Genetic control of susceptibility to carcinogen-induced colorectal cancer in mice: The Ccs3 and Ccs5 loci regulate different aspects of tumorigenesis

Colorectal cancer (CRC) is a multistep disease that involves a two-way interaction between a complex genetic pre-disposition component, and a set of poorly understood extrinsic environmental factors. In mice, CRC can be induced by treatment with azoxymethane (AOM). Using a set of AcB/BcA recombinant congenic strains derived from CRC-susceptible A/J and CRC-resistant C57Bl/6J (B6) progenitors, we previously detected the Ccs3 locus (colon cancer susceptibility locus 3) as a major regulator of CRC susceptibility. Phenotyping of additional AcB/BcA strains for susceptibility to AOM-induced CRC has refined the Ccs3 interval to a 6.7 Mb segment on chromosome 3. In addition, the presence of intermediate susceptibility phenotypes in individual AcB/BcA strains suggested additional gene effects regulating CRC susceptibility in A/J and B6 strains. Those were investigated by linkage analysis and whole genome scanning in a set of 208 informative (B6 x A/J)F2 progeny, using tumor multiplicity as a quantitative measure of susceptibility. This analysis validated the important role of Ccs3 in regulating this trait, and additionally detected contribution from a second locus on the distal portion of chromosome 9 (LOD = 3.76), that was given the temporary designation of Ccs5. Ccs5 modulates tumor multiplicity in F2 animals bearing at least one A/J-derived susceptibility allele at Ccs3, with A/J-derived Ccs5 susceptibility alleles being inherited in a recessive manner. There is a strong additive effect of Ccs3 and Ccs5 on tumor multiplicity in F2 mice: mice doubly homozygotes for A/J or B6 alleles at Ccs3 and Ccs5 show tumor numbers similar to those of parental A/J and B6, respectively. Interestingly, the Ccs5 region overlaps several quantitative trait loci previously reported to regulate intestinal homeostasis and susceptibility to intestinal colitis in mice and humans. Our findings identify a novel two-locus system regulating CRC susceptibility in mice, of which the relevance to human CRC can now be tested experimentally.     

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs^∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853^∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.     

Secreted Heat Shock Protein 90α (HSP90α) Induces Nuclear Factor-κB-mediated TCF12 Protein Expression to Down-regulate E-cadherin and to Enhance Colorectal Cancer Cell Migration and Invasion

Secreted levels of HSP90α and overexpression of TCF12 have been associated with the enhancement of colorectal cancer (CRC) cell migration and invasion. In this study, we observed that CRC patients with tumor TCF12 overexpression exhibited both a higher rate of metastatic occurrence and a higher average serum HSP90α level compared with patients without TCF12 overexpression. Therefore, we studied the relationship between the actions of secreted HSP90α and TCF12. Like overexpressed TCF12, secreted HSP90α or recombinant HSP90α (rHSP90α) induced fibronectin expression and repressed E-cadherin, connexin-26, connexin-43, and gap junction levels in CRC cells. Consistently, rHSP90α stimulated invasive outgrowths of CRC cells from spherical structures during three-dimensional culture. rHSP90α also induced TCF12 expression in CRC cells. Its effects on CRC cell epithelial-mesenchymal transition, migration, and invasion were drastically prevented when TCF12 was knocked down. This suggests that TCF12 expression is required for secreted HSP90α to enhance CRC cell spreading. Through the cellular receptor CD91, rHSP90α facilitated the complex formation of CD91 with IκB kinases (IKKs) α and β and increased the levels of phosphorylated (active) IKKα/β and NF-κB. Use of an IKKα/β inhibitor or ectopic overexpression of dominant-negative IκBα efficiently repressed rHSP90α-induced TCF12 expression. Moreover, κB motifs were recognized in the gene sequence of the TCF12 promoter, and a physical association between NF-κB and the TCF12 promoter was detected in rHSP90α-treated CRC cells. Together, these results suggest that the CD91/IKK/NF-κB signaling cascade is involved in secreted HSP90α-induced TCF12 expression, leading to E-cadherin down-regulation and enhanced CRC cell migration/invasion.     

Suppression of Experimental Choroidal Neovascularization by Curcumin in Mice

Purpose

To investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms.

Methods

C57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)−1α in the RPE-choroid was determined by Western blotting.

Results

Curcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF−1α (P<0.05).

Conclusion

Curcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF−1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.     

MicroRNA-146a Provides Feedback Regulation of Lyme Arthritis but Not Carditis during Infection with Borrelia burgdorferi

MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a^−/− mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a^−/− mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a^−/− mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a^−/− macrophages, and corresponded to elevated IL-1β, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a^−/− mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1β, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.     

The Effects of NF-κB and c-Jun/AP-1 on the Expression of Prothrombotic and Proinflammatory Molecules Induced by Anti-β2GPI in Mouse

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β₂GPI/β₂GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β₂GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β₂GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β₂GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β₂GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.     

Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia: ROLE OF MEK,ERK, AND p38*

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser^176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser^32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser^217/221), ERK1/2 (Thr²⁰²/Tyr²⁰⁴), and p38 (Thr¹⁸⁰/Tyr¹⁸²) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser⁵³⁶-phosphorylated (p65⁵³⁶) and Lys³¹⁰-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr³⁵⁹/Ser³⁶³ in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65⁵³⁶ kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection.     

An Interval of the Obesity QTL Nob3.38 within a QTL Hotspot on Chromosome 1 Modulates Behavioral Phenotypes

A region on mouse distal chromosome 1 (Chr. 1) that is highly enriched in quantitative trait loci (QTLs) controlling neural and behavioral phenotypes overlaps with the peak region of a major obesity QTL (Nob3.38), which we identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6). By positional cloning we recently identified a microdeletion within this locus causing the disruption of Ifi202b that protects from adiposity by suppressing expression of 11β-Hsd1. Here we show that the Nob3.38 segment also corresponds with the QTL rich region (Qrr1) on Chr. 1 and associates with increased voluntary running wheel activity, Rota-rod performance, decreased grip strength, and anxiety-related traits. The characterization of a subcongenic line carrying 14.2 Mbp of Nob3.38 with a polymorphic region of 4.4 Mbp indicates that the microdeletion and/or other polymorphisms in its proximity alter body weight, voluntary activity, and exploration. Since 27 out of 32 QTL were identified in crosses with B6, we hypothesized that the microdeletion and or adjacent SNPs are unique for B6 mice and responsible for some of the complex Qrr1-mediated effects. Indeed, a phylogenic study of 28 mouse strains revealed a NZO-like genotype for 22 and a B6-like genotype for NZW/LacJ and 4 other C57BL strains. Thus, we suggest that a Nob3.38 interval (173.0–177.4 Mbp) does not only modify adiposity but also neurobehavioral traits by a haplotype segregating with C57BL strains.     

Binding of NFκB Appears to Twist the Ankyrin Repeat Domain of IκBα

Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of IκBα, the temporally regulated inhibitor of canonical NFκB signaling. Under native conditions, most of the IκBα molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NFκB(RelA/p50)-IκBα complex. Similar high FRET efficiencies were found when the IκBα molecules were either free or in complex with NFκB(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound IκBα molecules (0.67) than for the free IκBα molecules (0.74), apparently indicating that binding of NFκB(RelA/p50) stretches the ARD of IκBα. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NFκB(RelA/p50) and IκBα encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on IκBα as well as IκBα bound to dimerization-domain-only constructs or full-length NFκB(RelA/p50). Although we expected the stretched IκBα to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NFκB NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the IκBα ARD induced by interactions of the IκBα proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.     

Airway Epithelial NF-κB Activation Promotes Mycoplasma pneumoniae Clearance in Mice

Background/Objective

Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.

Methodology/Main Results

Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-_CAIKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg−) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.

Conclusion

By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.