Hyunji Ryu

p53 Mediates TNF-Induced Epithelial Cell Apoptosis in IBD

Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS(-/-) mice. IEC p53 levels and apoptosis were reduced in IL-10(-/-) colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF-treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF-treated CUC patients.

Epithelial Phosphatidylinositol-3-Kinase Signaling Is Required for β-Catenin Activation and Host Defense against Citrobacter rodentium Infection

ABSTRACT Citrobacter rodentium infection of mice induces cell-mediated immune responses associated with crypt hyperplasia and epithelial β-catenin signaling. Recent data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin552). Our aim was to determine whether epithelial PI3K/Akt activation is required for β-catenin signaling and host defense against C. rodentium. C57BL/6 mice were infected with C. rodentium and treated with dimethyl sulfoxide (DMSO) (vehicle control) or with the PI3K inhibitor LY294002 or wortmannin. The effects of infection on PI3K activation and β-catenin signaling were analyzed by immunohistochemistry. The effects of PI3K inhibition on host defense were analyzed by the quantification of splenic and colon bacterial clearance, and adaptive immune responses were measured by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Increased numbers of P-β-catenin552-stained epithelial cells were found throughout expanded crypts in C. rodentium colitis. We show that the inhibition of PI3K signaling attenuates epithelial Akt activation, the Ser552 phosphorylation and activation of β-catenin, and epithelial cell proliferative responses during C. rodentium infection. PI3K inhibition impairs bacterial clearance despite having no impact on mucosal cytokine (gamma interferon [IFN-γ], tumor necrosis factor [TNF], interleukin-17 [IL-17], and IL-1β) or chemokine (CXCL1, CXCL5, CXCL9, and CXCL10) induction. The results suggest that the host defense against C. rodentium requires epithelial PI3K activation to induce Akt-mediated β-catenin signaling and the clearance of C. rodentium independent of adaptive immune responses.

PI3K/AKT Signaling Is Essential for Communication between Tissue-Infiltrating Mast Cells, Macrophages, and Epithelial Cells in Colitis-Induced Cancer

Purpose: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models. Experimental Design: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10−/− mouse model of progressive colitis, and use of the PI3K inhibitor LY294002. Results: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer. Conclusion: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10−/− mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002. Clin Cancer Res; 19(9); 2342–54. ©2013 AACR.

P-selectin glycoprotein ligand-1 is needed for sequential recruitment of T-helper 1 (Th1) and local generation of Th17 T cells in dextran sodium sulfate (DSS) colitis.

Background: Activated effector T cells contribute to tissue injury observed in inflammatory bowel disease. T cells are recruited to effector sites after activation in peripheral lymph nodes directs expression of tissue‐specific homing receptors. One such mechanism for effector T cell recruitment employs activation‐induced fucosylation of P‐selectin glycoprotein ligand (PSGL)‐1 that mediates binding to endothelial P‐selectin. Here we examine the differential role of PSGL‐1 in recruiting effector T‐cell subsets in colitis. Methods: C57BL/6 wildtype and PSGL‐1−/− mice received 2.5% dextran sodium sulfate (DSS) for 6 days and were euthanized 7 and 14 days after the initiation of DSS. Disease activity was monitored throughout. Histologic colitis scores, colonic CD4+ accumulation, and cytokine production were assessed at days 7 and 14. Recruitment of T‐helper (Th) subsets was assessed by enumerating adoptively transferred Th1 or Th17 CD4+ cells 2 days after transfer to DSS‐treated mice. Results: DSS colitis increases CD4+ T cells in colonic tissue and induces Th1 (interferon gamma [IFN‐&ggr;], tumor necrosis factor [TNF]) and Th17 (interleukin [IL]‐17, IL‐22) cytokines. Loss of PSGL‐1 attenuates DSS colitis, decreases colonic CD4+ T cell numbers, and reduces both Th1 and Th17 cytokine production. Colitis increases recruitment of Th1 (19‐fold) and Th17 (2.5‐fold) cells. PSGL‐1 deficiency in transferred T cells abrogates colonic recruitment of Th1 cells in DSS colitis, whereas Th17 recruitment is unaffected. Conclusions: PSGL‐1 selectively controls Th1 recruitment in colitis. Whereas Th17 recruitment is independent of PSGL‐1, generation of colonic Th17 cytokine requires initial Th1 recruitment. Therefore, attenuating PSGL‐1 binding may prevent colonic recruitment of disease‐causing Th1 cells that promote local Th17 generation. (Inflamm Bowel Dis 2011;)

Bin1: A New Player in IBD Barrier Dysfunction

The ability of the host to regulate permeability of theintestinal barrier is a critical determinant of host defense.Regulation of permeability occurs at the level of epithelialtight junctions (TJ). TJ are composed of transmembraneproteins, such as occludin, claudin, and JAM family pro-teins. Plaque proteins, such as zonula occludins (ZO),connect the TJ complex to F-actin and actomyosin ringsregulating cytoskeletal reorganization (Fig. 1, reviewed in[1]). Several signaling pathways, such as myosin lightchain kinase (MLCK), protein kinase C (PKC), mitogen-activated protein kinases (MAPK), and the Rho family ofsmall GTPases, control the remodeling and maintenance ofTJ. TJ not only regulate paracellular transport of nutrientsand water, but they also provide a barrier against entericmicrobes. Although the exact etiology is unknown, there isample evidence that epithelial barrier function is compro-mised in inflammatory bowel disease (IBD) patients. Datafrom various studies using inert non-metabolized probesindicate that paracellular permeability is increased inCrohn’s Disease (CD) [2]. Furthermore, increased perme-ability may precede the onset of inflammation [3].Researchers also found that enteric permeability isincreased in 10–54 % of healthy, first-degree relatives ofIBD patients [4, 5]. To date, several studies have associatedIBD with mutations in the NOD2 gene [6, 7]. However,NOD2 penetrance of the most at-risk genotypes is low [8],and it is likely that cooperation with other genetic factors,such as TJ defects, is required for disease development.Interestingly, Buhner et al. detected increased permeabilityin IBD patients and family members with NOD2 mutations[9]. Altogether, these findings support the speculation thataltered permeability constitutes one of several geneticallydetermined variables that enhances the risk for developingIBD.The notion that altered epithelial permeability predis-poses patients to IBD is supported by data from severalanimal models with transmucosal barrier defects. Usingtransgenic mice that express constitutively active MLCK,Su et al. demonstrated that, while insufficient to fullyinduce experimental colitis alone, TJ defects exacerbateddisease in the adoptive transfer colitis model [10]. Othermodels of barrier disruption, such as dominant negativeN-cadherin expression, resulted in spontaneous inflamma-tion [11], further enforcing the link between barrier dys-function and immune homeostasis. Altered permeabilitymay also occur due to effects of local cytokines. Tumornecrosis factor, a cytokine critical for the pathogenesis ofIBD, activates MLCK, resulting in TJ disruption viainternalization of occludin from the cell membrane [12].Also, interleukin-13, a cytokine upregulated in ulcerativecolitis, induces expression of the pore-forming TJ proteinclaudin-2, leading to barrier dysfunction [13]. Altogether,these data represent the dynamic relationship betweeninflammation and permeability and how their imbalancemay result in the development of IBD.In this issue of Digestive Diseases and Sciences, Changet al. present data that further support the tie between TJpermeability and IBD pathogenesis via the protein Bridg-ing integrator 1 (Bin1)[14]. Bin1 is a member of the Binamphiphysin rys (BAR) adaptor family with myriad

A Cytosolic Multiprotein Complex Containing p85α Is Required for β-Catenin Activation in Colitis and Colitis-associated Cancer

Wnt/β-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated β-catenin (pβ-CatSer-552) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in β-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with β-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85ΔIEC) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial β-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces β-catenin signaling. Despite worse colitis, p85ΔIEC mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the β-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.

Su1992 TNF Mediates β-Catenin Activation in Intestinal Stem Cells During Colitis

G A A b st ra ct s in colonoid culture and we posit that activation of the TRIF signaling pathway is the main mediator of these changes. Understanding how innate immune recognition pathways crosstalk with epithelial stem cell self-renewal and differentiation pathways will be important for future studies examining the role of stem cells in homeostasis and tumor promotion. We would like to acknowledge the technical help and provision of reagents by Richard J. von Furstenberg and Susan J. Henning.

177 The Roles of TNFR Signaling and NOX1 in Epithelial ß-Catenin Activation During Colitis

TNF blockade in Crohns disease (CD) and ulcerative colitis (UC) patients reveals that TNF plays a central role in disease pathogenesis. Here we examine the potential that TNF activates epithelial stem and progenitor cell (ISC/PC) β-catenin through intestinal epithelial cell (IEC) TNFR1 and TNFR2 signaling. Tissue sections and purified IEC protein isolates were analyzed using Akt-phosphorylated β-catenin (P-β-catenin552 or P-β-cat) Ab. Results: IHC staining of biopsy tissue from control (uninflamed) and inflamed CD/UC patients revealed that Pβ-cat levels increased 2.8-fold in untreated colitis. However, in anti-TNF treated patients selected for ongoing active inflammation, P-β-cat levels were reduced by over 50% (p host) revealed that T cell activation increased numbers of P-βcat-stained IECs by 110% in WT->WT BMC with no increases seen in anti-CD3-treated WT-> TNFR1/2 KO mice. These findings suggest epithelial TNFR signaling regulates IEC β-catenin activation. In studies comparing colitic IL-10 KO (colitis score >3/4) mice to WT healthy controls (n=5 in each group) mRNA from CD44+-sorted IECs was used for genomewide expression array analysis revealing that levels of NADPH oxidase 1 (Nox1) were significantly induced (3.22-fold, p-value: 3 x106). We next tested whether Nox1 may participate in TNF-mediated activation of β-catenin in ISC. We confirmed Nox1 mRNA induction in IECs from IL-10 KO colitis mice (17-fold). Furthermore Nox1 mRNA was induced in purified IECs by anti-CD3 mAb treatment of WT mice (6-fold). Importantly, Nox1 mRNA induction was reduced by over 50% in T cell stimulated TNF-R1/R2 KO mice. Lastly, mRNA from purified CD44+-sorted IECs from Nox1 KO mice revealed that baseline levels of cMyc, cyclinD1, Ascl2 and Lrig1 expression were reduced compared to WT mice. Conclusion: Together the data suggest that epithelial TNF receptor signaling is required for optimal levels of IEC β-catenin activation in patients with active colitis. Data in IL-10 ko and WT mice suggest that epithelial TNFR signaling induces Nox1 which has recently been shown to play a role in Wnt/β-catenin signaling in the intestine (Coant et al. MCB 2010). Together these data are consistent with the hypothesis that Nox1 is a key mediator of TNFinduced epithelial β-catenin activation in UC and CD.