryoji Yao [ApcΔ716 mice was described previously [http://www.mext.go.jp/b_menu/hakusho/nc/06060904.htm).yoji Yao . Generateviously . All aniApcflox/flox mice as described previously [Apc-deficient organoids (Apc KO) were generated by infecting Apcflox/flox organoids with Cre-expressing lentivirus from the LV-Cre pLKO.1 plasmid encoding Cre-recombinase . Organoids were seeded on Matrigel . Apcflox/flox organoids were cultured in Advanced DMEM/F12  supplemented with L-WRN cell conditioned medium, 1% penicillin/streptomycin , EGF  and Y-27632 (a Rock inhibitor) . L-WRN cell conditioned medium was prepared as described in [Apc KO organoids were cultured in Advanced DMEM/F12 supplemented with 1% penicillin/streptomycin, EGF, Y-27632 and L-glutamine  in a humidified incubator at 5% CO2 and 37 °C.Mouse intestine-derived organoids were generated from eviously . Apc-defribed in . Apc KO The transfection of short interfering RNA (siRNA) was performed using RNAiMAX  according to the manufacturer’s protocols. Sequences of siRNAs targeting human mRNAs are listed in β-actin or Gapdh expression. The sequences of the primers used are listed in Total RNA was prepared using ISOGENII . One microgram of total RNA was utilized for cDNA synthesis using random hexamer primers  and Moloney murine leukemia virus (M-MLV) reverse transcriptase . qRT-PCR was conducted using SYBR Premix Ex Taq II  and a Thermal Cycler Dice Real Time System II . The PCR conditions were 95 °C for 30 sec, 30 cycles of 95 °C for 5 sec, 60 °C for 30 sec and 95 °C for 15 sec, 60 °C for 30 sec and 95 °C for 15 sec. All target gene expression levels were normalized to 2), 1x protease inhibitor , 10 µM MG132  and 10% NP-40 . After centrifugation, the precipitated nuclei were lysed with SDS sample buffer. The protein concentrations of the cell extracts were measured using a bicinchoninic acid (BCA) kit . Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes . The blots were incubated with the primary antibodies indicated on the figures and then with a horseradish peroxidase-conjugated secondary antibody . The protein bands were visualized using enhanced chemiluminescence .To prepare whole cell extracts, cells were lysed with SDS sample buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol and 1% SDS). For nuclear extracts, cells were lysed in buffer A (10 mM Tris-HCl (pH 8.0), 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgClHCT116 cells grown on a 10-cm dish were cross-linked in 1% formaldehyde for 10 min, followed by quenching with 0.125 M glycine for 5 min at room temperature and two washes with ice-cold phosphate-buffered saline (PBS). ChIP analysis was conducted as described previously , with mi5 cells per well), transfected with the indicated siRNA and cultured for the indicated number of days. The cells were detached from plates with 0.05% trypsin and gently suspended in ice-cold PBS. Cell counting was performed using a hemocytometer.HCT116 cells were plated onto 6-well dishes  of at least three independent experiments.The statistical significance of repeated measurements was evaluated using ANOVA-Tukey’s t-test and Student’s


Tg) of the nanocomposites were improved with organoclay loading up to 1 wt %. Beyond this loading limit, the deterioration of properties was observed. A similar trend was also observed on tensile strength and modulus. We concluded from this study that organoclay loading up to 1 wt % is suitable for further study to fabricate hybrid nanocomposites for various applications.In this work, the optimum filler loading to prepare epoxy/organoclay nanocomposites by the in-situ polymerization method was studied. Bi-functionalized montmorillonite at different filler loading  was dispersed in epoxy resin by using a high shear speed homogenizer. The effect on morphology, thermal, dynamic mechanical, and tensile properties of the epoxy/organoclay nanocomposites were studied in this work. Wide-angle X-ray scattering (WAXS) and field emission scanning electron microscope (FESEM) studies revealed that possible intercalated structures were obtained in epoxy/organoclay nanocomposites. Thermogravimetric analysis (TGA) shows that epoxy/organoclay nanocomposites exhibit higher thermal stability at the maximum and final decomposition temperature, as well as higher char content, compared to pristine epoxy. The dynamic mechanical analysis (DMA) indicate that storage modulus (E′), loss modulus (E″), cross-link density and glass transition temperature ( Epoxy resin falls under the category of thermosetting polymer which contained one or more oxirane ring structure in the molecule . Epoxy rOne of the approaches to overcome the shortage of pure epoxy is the incorporation of fillers. The technology advances in the field of nanotechnology and nanomaterials have brought new and interesting possibilities into the materials field with the development of polymer/clay nanocomposite where clay as a nano-sized layered silicate is dispersed in the polymer matrix. Many researchers have reported that polymer nanocomposites (PNCs) filled with small amount of nano-sized particles (< 5% by weight) show enhanced properties in several perspectives, such as improvement of mechanical properties, enhanced barrier properties, heat resistance, and fire performance ,7. The g4N+X−) [4P+X−) [+2X−) [Despite the success story achieved by the Toyota group, it is still challenging to obtain a fully exfoliated epoxy-clay nanocomposite which is able to meet the industrial applications requirement. The challenges lie in two main areas, which are enhancing the interactions between clay sheets by a suitable organic modifier and exfoliating the clay particles. The surface modification on nanoclay by using organic cations such as alkylammonium (R4N+X−) , alkylph [4P+X−)  and diaz) [+2X−)  is often) [+2X−) ,13. The ) [+2X−) ,14,15. H) [+2X−)  or ultra) [+2X−)  is often) [+2X−) .In recent years, utilizing a bi-functionalized nanoclay in preparation of PNCs is gaining more interest from the research community. Shakeri et al.  reportedAlthough bi-functionalized nanoclay seems to be more efficient, to date only limited work has been reported on the preparation of epoxy nanocomposites via this approach. Therefore, the aim of this work is to carry out studies on bi-functionalized MMT reinforced epoxy nanocomposites prepared via in-situ polymerization. The MMT inner gallery was modified by 18 carbon atoms, cationic ammonium salt (octadecylamine), and a silane coupling agent (3-aminopropyltriethoxysilane) was used to treat the surface and edges of the nanoclay to enhance dispersion in polymer resin. The dispersion method employed in this study is by mechanical stirring followed by high shear speed mixing. A set of epoxy/OMMT nanocomposites with different filler loading  was prepared at room temperature curing for 24 hours. The effects of the bi-functionalized MMT on the morphological, thermal stability, dynamic, mechanical, and tensile properties of the epoxy/OMMT nanocomposites were studied. As part of our previous work ,23,24, tOrgano-modified MMT (OMMT) used in this study  was procured from Sigma-Aldrich, Selangor, Malaysia and was used as received. The matrix used in the present study is a diglycidyl ether of bisphenol-A (DGEBA) epoxy resin with a trade name of DER-331 and a modified cycloaliphatic amine hardener with a trade name of JOINTMINE 905-3s. Both epoxy and hardener were obtained from Tazdiq Engineering Sdn. Bhd. . The epoxy/OMMT nanocomposites were prepared by in-situ polymerization. The OMMT at different weight fractions  were dispersed slowly into epoxy resin and mixed homogeneously by mechanical stirring at 500 rpm for 15 mins. The mixture is further mixed using a high shear speed homogenizer  with a rotation speed of 10,000 rpm for 30 min, according to a modified method reported by Al-Qadhi et al. [001) between the interlayer was calculated according to Bragg’s Law as per Equation (1) [Wide-angle X-ray scattering (WAXS) was conducted on the OMMT powder and epoxy/OMMT nanocomposites using an X−ray diffraction unit  with Cu-Kα radiation (wavelength = 0.1542 nm) under voltage of 50 kV and current of 1 mA. The epoxy/OMMT samples were prepared by casting a thin layer of the mixture of epoxy/nanoclay/hardener onto an aluminum pan  and allotion (1) ,27,28.(The morphology and the dispersion state of the nanoclay in the epoxy matrix were studied using an Ultra High-Resolution Scanning Electron Microscope (FESEM) . The exposure surface was sputter coated with gold to achieve better visualization.The thermal decomposition profile of the OMMT powder and epoxy/OMMT nanocomposites were examined using a thermogravimetry analyzer  according to ASTM E1131-03. The samples were subjected to a temperature profile heating from 30 to 800 °C with a heating rate of 20 °C/min. All measurements were carried out under a nitrogen atmosphere with a gas flow rate of 50 mL/min.The viscous elastic behavior of the nanocomposites was studied with a dynamic mechanical analyzer  according to ASTM D5023-01. The specimens were clamped on a single cantilever measurement configuration. The specimens were pre-cut into the desired dimension with a clamping free length of 10 mm and width of 10 mm. The specimens were subjected to a heating profile scanning from 25 to 150 °C with heating rate of 5 °C/min under a frequency of 1 Hz and a displacement amplitude of 10 μm. The glass transition temperature was determined from the peak of the tan delta curve according to ASTM E1640-99.The tensile properties of the nanocomposites were tested by using a 10 kN capacity Universal Testing Machine model 5566 INSTRON  according to ASTM D3039. The specimens were prepared in 120 mm × 20 mm × 3 mm. The gauge length used was 60 mm and a constant cross head speed of 2 mm/min was employed on the testing. The samples were placed in a preconditioning chamber for a day with a temperature of 23 ± 3 °C and relative humidity of 50% ± 10% prior to analysis. Five replicate samples were tested for each group and the average values were reported.Due to the fact that pristine MMTs are hydrophilic and the stacks of clay platelets are held tightly together by electrostatic forces, this led to a poor dispersion of nanoclay within the epoxy resin . Surface2) of APTES can react with the oxirane ring of the monomer of DGEBA . The TGA and DTG (derivative thermo gravimetric) curves of OMMT powder are shown in TIDT), maximum decomposition (TMAX), and final decomposition (TFDT) temperature of the composites are summarized in TIDT of epoxy is 365 °C, while the TIDT for nanocomposites with 0.5, 1, 2 and 4 wt % of OMMT are 353, 356, 347 and 336 °C, respectively. The reduction in thermal stability at a low temperature may be due to the existence of the organic surface modifier on clay which exhibits lower thermal stability. In addition, the ammonium salt that attached to the clay may act as a catalyzer towards the degradation of the polymer matrix through the Hoffman degradation mechanism; a similar observation has been reported by several researches [TIDT of the epoxy nanocomposites decreases as the concentration of the OMMT increases. This may be due to the increased amount of surface modifier in the epoxy matrix leading to the acceleration of the decomposition reaction of the polymer matrix.The thermal decomposition profile of epoxy nanocomposites with 0.5, 1.0, 2 and 4 wt % of OMMT is shown in searches ,42,43. TTMAX and TFDT among all the composites. With increasing OMMT concentration, the TMAX and TFDT of the corresponding nanocomposites decrease, yet it is still higher compared to pristine epoxy. In addition, the char formation of epoxy nanocomposites was also found to be higher compared to pristine epoxy. The decomposition temperature at different weight loss percentages of 10%, 30%, 50%, 80% and 90% and char residue at 800 °C are reported in The presence of the nanoclay has improved the thermal stability of the epoxy nanocomposites at a higher temperature range compared to pristine epoxy as illustrated in gT) region or α relaxation of the polymer whereby it represents the movement of the polymer main chain. Below the glass transition region, the movement of the polymer chain was restricted due to the low mobility of the frozen and closely packed molecules arrangement. As a result, the E′ is high in the glassy state. With temperature increases, the closely packed molecule arrangement collapses resulting in high molecular mobility and increases the free volume components, thus resulting in a drastic fall of modulus and moving into the rubbery region of the material.The effect of the organoclay loading on the viscoelastic behavior of the nanocomposites as a function of temperature and frequency under an oscillating force was studied by the DMA. The storage modulus (E′), loss modulus (E″), and tan delta curves of the pristine epoxy and epoxy nanocomposites are shown in Tg + 30) °C. With 0.5 and 1.0 wt % of OMMT loading, the E′ at the glassy region improved by 50% and 87% respectively compared to pristine epoxy. With filler loading at 2 wt %, we observed a decrease in modulus with E′ recorded at 615MPa and a further reduction in E′ was observed on epoxy/4% OMMT nanocomposites with the recorded E′ of 466 MPa. A similar trend was also observed on the storage modulus in the rubbery region. The improvement of the E′ in both glassy and rubbery region can be attributed to the incorporation of the OMMT limiting the movement of the epoxy polymer chain as they intercalated in between the clay layers of OMMT [3), T = (Tg + 30) °C, R is the gas constant (8.3145 J/K mol) and T is temperature in Kelvin. It is evident that the increasing loading of the OMMT improved the crosslink density compared to pristine epoxy. The crosslink density of epoxy raised from 6.1 × 10−4 to 10.2 × 10−4 (mol/m3) (An increasing loading of OMMT increases the E′ in both glassy and rubbery regions as shown in  of OMMT . The dec of OMMT .(2)νe=È(mol/m3) . The obtThe obtained loss modulus (E″) was summarized in Tg) of the material. Tg by the peak of tan delta. The tan delta curve of pristine epoxy appears to be a sharp and narrow peak with the highest tan delta value recorded at 1.15 with Tg recorded at 77.8 °C. A high tan delta value indicates high damping behavior or a high degree of non-elastic deformation. On the contrary, a low value indicates that the material is more elastic. With the incorporation of organoclay, the tan delta curve of epoxy nanocomposites appears to be broader and decreases in peak intensity with the Tg also found to be shifted to a higher temperature. The lower tan delta value and broad peak is evidence that the improving damping behavior is due to the presence of the intercalated layers of OMMT modifying the network structure, effecting the relaxation mechanisms and arresting the segmental motion near the organic-inorganic interface. These findings are in line with other research findings [Tg higher. The increase of Tg can be attributed to the constraint of relaxation mobility in the polymer segments caused by the chemical bonding around the interface of the clay layer and epoxy matrix [−4 mol/m3) with the highest Tg (82.6 °C) among all composites. This is followed by the Epoxy/0.5% OMMT nanocomposite with a crosslink density of 9.2 × 10−4 mol/m3 and its Tg recorded at 81 °C. However, lacking surrounding entanglements and reduced crosslink density at the interface decreases the Tg. This trend was observed on epoxy nanocomposites with 2 and 4 wt % of OMMT loading.Lastly, the loss factor or Tan delta curve corresponds to the ratio of loss modulus to storage modulus and its maximum peak height can be denoted as the glass transition temperature (findings ,51,54,55The tensile properties of epoxy organoclay nanocomposites strongly depends on the structure or configuration of nanocomposites, i.e., conventional/intercalated/exfoliated and interfacial interaction between polymer and clay layers . The tenBeyond 1 wt % of OMMT loading, the tensile properties of the nanocomposites start to decline. A similar phenomena is also reported elsewhere ,45,58. STg value among all the composites. A similar observation is also observed on the tensile properties of the nanocomposites with the highest tensile strength and modulus was observed on epoxy/1% OMMT nanocomposite. Overall, it can be concluded that 1.0 wt % OMMT filler loading epoxy nanocomposites shows considerable higher thermal stability, viscous elastic properties, crosslink density, Tg, and tensile properties.Epoxy/organoclay nanocomposites with a different organoclay concentration were prepared by high shear speed homogenizer through in-situ polymerization in the presence of bi-functionalized MMT. The WAXS and FESEM suggested that intercalated epoxy/organoclay nanocomposites were obtained on epoxy/0.5% OMMT and epoxy/1.0% OMMT nanocomposites. The FESEM images confirmed that nanocomposites with OMMT loading beyond 1 wt % exhibits large tactoids particles and agglomeration. The TGA analysis shows that the thermal stability of the nanocomposites was improved at a higher temperature range. The initial decomposition of all nanocomposites was lower compared to pristine epoxy due to the existence of the organic surface modifier which possesses lower thermal stability. The E′, and E″ of the nanocomposites were greatly improved with the incorporation of organoclay up to 1 wt % loading of OMMT. The DMA results also reveal that epoxy/1% OMMT nanocomposites exhibit the highest crosslink density and


It is well-established that aberrant WNT expression and signaling is associated with developmental defects, malignant transformation and carcinogenesis. More recently, WNT ligands have emerged as integral components of host responses to infection but their functions in the context of immune responses are incompletely understood. Roles in the modulation of inflammatory cytokine production, host cell intrinsic innate defense mechanisms, as well as the bridging of innate and adaptive immunity have been described. To what degree WNT responses are defined by the nature of the invading pathogen or are specific for subsets of host cells is currently not well-understood. Here we provide an overview of WNT responses during infection with phylogenetically diverse pathogens and highlight functions of WNT ligands in the host defense against infection. Detailed understanding of how the WNT network orchestrates immune cell functions will not only improve our understanding of the fundamental principles underlying complex immune response, but also help identify therapeutic opportunities or potential risks associated with the pharmacological targeting of the WNT network, as currently pursued for novel therapeutics in cancer and bone disorders. The WNT signaling network is a central regulator of embryonic development and tissue homeostasis. WNT proteins are phylogenetically highly conserved secreted, cysteine-rich glyco-lipoproteins . NineteeWNT proteins are highly hydrophobic due to post-translational modification by palmitoleic and palmitic acid at conserved cysteine residues. This is afforded through action of the acyltransferase Porcupine (PORCN) in the endoplasmic reticulum . WNT acyDrosophila] and WNT/Ca2+-signaling are modes of β-catenin-independent WNT signaling. WNT/JNK signaling results in FZD/DVL-mediated activation of the small GTPases RAC1 and RHOA, directing cytoskeletal rearrangements, cell polarization and motility. Activation of JNK can drive c-Jun- and AP-1-controlled transcription  activation and enhanced levels of cytosolic Ca2+, which activates calmodulin/calmodulin-dependent kinase II and NFAT-regulated transcriptional responses  7-transmembrane domain receptors, low-density lipoprotein-related proteins LRP5, LRP6), as well as receptor tyrosine kinases ROR and RYK . Cytopla, LRP6, acription . WNT/Ca2esponses .Tight regulation and precise targeting of WNT signaling is essential, as emphasized by the evolutionary investment in multiple layers and modes of WNT pathway modulation. WNT signaling is negatively regulated by secreted Frizzled-related proteins (sFRP) and WNT inhibitory factor 1 (WIF-1), which directly bind WNT proteins interfering with receptor interactions . The palWNT5A as a highly responsive gene in human macrophages upon microbial encounter  enterica serovar Typhimurium infection of streptomycin-pretreated mice increased mRNA expression of Wnt3, Wnt6, Wnt9a, and protein expression of Wnt2 and Wnt11 in intestinal tissues , Fzd2, Fzd4, Fzd6, Fzd7, Fzd8, Fzd9, with limited or no effects on the expression of other Wnt and Fzd genes  chaffeensis infection of human THP-1 macrophage-like cells transiently increased mRNA expression of WNT6, WNT10A, FZD5, and FZD9, while decreasing expression of WNT5B, WNT7B, and FZD7, as determined by pathway-specific qPCR arrays  coli have been investigated to some extent in mouse models in vivo. Mono-colonization of mice with E. coli F18 enhanced expression of Wnt2 in the intestine compared to germ-free mice (E. coli (UPEC) induced rapid downregulation of Wnt5a expression in the urothelium of infected mice, which was partially attributed to the bacterial virulence and adhesion factor, FimH  aeruginosa ..EschericWNT5A expression reflect pathogen-specific responses, cell-type-dependent variations, or species-specific differences between humans and mice. Some indication that the latter apect might indeed be of importance comes from studies of macrophages stimulated with lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Increased WNT5A expression has been noted in LPS-stimulated human monocytes, the human monocytic THP-1 cell line, as well as human bronchial epithelial BEAS-2B cells  activation, and was amplified by inflammatory cytokines such as tumor necrosis factor (TNF) and interferon γ (IFNγ)  , 34, 60.rophages , yet LPSrophages . NeverthWNT3 in human monocyte-derived macrophages , more detailed insight is required to delineate the impact of cell type-, species-, and pathogen-specific contributions to the amplitude and composition of infection-associated WNT responses.Staphylococcus (S.) aureus infection of Drosophila melanogaster led to enhanced expression of Wnt4 . pneumoniae has been reported to suppress Wnt5a protein expression  tuberculosis infection of C57BL/6 mice enhanced lung mRNA expression of Wnt1, Wnt6, Wnt10a, Fzd1, and Fzd5, while reducing expression of Wnt2, Wnt2b, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt8a, Wnt10b, as well as Fzd3, Fzd7, Fzd8, Fzd9, and Fzd10  greatly enhanced expression of WNT5A  cruzi, protein expression of Wnt3a, Wnt5a, and β-catenin in splenic mononuclear cells increased with disease progression  fumigatus, WNT5A expression was found to be significantly higher than in uninfected corneal tissues. WNT5A mRNA and protein expression were also enhanced by A. fumigatus infection of human THP-1 macrophages - infected nasopharyngeal carcinoma epithelial cells  contribute to the host control of phylogenetically diverse pathogens in non-vertebrates and vertebrates , 88, 89.in vitro cell-based studies. Conditioned media from WNT-overexpressing cells and recombinant WNT proteins have also been proven as valuable tools for deciphering WNT functions. Of note, some biological responses of innate immune cells observed upon exposure to recombinant WNT protein preparations have been attributed to Toll-like receptor activation, rather than known WNT receptors  by S2 phagocytes. Functional interactions of dally with frizzled 2 and wnt2 in this process were deduced from gene expression analyses  and P. aeruginosa (Gram-negative) mediated by Rac-1 and Dvl  are secreted effectors of E. chaffeensis that have been shown to interact with host cell proteins, including components of the WNT signaling network   . With ev12, p62) –135, andL. donovani  (cfms-icre Wlsfl/fl mice (Nos2 expression and nitric oxide production were shown to be positively regulated by β-catenin and TCF-4 (Wls-deficient macrophages may indicate compensatory mechanisms associated with the inability to release WNT proteins from producing cells and significant elevation of WNT gene expression observed in these cells (+ liver macrophages of Wlsfl/fl-Lyz2-Cre mice did not show significant differences in Wnt4 and Wnt6 expression (by Nos2) , a pheno/fl mice . This mand TCF-4 . These ose cells . Howeverpression .Nos2−/− mice showed lower induction of Wnt5a, Fzd4, and Lrp5 mRNA expression upon M. bovis BCG infection compared to wild type control cells. Treatment with an NO-donor restored Wnt5a, Fzd4, and Lrp5 expression in Nos2-deficient macrophages  and β-defensin 1 in RAW264.7 mouse macrophages, which correlated with a small increase in bacterial killing by these cells  upon S. aureus infection is dependent upon the β-catenin homolog bar-1  or the immune responses (parasite infection vs. sterile inflammation) are worth further investigations.Indoleamine 2,3dioxygenase (IDO) catalyzes the first rate-limiting step in the catabolism of the tryptophan for the formation of active metabolites . IDO actarasites . This sue in DCs , 145. WhIFNβ1) expression, and inhibition of GSK3β-controlled virus-induced type I IFN responses in a β-catenin-dependent manner in a range of human cell lines and primary cells  impaired infection of HeLa cells by Dengue virus (GSK3β activity and β-catenin functions have been implicated in the positive or negative regulation of type I interferon (IFN) responses associated with protection or susceptibility of cells to viral infection , 146–152ry cells . SiRNA-mry cells . Knock-due virus . Enhanceue virus . Howeverue virus .WNT signaling has been ascribed both pro-inflammatory and immune-regulatory properties. The paradigm developed over the past decade or so suggests that WNT ligands triggering β-catenin-independent signaling exert pro-inflammatory functions, whereas WNT ligands driving β-catenin stabilization have anti-inflammatory or immune-modulatory effects. These emerging concepts of WNT ligands orchestrating inflammation and immune cell functions have been reviewed and commented on extensively over time , 155–163E. coli-induced bacterial peritonitis  and β-catenin/TCF inhibitors (iCRT14) (T. cruzi-induced IL-10 production by infected macrophages in vitro (in vivo LPS-induced endotoxemia model (Evidence for net anti-inflammatory functions of WNT ligands can be deduced from enhanced pro-inflammatory cytokine release and decreased release of regulatory TGF-β by (iCRT14) . In thisin vitro . Similaria model . These oM. tuberculosis-infected mouse macrophages. Bone marrow-derived macrophages from Wnt6-deficient mice displayed elevated TNF expression and secretion upon M. tuberculosis infection .Wls deletion in resting mouse bone marrow-derived macrophages was accompanied by elevated expression of Nos2, Tnf, and Il6, and reduced expression of the M2-associated gene Mrc1 (macrophage mannose receptor), without affecting Arg1 expression (Wls activity (and by inference the net impact of released WNT ligands) contributed toward M2 polarization of these macrophages. In contrast, several studies indicated that Arg1 expression is regulated by WNT ligands in macrophages upon pathogen encounter. For example, the PORCN inhibitor IWP-L6, but not the β-catenin inhibitor iCRT14, decreased Arg1 expression in T. cruzi-infected mouse macrophages, yet without impacting production of reactive nitrogen intermediates  have returned varying results on their ability to elicit phenotypic changes indicative of alternatively activated macrophages or macrophages tolerized against LPS activation  and DC endocytic capacity has been analyzed in a number of studies returning varying results , 174–182in vivo models of autoimmunity, gut mucosal homeostasis and cancer  rendered mice more susceptible to dextran sodium sulfate (DSS)-induced colitis , collagen-induced arthritis, and tumorigenesis  infection, TCF-1-deficiency had no effect on the expansion and functions  of effector CD8 T cells , 195, whpression . In conthallenge –196. Hownfection . Yet, inreported . These sin vitro system, depletion of WNT1, 2B, 3 and 5B from astrocyte-conditioned medium reduced the differentiation of CD8+ T cells toward a CD4dimCD8bright T phenotype in cultures of human peripheral blood mononuclear cells. CD4dimCD8bright T cells in the central nervous system are thought to be effector memory T cells important in the control of HIV . To our knowledge, there are thus far only very few links between WNT ligands and CD4+ T cell functions during infection. In a susceptible mouse model of Leishmania major infection, an inhibitor of Dkk1, which should increase WNT/β-catenin signaling, exhibited reduced numbers of CD4+ T cells in the draining lymph node, with subsequent reduced IL-4 and IL-10 expression after ex vivo stimulation , it remains largely unclear whether stereotypical WNT responses to infection exist regardless of the invading pathogen, or whether the nature of the pathogen dictates the WNT response. Comparative studies using phylogenetically diverse pathogens covering spectra of virulence and pathogenesis mechanisms will be essential to distinguish stereotypical and selective responses to microbial infection. In depth understanding of the molecular drivers and regulators of WNT ligand and receptor expression during infection will be invaluable in delineating which microbial factors drive WNT responses. Whereas our understanding of WNT responses and functions during viral and bacterial infections is taking shape, WNT contributions to parasitic and fungal infections remain to be explored in more breadth and depth. Knowledge of the investment of pathogens into actively manipulating the WNT signaling network –204 willDue to the central role of WNT signaling in maintaining tissue homeostasis, including epithelial barrier functions, consequences of immune-related WNT responses reach beyond leukocyte functions. Indications that WNT/WNT receptor interactions shape chemokine responses  and cellJL, JK, and AB wrote the manuscript. TT contributed sections to the manuscript. JL and AB conceived and designed figures and tables. All authors read and approved the final version of the manuscript.We apologize to colleagues whose primary research work was not integrated into this review due to space constrains or whose contributions to this immense and rapidly growing field was inadvertently overlooked.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alzheimer’s disease (AD) is an aging-related neurological disorder characterized by synaptic loss and dementia. Wnt/β-catenin signaling is an essential signal transduction pathway that regulates numerous cellular processes including cell survival. In brain, Wnt/β-catenin signaling is not only crucial for neuronal survival and neurogenesis, but it plays important roles in regulating synaptic plasticity and blood-brain barrier integrity and function. Moreover, activation of Wnt/β-catenin signaling inhibits amyloid-β production and tau protein hyperphosphorylation in the brain. Critically, Wnt/β-catenin signaling is greatly suppressed in AD brain via multiple pathogenic mechanisms. As such, restoring Wnt/β-catenin signaling represents a unique opportunity for the rational design of novel AD therapies. Alzheimer’s disease (AD) is the most common form of dementia accompanied by detrimental cognitive deficits and pathological accumulation of amyloid-β (Aβ) plaques and tau-containing neurofibrillary tangles . As one The Wnt/β-catenin signaling pathway is a significant pathway regulating cell proliferation, migration and differentiation, and Wnt proteins are key drivers of adult stem cells in mammals . StudiesWnt proteins are secreted glycoproteins that bind to the extracellular cysteine-rich domain of the Frizzled (Fzd) receptor family and Wnt co-receptor low density lipoprotein receptor-related protein 5 (LRP5) or LRP6 to activate the canonical Wnt/β-catenin signaling pathway. Binding of Wnt to the Fzd/LRP5/6 receptor complex results in inhibition of glycogen synthase kinase 3β (GSK3β) and stabilization of cytosolic β-catenin. Stabilized β-catenin then translocates into the nucleus, interacts with T-cell factor/lymphoid enhancing factor (TCF/LEF), and induces the expression of specific target genes Fig.  [6]. WntIn the rest of this section, we summarize our current understanding of the roles of Wnt/β-catenin signaling on multiple physiological and pathophysiological processes in AD brain Fig. .Fig. 2RThe neurodegenerative process in AD is initially characterized by synaptic damage followed by neuronal loss . The WntWhile there is a debate of the presence of neurogenesis in human adult brain , emerginSynaptic plasticity is associated with higher brain functions such as learning and memory. Synapse loss, which occurs prior to neuronal death at early stages in AD brain, is a major correlate of cognitive impairment in AD brain , 37. RecLRP6 is an essential Wnt co-receptor for activation of Wnt/β-catenin signaling on the cell surface. LRP6 is selectively localized to excitatory synapses, and is required for excitatory synapse development in vitro and in vivo . MoreoveDKK1 binds to LRP6 and blocks Wnt/β-catenin signaling on the cell surface. Mice with a dorsal hippocampal infusion of DKK1 exhibited impaired hippocampal-dependent novel object recognition memory with rapidly decreasing levels of key Wnt/β-catenin signaling proteins, including β-catenin, Cyclin D1, c-myc, Wnt7a, and PSD95 . InductiThe BBB protects the brain from exposure to neurotoxic blood-derived debris, cells and microbial pathogens. Therefore, BBB disruption allows influx of harmful substances into the brain, induces inflammatory and immune responses, and may subsequently initiate multiple pathways of neurodegeneration , 52. BBBIn the past decade, studies have established that the Wnt/β-catenin pathway is a key pathway required not only for BBB formation but also for BBB integrity and function , 58. By Brain ECs are held together by tight junctions, in which claudins are the main constituent. In addition, glucose transporter 1 (GLUT1), which is specifically expressed in BBB ECs, is responsible for the transport of glucose from the blood into the brain; and p-glycoprotein (Pgp-1) is an active efflux transporter highly expressed on the luminal surface of BBB endothelial cells. Mechanically, claudin-1, − 3 and − 5, the three major claudins expressed in brain ECs , are theOne of the two major hallmarks of AD is the accumulation of amyloid plaques between neurons in the brain , 73. RecAnother major hallmark of AD is the presence of intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated forms of the microtubule-associated protein tau (MAPT) in neurons , 73, 77.Trem2−/− microglia and Trem2−/− mouse brain [Glia-mediated neuroinflammation is another pathological hallmark of AD –83. Genese brain . In addise brain . Howeverse brain . Wnt/β-cse brain , and furWhile the Wnt/β-catenin signaling pathway is essential for brain function, this pathway is greatly suppressed via multiple pathogenic mechanisms in AD brain.It is well established that increasing age is the greatest risk factor for AD , 96. MouA growing body of evidence shows dysregulation and loss of function of Wnt co-receptor LRP6 contributes to down-regulation of Wnt/β-catenin signaling in AD. Firstly, two LRP6 SNPs and an alternatively splice variant that display impaired Wnt/β-catenin signaling activity, are associated with increased risk of developing AD , 102. SeAβ peptides can induce DKK1 expression and inhibit Wnt/β-catenin signaling in primary cortical neurons , and DKKThe binding of Wnt protein to Fzd/LRP results in inhibition of GSK3β and consequent activation of Wnt/β-catenin signaling , 109. GSAPP mutations can cause early-onset FAD , 116. WhPSEN1 are among the major causes of early-onset FAD [2+ homeostasis [Drosophila PSEN was identified as a suppressor of wingless/Wnt signaling [PSEN1 mutations [PSEN1 mutations associated with AD cause a perturbation in the intracellular trafficking of β-catenin [L286V and -PSEN1M146L fail to induce β-catenin degradation [L286V can upregulate a subset of TCF/β-catenin transcription by enhancing the level of cAMP-response element-binding protein (CREB)-binding protein (CBP) [Mutations in nset FAD , 119. Ineostasis , 121. Maeostasis , 122–128eostasis . In a geignaling . PSEN deignaling , and facignaling . Moreoveutations , and PSE-catenin , 124. Horadation , 132. Inin (CBP) .Giving that the Wnt/β-catenin pathway is greatly suppressed in the brain of AD patients, restoring Wnt/β-catenin signaling represents a unique opportunity for rational AD therapy Fig. .A physically active lifestyle in adults and the elderly can improve brain health and reduce cognitive impairment associated with aging . It has Estrogens can exert numerous protective actions in the adult brain, and reduced estrogen levels in adulthood are associated with increased risk of AD in women , 137. InThe activity of GSK3β is negatively regulated by Wnt/β-catenin signaling , 109. GiSuppression of Wnt/β-catenin signaling by Aβ-induced up-regulation of DKK1 expression in AD brain suggests DKK1 inhibition is a potential therapeutic strategy for restoring Wnt/β-catenin signaling in AD . Indeed,50 of 3 μM [A virtual screen of the National Cancer Institute database for chemical compounds identified a small molecule, IIIC3 , as a DKK1 inhibitor . IIIC3 c of 3 μM , and rev of 3 μM , 144. Mo of 3 μM . Interes of 3 μM , 146. Ho50 of about 250 nM in the Wnt reporter assays [WASP-1 is a small molecule Wnt activator with an ECr assays . Althougr assays , 149.Curcuma longa), displays protective effects in various animal models of AD [Curcumin, a natural compound found in the plant turmeric (ls of AD , 151. Stls of AD , 153. Hols of AD , 151. ReStatins are a class of drugs typically used to lower blood levels of cholesterol by reducing the production of cholesterol by the liver, and many studies suggest that statin use might protect against AD pathology –158. SevCompared to a large number of Wnt inhibitors as potential agents for cancer prevention and treatment, there are only a few Wnt activators reported in the literature , 165. PaIt is well established that Wnt/β-catenin signaling plays a key role in the regulation of bone mineral density, and that the Wnt/β-catenin signaling pathway is an attractive target for therapeutic intervention to restore bone strength in patients with osteoporosis disorders , 173. InLRP5 gain-of-function mutations, and Sost- or Dkk1-deficient animals do not have an increased risk of tumor developments [There is always a concern that overstimulation of Wnt/β-catenin signaling can promote cancer because aberrant activation of Wnt/β-catenin signaling can lead to tumor formation , 109. Holopments . Neverth