Rebecca B. Katzman

Phosphoinositide 3-Kinase Signaling Mediates β-Catenin Activation in Intestinal Epithelial Stem and Progenitor Cells in Colitis

BACKGROUND & AIMS Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation. METHODS The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice named I-pik3r1KO. Acute inflammation was induced in mice and intestines were analyzed by biochemical and histologic methods. The effects of chemically blocking PI3K in colitic interleukin-10(-/-) mice were examined. Biopsy samples from patients were examined. RESULTS Compared with wild-type, I-pik3r1KO mice had reduced T-cell-mediated Akt and beta-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K-Akt signaling increased nuclear total beta-catenin and P-beta-catenin(552) levels and reduced N-terminal beta-catenin phosphorylation, which is associated with degradation. PI3K inhibition in interleukin-10(-/-) mice impaired colitis-induced epithelial Akt and beta-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-beta-catenin(552) throughout expanded crypts and increased messenger RNA expression of beta-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples. CONCLUSIONS PI3K-Akt signaling cooperates with Wnt to increase beta-catenin signaling during inflammation. PI3K-induced and Akt-mediated beta-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.

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 NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

In inflammatory bowel disease (IBD), aberrant activation of innate and adaptive immune responses enhances mucosal permeability through mechanisms not completely understood. To examine the role of epithelial nuclear factor (NF-kappaB) in IBD-induced enhanced permeability, epithelial-specific IkappaBalpha mutant (NF-kappaB super repressor) transgenic (TG) mice were generated. NF-kB activation was inhibited in TG mice, relative to wild-type mice, following T cell-mediated immune cell activation using an anti-CD3 monoclonal antibody. Furthermore, epithelial NF-kappaB super repressor protein inhibited diarrhea and blocked changes in transepithelial resistance and transmucosal flux of alexa350 (0.35 kDa) and dextran3000 (3 kDa). In vivo perfusion loop studies in TG mice revealed reversed net water secretion and reduced lumenal flux of different molecular probes (bovine serum albumin, alexa350, and dextran3000). Cell-imaging and immunoblotting of low-density, detergent-insoluble membrane fractions confirmed that tight junction proteins (occludin, claudin-1 and zona occludens-1) are internalized through an NF-kappaB-dependent pathway. Taken together, these data suggest that IBD-associated diarrhea results from NF-kappaB-mediated tight junction protein internalization and increased paracellular permeability. Thus, reduction of epithelial NF-kappaB activation in IBD may repair defects in epithelial barrier function, reduce diarrhea, and limit protein (eg, serum albumin) losses. Epithelial NF-kappaB activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid effux into the intestinal lumen.

The Effects of the Epstein-Barr Virus Latent Membrane Protein 2a on B Cell Function

Epstein-Barr Virus (EBV) infects B-lymphocytes circulating through the oral epithelium and establishes a lifelong latent infection in a subset of mature-memory B cells. In these latently infected B cells, EBV exhibits limited gene expression with the latent membrane protein 2A (LMP2A) being the most consistently detected transcript. This persistent expression, coupled with many studies of the function of LMP2A in vitro and in vivo, indicates that LMP2A is functioning to control some aspect of viral latency. Establishment and maintenance of viral latency requires exquisite manipulation of normal B cell signaling and function. LMP2A is capable of blocking normal B cell signal transduction in vitro, suggesting that LMP2A may act to regulate lytic activation from latency in vivo. Furthermore, LMP2A is capable of providing B cells with survival signals in the absence of normal BCR signaling. These data show that LMP2A may help EBV-infected cells to persist in vivo. This review discusses the advances that have been made in our understanding of LMP2A and the effects it has on B cell development, activation, and viral latency.

p53 and PUMA independently regulate apoptosis of intestinal epithelial cells in patients and mice with colitis.

BACKGROUND & AIMS Inflammatory bowel disease (IBD) is associated with increased apoptosis of intestinal epithelial cells (IECs). Mutations in the tumor suppressor p53 appear during early stages of progression from colitis to cancer. We investigated the role of p53 and its target, p53-upregulated modulator of apoptosis (PUMA), in inflammation-induced apoptosis of IECs. METHODS Apoptosis was induced in mouse models of mucosal inflammation. Responses of IECs to acute, T-cell activation were assessed in wild-type, p53⁻/⁻, Bid⁻/⁻, Bim⁻/⁻, Bax3⁻/⁻, Bak⁻/⁻, PUMA⁻/⁻, and Noxa⁻/⁻ mice. Responses of IECs to acute and chronic colitis were measured in mice following 1 or 3 cycles of dextran sulfate sodium (DSS), respectively. Apoptosis was assessed by TUNEL staining and measuring activity of caspases 3 and 9; levels of p53 and PUMA were assessed in colon tissue from patients with and without ulcerative colitis. RESULTS Apoptosis of IECs occurred in the lower crypts of colitic tissue from humans and mice. Colitis induction with anti-CD3 or 3 cycles of DSS increased apoptosis and protein levels of p53 and PUMA in colonic crypt IECs. In p53⁻/⁻ and PUMA⁻/⁻ mice, apoptosis of IECs was significantly reduced but inflammation was not. Levels of p53 and PUMA were increased in inflamed mucosal tissues of mice with colitis and in patients with UC, compared with controls. Induction of PUMA in IECs of p53⁻/⁻ mice indicated that PUMA-mediated apoptosis was independent of p53. CONCLUSIONS In mice and humans, colon inflammation induces apoptosis of IECs via p53-dependent and - independent mechanisms; PUMA also activates an intrinsic apoptosis pathway associated with colitis.

LMP2A Does Not Require Palmitoylation To Localize to Buoyant Complexes or for Function

ABSTRACT Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed constitutively in lipid rafts in latently infected B lymphocytes. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids selective for specific protein association. Lipid rafts have been shown to be necessary for B-cell receptor (BCR) signal transduction. LMP2A prevents BCR recruitment to lipid rafts, thereby abrogating BCR function. As LMP2A is palmitoylated, whether this fatty acid modification is necessary for LMP2A to localize to lipid rafts and for protein function was investigated. LMP2A palmitoylation was confirmed in latently infected B cells. LMP2A was found to be palmitoylated on multiple cysteines only by S acylation. An LMP2A mutant that was not palmitoylated was identified and functioned similar to wild-type LMP2A; unmodified LMP2A localized to lipid rafts, was tyrosine phosphorylated, was associated with LMP2A-associated proteins, was ubiquitinated, and was able to block calcium mobilization following BCR cross-linking. Therefore, palmitoylation of LMP2A is not required for LMP2A targeting to buoyant complexes or for function.

Antioxidant Properties of Mesalamine in Colitis Inhibit Phosphoinositide 3-Kinase Signaling in Progenitor Cells

Background: Mesalamine, 5-aminosalicylic acid (5-ASA), is a potent antioxidant and is known to enhance peroxisome proliferator–activated receptor &ggr; activity in the intestine. Our previous studies suggested reduced Phosphoinositide 3-Kinase (PI3K)/&bgr;-catenin signaling as a mechanism for 5-ASA chemoprevention in chronic ulcerative colitis (CUC). We now hypothesize that 5-ASA mediates changes in intestinal epithelial cell (IEC) reactive oxygen species during colitis to affect phosphatase and tensin homolog (PTEN), PI3K, and &bgr;-catenin signaling. Methods: Here, we examined effects of 5-ASA on oxidant-induced cell signaling pathways in HT-29 cells, IECs from mice, and biopsy tissue from control and CUC patients. Samples were selected to control for inflammation between untreated and 5-ASA–treated CUC patients. Results: Direct evaluation of IEC in H2O2-stimulated whole colonic crypts indicated that 5-ASA reduces reactive oxygen species levels in lower crypt IECs where long-lived progenitor cells reside. Analysis of biopsies from patient samples revealed that 5-ASA increases expression of the antioxidant catalase in CUC patients. Also, 5-ASA increased nuclear peroxisome proliferator–activated receptor &ggr; protein and target gene expression. Data showed 5-ASA–induced peroxisome proliferator–activated receptor &ggr; DNA binding to the PTEN promoter (chromatin immunoprecipitation) and reduced both phosphorylated and oxidized (inactive) PTEN protein levels. Analysis of patient samples revealed 5-ASA that also reduced levels of active phosphorylated Akt in inflamed colitis tissue. Reduced PI3K/Akt signaling and expression of &bgr;-catenin target genes in 5-ASA–treated CUC patients additionally suggests enhanced PTEN activity as well. Conclusions: Therefore, 5-ASA reduces CUC-induced reactive oxygen species in colonic progenitor cells and enhances PTEN activity, thus attenuating PI3K/Akt signaling. These data suggest that the antioxidant properties of 5-ASA may be the predominant mechanism for 5-ASA chemoprevention.

Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection

Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of β-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome.

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.

Genetic Evidence That Phosphoinositol 3 – Kinase (PI3K) is Required for the Epidermal Growth Factor (EGF) Induced Adaptive Response After Ileocecal Resection (ICR)

Background: Growth factors including EGF have emerged as potential therapeutic options for patients with surgical short gut syndrome. The potential augmentation of the adaptive response by EGF makes it an appealing therapy to promote intestinal autonomy. Crypt epithelium responds to small bowel (SB) resection with increased proliferation and fissioning. EGF augments the intestinal epithelial cell (IEC) responses to intestinal resection although its role in regulating β-catenin is unclear. To examine the potential that EGF-induced PI3K mediates the epithelial response to β-catenin activation in ICR, we utilized an inducible genetic model of intestinal epithelial specific deletion of Class IA PI3K p85α AhCre/pi3kr1 ). Our laboratory demonstrated cross-talk between PI3K and the Wnt/β-catenin signaling pathways in inflammatory bowel disease tissue. Previous data from Clevers and colleagues showed that the proliferative responses originate in intestinal stem cells (ISC) and are dependent on Wnt/β-catenin signaling. Here we test the notion that EGF stimulates IEC via β-catenin activation after ICR through PI3K pathway. Design/Methods: C57BL/6 wild type (WT) mice and AhCre/pi3kr1 animals underwent sham surgery, ICR, or no surgery. All animals were given 50 ug/kg/day of rEGF in osmotic pumps immediately after the surgery. Five days later, SB morphometrics, immunohistochemistry (IHC) for BrdU incorporation (2hr), phospho(p) AKT(Thr308), and pBcatenin(ser552) and western blots of purified IEC proteins including cleaved caspase-3 and nuclear total β-catenin were performed. Results: Compared to EGF-treated sham, EGF-treated ICR mice have increased crypt length (63.3 ± 7 vs 118.3 ±2 8.9 μm, p< 0.05), BrdU incorporation, pAKT(Thr308), pβ-catenin(ser552) by IHC, and nuclear total β-catenin (WB). Data on AhCre/pi3kr1 ICR mice revealed reduced crypt length (118.3±28.9 vs. 67.1 ± 15.5 μm, p< 0.05 ), incorporation of pBcatenin(ser552) (1.43 ± 0.58 vs. 0.85 ± 0.19 counts/crypt, p< 0.05), and a 90% reduction of nuclear total β-catenin by WB. Interestingly, BrdU incorporation in AhCre/pi3kr1 ICR mice was similar to EGF-treated ICR alone, whereas cleaved caspase-3 (WB) doubled, suggesting enhanced apoptosis attenuated the observed induction of IEC proliferation. Conclusions: These data indicate that the epithelial response to ICR in EGF-treated mice enhanced crypt length and IEC proliferation along with PI3K and β-catenin signaling. Furthermore, data indicate that PI3K enhanced IEC crypt lengths, β-catenin activation, and cell survival. Thus, we propose that enhanced IEC PI3K signaling in short bowel patients may facilitate the recovery of absorptive surface through epithelial restitution.