Tracy S. Obertone

TNF-α upregulates adenosine 2b (A2b) receptor expression and signaling in intestinal epithelial cells: a basis for A2bR overexpression in colitis

Abstract.Adenosine is an endogenous signaling molecule upregulated during inflammatory conditions. Acting through the A2b receptor (A2bR), the predominant adenosine receptor in human colonic epithelia, adenosine has been directly implicated in immune and inflammatory responses in the intestine. Little is known about expression and regulation of A2bR during inflammation. Tumor necrosis factor alpha (TNF-α) is highly upregulated during chronic and acute inflammatory diseases. This study examined the expression of A2bR during colitis and studied effects of TNF-α on A2bR expression, signaling and function. Results demonstrated that A2bR expression increases during active colitis. TNF-α pretreatment of intestinal epithelial cells increased A2bR messenger RNA and protein expression. TNF-α significantly increased adenosine-induced membrane recruitment of A2bR and cyclic adenosine monophosphate downstream signaling. Further, TNF-α potentiated adenosine-induced shortcircuit current and fibronectin secretion. In conclusion, we demonstrated that TNF-α is an important regulator of A2bR, and during inflammation, upregulation of TNF-α may potentiate adenosine-mediated responses.

Prohibitin Is a Novel Regulator of Antioxidant Response That Attenuates Colonic Inflammation in Mice

BACKGROUND & AIMS Increased free radicals and/or impaired antioxidant defenses have been shown to play a pathogenetic role in human and animal models of inflammatory bowel disease. Our previous studies showed that prohibitin (PHB) levels are decreased during colitis and that cultured intestinal epithelial cells overexpressing PHB are protected from oxidative stress. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on oxidative stress associated with experimental colitis and the potential mechanism by which PHB functions as an antioxidant using PHB transgenic mice. METHODS Colitis was induced using 2 established mouse models (Salmonella typhimurium and dextran sodium sulfate) in PHB transgenic mice and wild-type littermates. Oxidative stress was determined by measuring glutathione and protein carbonyl levels in the cecum or colon. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of oxidant responses, expression, and activation, was assessed in colon mucosa and cultured intestinal epithelial cells overexpressing PHB. RESULTS Cells overexpressing PHB showed sustained Nrf2 nuclear accumulation and DNA binding during oxidant stress. PHB transgenic mice exhibited decreased oxidative stress and colitis and increased Nrf2 messenger RNA expression, nuclear protein translocation, and DNA binding compared with wild-type littermates during colitis. CONCLUSIONS These results show that PHB is a regulator of Nrf2 expression in intestinal epithelial cells during oxidative conditions and prevents inflammation-associated oxidative stress and injury through sustained activation of Nrf2. Our data show that PHB is a novel regulator of antioxidants and suggest that restoration of PHB levels represents a potential therapeutic approach in inflammatory bowel disease.

Nanoparticle-based therapeutic delivery of prohibitin to the colonic epithelial cells ameliorates acute murine colitis

Background: Intestinal epithelial expression of antioxidants and nuclear factor kappa B (NF‐&kgr;B) contribute to mucosal barrier integrity and epithelial homeostasis, two key events in the pathogenesis of inflammatory bowel disease (IBD). Genetic restoration of intestinal epithelial prohibitin 1 (PHB) levels during experimental colitis reduces the severity of disease through sustained epithelial antioxidant expression and reduced NF‐&kgr;B activation. To determine the therapeutic potential of restoring epithelial PHB during experimental colitis in mice, we assessed two methods of PHB colonic mucosal delivery: adenovirus‐directed administration by enema and poly(lactic acid) nanoparticle (NPs) delivery by gavage. Methods: As a proof‐of‐principle to demonstrate the therapeutic efficacy of PHB, we utilized adenovirus‐directed administration by enema. Second, we used NPs‐based colonic delivery of biologically active PHB to demonstrate therapeutic use for human IBD. Colitis was induced by oral administration of dextran sodium sulfate (DSS) in water for 6‐7 days. Wildtype mice receiving normal tap water served as controls. Results: Both methods of delivery resulted in increased levels of PHB in the surface epithelial cells of the colon and reduced severity of DSS‐induced colitis in mice as measured by body weight loss, clinical score, myeloperoxidase activity, proinflammatory cytokine expression, histological score, and protein carbonyl content. Conclusions: This is the first study to show oral delivery of a biologically active protein by NPs encapsulated in hydrogel to the colon. Here we show that therapeutic delivery of PHB to the colon reduces the severity of DSS‐induced colitis in mice. PHB may represent a novel therapeutic target in IBD. (Inflamm Bowel Dis 2010)

Overexpression of Ste20-Related Proline/Alanine-Rich Kinase Exacerbates Experimental Colitis in Mice

Inflammatory bowel disease, mainly Crohn’s disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease.

Adenosine 2B receptor expression is post-transcriptionally regulated by microRNA.

We have reported that epithelial adenosine 2B receptor (A2BAR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor α (TNF-α). Here, we examined the mechanism that governs A2BAR expression during colitis. A 1.4-kb sequence of the A2BAR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3′-untranslated region (UTR) of A2BAR mRNA was examined by cloning this 3′-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-α induced a 35-fold increase in A2BAR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A2BAR mRNA following TNF-α treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3′-UTR of the A2BAR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-α reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A2BAR levels by >60%. Blockade of miR27b increased A2BAR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A2BAR mRNA. Collectively, these data demonstrate that TNF-α-induced A2BAR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A2BAR expression in murine colitis.

Interleukin-6 Transcriptionally Regulates Prohibitin Expression in Intestinal Epithelial Cells

Prohibitin (PHB) is a highly conserved protein that has multiple functions in the cell. We recently demonstrated that PHB plays an important role in combating oxidative stress and its expression is down-regulated in human and animal models of inflammatory bowel disease. Little is known regarding the regulation of PHB expression in intestine or other tissues. In this study we examined the regulation of PHB expression in intestinal epithelial cells using the model cell line Caco2-BBE. We successfully cloned the 1192-bp human PHB promoter region and identified the transcription start site 1594 bp upstream from the translation start site due to an intervening intron. We show that the acute phase cytokine interleukin-6 (IL-6) increases PHB protein and mRNA abundance and induces PHB promoter activation. The IL-6 response element site in the PHB promoter is required for maximal basal promoter activity and responsiveness to IL-6. IL-6 also increases binding of nuclear proteins to the IL-6 response element in the PHB promoter that are supershifted by a STAT3 antibody. Both basal promoter activity and IL-6 responsiveness are attenuated by signal transducer and activator of transcription 3 short interference RNA, suggesting that signal transducer and activator of transcription 3 mediates PHB activity by IL-6. Confirming these in vitro results, IL-6-/- mice exhibit reduced PHB expression in the colon compared with wild-type mice. These results suggest that IL-6 modulates PHB expression in cultured intestinal epithelial cells and in the intestine in vivo.

Ca2+-Dependent Permeability Transition and Complex I Activity in Lymphoblast Mitochondria from Normal Individuals and Patients with Huntington's or Alzheimer's Disease

Alzheimer’s disease (AD) and Huntington’s disease (HD) are neurodegenerative diseases with distinct clinical and morphological manifestations and genetic etiologies. Presently it is hypothesized that a common feature of different neurodegenerative diseases is impairment of mitochondrial energy metabolism in brain cells. Mitochondria play a key role in glutamate excitotoxicity and other forms of cell death via apoptotic or necrotic pathways.1,2 The proposed mechanisms by which mitochondria induce cell death are the Ca2+-dependent disruption of mitochondrial electrical membrane potential (∆Ψ)1,3 and opening of the pore with nonspecific permeability, known as the mitochondrial permeability transition pore (mPTP).4 Studies on mitochondrial neuromuscular diseases5 have shown the usefulness of evaluating mitochondrial function in mitochondria isolated from muscle biopsies or from patient-derived lymphoblasts grown in culture. The latter approach may be particularly useful for understanding pathogenesis of neurodegenerative diseases (NDD). The purpose of this work was to study the functional parameters of mitochondria from human lymphoblasts, particularly the Ca2+-dependent mPTP in patients with HD or AD, and elderly individuals without NDD.

Ecto-Phosphorylation of CD98 Regulates Cell-Cell Interactions

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.

189 Treatment of Experimental Colitis Using Adenovirus-Directed Expression of Prohibitin

associated with an increased risk of neoplastic progression. Aim: To compare the prevalence of DNA promoter hypermethylation in esophageal biopsies of BE patients with residual BE (ND or LGD) after endoscopic ablation therapy versus patients who present with BE and ND or LGD at baseline and have never been treated. Methods: Using a prospective large BE cohort of 220 patients followed over the past 6 years, we performed a case-control study comparing two groups of patients: 1) patients with BE and high-grade dysplasia and/or intramucosal adenocarcinoma who were treated with photodynamic therapy and had residual BE [ND or LGD as the highest grade of dysplasia on follow-up upper endoscopies], and 2) patients with a baseline diagnosis of BE (ND or LGD) who had never undergone endoscopic therapy. All patients had undergone endoscopic surveillance with the Seattle biopsy protocol. Esophageal biopsies of BE from residual disease in group 1 and at baseline in group 2 were analyzed using methylation-specific PCR, and the prevalence of p16 and APC promoter methylation was compared. Results: There were 15 patients with residual BE (ND or LGD) after photodynamic therapy and 82 patients who presented with BE and ND or LGD at baseline and had never been treated. Patients with residual BE (ND or LGD) after ablation had a significantly higher prevalence of DNAmethylation compared to patients who presented with BE and ND or LGD at baseline for both p16 (73% vs. 31%; OR [95% CI]=6.1 [1.7521.2]; p<0.01) and APC (80% vs. 50%; OR [95% CI]=4.0 [1.0-15.3]; p=0.04). Conclusions: Since DNA methylation is a strong predictor of subsequent neoplastic progression to highgrade dysplasia or esophageal adenocarcinoma, the high prevalence of DNA methylation seen in patients with residual BE (ND or LGD) after endoscopic ablation suggest that these patients may have a higher risk of neoplastic progression compared to patients who have an initial presentation of BE and ND or LGD at baseline and have never been treated. Therefore, residual BE (ND or LGD) after endoscopic ablation should likely be managed more aggressively than de novo BE with ND or LGD.