Fatty Acid Binding Protein Promoter Research Articles

Peroxisome proliferator-activated receptor γ isoforms differentially regulate preadipocyte proliferation, apoptosis, and differentiation in chickens

Peroxisome proliferator-activated receptor γ (PPARγ) has 2 protein isoforms (PPARγ1 and PPARγ2) generated by alternative promoter usage and alternative splicing. However, their functional uniqueness and similarity remain unclear. In the study, we investigated the effects of lentivirus-mediated overexpression of PPARγ1 and PPARγ2 on proliferation, apoptosis, and differentiation of the immortalized chicken preadipocytes. Cell Counting Kit–8 assay showed PPARγ1 and PPARγ2 overexpression markedly suppressed cell proliferation, and fluorescence activated cell sorting analysis showed that PPARγ1 and PPARγ2 overexpression caused cell cycle arrest at G0/G1 phase. Cell death detection ELISA analysis showed both PPARγ1 and PPARγ2 overexpression induced cell apoptosis. Oil red O staining and gene expression analysis showed both PPARγ1 and PPARγ2 overexpression promoted preadipocyte differentiation. In the presence of PPARγ ligand, rosiglitazone, PPARγ2 overexpression was more potent in inducing apoptosis, promoting adipogenesis, and suppressing cell proliferation than PPARγ1 overexpression. We further explored the molecular basis for their functional differences. Reporter gene assay showed that under ligand conditions, PPARγ2 overexpression resulted in 1.68-fold increase in transcription activity compared with PPARγ1. Electrophoretic mobility shift assay showed both PPARγ1 and PPARγ2 could bind to PPAR response element (PPRE) as heterodimer with retinoid X receptor alpha, and by comparison, PPARγ2 had a higher affinity for PPRE than PPARγ1. Reporter gene assay showed expression PPARγ1 and PPARγ2 similarly induced fatty acid synthase and adipocyte fatty acid–binding protein promoter activity but differentially induced lipoprotein lipase and perilipin 1 promoter activities. Coimmunoprecipitation analysis showed that PPARγ1 and PPARγ2 interacted similarly with the coactivators, Tat-interacting protein 60. Taken together, our results demonstrate that PPARγ1 and PPARγ2 differentially regulate preadipocyte proliferation, apoptosis, and differentiation as a result of their distinct and overlapping molecular functions.

205 Towards the correction of meconium ileus with cystic fibrosis transmembrane conductance regulator (CFTR) intestinal expression in CFTR knockout sheep

Cystic fibrosis (CF) is a human autosomal genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is responsible for Cl − anion transport in epithelial cells. We previously generated CFTR +/− and CFTR −/− lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. The CFTR −/− lambs display many features similar to humans with CF, including meconium ileus (MI), pancreatic fibrosis, portal fibrosis and biliary hyperplasia, small gallbladder, and absence of the vas deferens. Although MI affects only 15 to 20% of human babies with CF, it was observed in 100% of newborn CFTR −/− lambs and was the primary cause of death. We here hypothesized that the transgenic expression of ovine CFTR cDNA under regulation of the rat intestinal fatty acid binding protein (iFABP) promoter would promote the correction of MI in CFTR −/− sheep. In this study, we constructed the pcDNA3.1>iFABP>CFTR expression vector in order to generate iFABP>CFTR transgenic CFTR −/− sheep. PCR and sequence analysis of all junction regions confirmed the presence of the inserts in the vector. We transfected male and female CFTR −/− sheep fetal fibroblasts with the construct. Three days after transfection, limiting dilution and G418 selection were used for the isolation of resistant single cell derived colonies. A total of 15 male colonies containing the pcDNA3.1>iFABP>CFTR vector was confirmed by PCR and sequence analysis. Three male cell colonies were used as nuclear donors for SCNT. In total, 129 one-cell-stage cloned embryos were generated and transferred into 11 estrus-synchronized recipients. Four recipients (4/11; 36.3%) were confirmed pregnant at Day 40 to 45 of gestation. One pregnancy went to term and resulted in the birth of a single lamb. The necropsy indicated the same abnormalities, including MI, seen in CFTR −/− lambs. A PCR assay confirmed the presence of the iFABP>CFTR transgene in the genome of the cloned offspring. The lack of MI correction is likely due to the low expression of CFTR in this particular colony. Western blot analysis is in progress. Additional embryo transfers will be performed using different single-cell-derived colonies.

AP2-NR4A3 transgenic mice display reduced serum epinephrine because of increased catecholamine catabolism in adipose tissue.

The NR4A orphan nuclear receptors function as early response genes to numerous stimuli. Our laboratory has previously demonstrated that overexpression of NR4A3 (NOR-1, MINOR) in 3T3-L1 adipocytes enhances insulin-stimulated glucose uptake. To assess the in vivo effect of NR4A3 on adipocytes, we generated transgenic mice with NR4A3 overexpression driven by the adipocyte fatty acid-binding protein (AP2) promoter (AP2-NR4A3 mice). We hypothesized that AP2-NR4A3 mice would display enhanced glucose tolerance and insulin sensitivity. However, AP2-NR4A3 mice exhibit metabolic impairment, including increased fasting glucose and insulin, impaired glucose tolerance, insulin resistance, decreased serum free fatty acids, and increased low-density lipoprotein-cholesterol. AP2-NR4A3 mice also display a significant reduction in serum epinephrine due to increased expression of catecholamine-catabolizing enzymes in adipose tissue, including monoamine oxidase-A. Furthermore, enhanced expression of monoamine oxidase-A is due to direct transcriptional activation by NR4A3. Finally, AP2-NR4A3 mice display cardiac and behavioral alterations consistent with chronically low circulating epinephrine levels. In conclusion, overexpression of NR4A3 in adipocytes produces a complex phenotype characterized by impaired glucose metabolism and low serum catecholamines due to enhanced degradation by adipose tissue.

Abstract C68: Decreased antitumor cytotoxic immunity in African American colon cancer

Abstract Background: African Americans (AA) have a higher incidence of colorectal cancer (CRC) and more aggressive disease than patients of other ethnicities. Several studies have suggested that biological/genetic factors contribute to this health disparity. Recent work has shown the importance of the immune system to cancer patient prognosis, for CRC in particular. Anti-tumor T helper 1 (TH1) cell activation of cytotoxic T lymphocytes (CTL) increases CRC patient disease-free survival while deficiencies in these responses correlate with a worse prognosis. We hypothesize that differences in anti-tumor immunity by race contribute to the disparity and lead to more aggressive CRC in AA patients. Methods: Over 250 human colon tumors samples were stained using immunohistochemistry to evaluate in situ immune cell infiltration (CD8 and CD57), CTL activity [granzyme B (GzmB)], and IL-17 production. Also, using GzmB immunostaining, we analyzed the histological location of CTL/Natural Killer (NK) cell activity at intratumoral vs. invasive border sites of the CRC samples. In addition, a colon cancer mouse model is being used to identify mechanisms that may alter TH1 and CTL activity in developing tumors. The mouse model is the TS4Cre x APCfl-468 conditional Adenomatous Polyposis Coli (APC) knockout. Exons 11 and 12 are deleted from one APC gene in the colon and distal ileum due to the expression of CRE under a Fatty acid binding protein promoter leading to polyposis. These colonic tumors are being evaluated using qPCR on immune and inflammatory gene products. Results: Our studies on human microsatellite stable colon cancer samples showed that the number of CD8+ and CD57+ tumor-infiltrating cells doesn't differ by race (p=0.83 and 0.32, respectively). The level of GzmB+ cells, i.e. CTLs and activated NK cells, however, does vary significantly by race (p=0.01), particularly at the invasive border (p=0.0006). The numbers of IL-17A producing cells do not vary (p=0.63) but IL-17 may modulate GzmB correlation with CD8 and CD57 by race. For example, in AA, the correlation between GzmB and CD8 drops from 0.80 in IL-17Ahigh samples to 0.53 in IL-17Alow samples. For GzmB and CD57, the correlation level drops from 0.51 to 0.28 between IL-17A-high and -low samples, respectively. No such drop in correlation is observed in Caucasian American samples. In our mouse model, our preliminary studies show the TS4CRExAPCfl-468 mice initiate colonic polyposis at ~18 weeks of age and invasive cancer is observed in 20-25% of mice older than 31 weeks. The mean number of colonic polyps significantly increases between 18 and 24 weeks (from 2.3±0.9 to 8.0±1.9 tumors/mouse). Interestingly, the number declines by 36 weeks of age (5.5±0.5 tumors/mouse). However, the mean size of the colonic polyps significantly increases from 18 weeks to 36 weeks of age (from 1.54mm±0.55 to 4.23mm±0.56). To determine what mechanisms might result in lower anti-tumor CTL/NK activity, mouse tumors at different stages of development are currently being evaluated for IL-27, GzmA, IL-23, ƔIFN, CD8, Perforin, CX3CL1, IL-17a, H2DMB2, and Tbet gene expression using qPCR. Primers sets are also being developed to include IL-4, IL-6, IL-10, IL-12p40, and FoxP3 in our analyses. Conclusion: Our data to date indicate that the number of AA patients with high numbers of GzmB expressing cells infiltrating their tumors is significantly lower than Caucasians. In addition, IL-17 may have a modulatory effect on anti-tumor cytotoxicity by race. These observations support our hypothesis that immune processes contribute to the racial disparities observed in CRC. Current studies in our mouse model of CRC should identify mechanisms decreasing anti-tumor CTL/NK cell activity that will be tested in our Caucasian and AA colon cancer patient tumor samples in future studies. Note: This abstract was not presented at the conference. Citation Format: Mohammad W. Khan, MengXi Tian, John M. Carethers, Kathleen L. McGuire. Decreased antitumor cytotoxic immunity in African American colon cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C68.

Abstract 3925: The PIK3CA H1047R mutation can initiate tumorigenesis in the colon

Abstract Background: Our understanding of how mutations within the intestinal epithelium result in tumor development and progression is continuously evolving. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K resulted in invasive mucinous adenocarcinomas in the proximal colon through a non-canonical mechanism. We sought to determine if the hotspot PIK3CA H1047R mutation, commonly found in human colorectal cancers, can also induce intestinal tumorigenesis. Methods: Female FVB mice carrying a conditional PIK3CA H1047R mutation (PIK3CAH1047R) were crossed to B6 males carrying atransgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3CAH1047R progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon. 22 F1 FC PIK3CAH1047R mice were examined. Necropsy was performed at approximately 200 days of age or once moribund. Results: 50% of FC PIK3CAH1047R mice develop invasive adenocarcinomas within the colon. These cancers are highly mucinous and often penetrate through the serosal layer of the colon. These lesions can be found throughout the colon, but are more common proximally. No tumors were identified within the small intestine. These cancers can have associated invasion into the regional lymphatics and metastases to the mesentery. Interestingly these cancers have a minimal luminal component compared to the amount of submucosal extension. Precursor lesions are able to be identified and are not polypoid. These cancers demonstrate activation of the AKT signaling pathway, but do not demonstrate nuclear localization of β-catenin. Conclusion: This is the first report of the hotspot PIK3CA H1047R mutation leading to intestinal tumorigenesis through a non-canonical mechanism, resulting in flat mucinous invasive adenocarcinomas. Citation Format: Cheri Pasch, Molly Maher, Dustin Deming. The PIK3CAH1047R mutation can initiate tumorigenesis in the colon. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3925. doi:10.1158/1538-7445.AM2014-3925

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

Abstract 2738: PIK3CA and APC mutations are synergistic in the development of intestinal cancers.

Abstract Background: Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been thoroughly investigated. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas in the proximal colon. These cancers form via a non-canonical mechanism without aberrations in Wnt signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the vast majority of human colon cancers and often occurs concurrently with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine using novel murine models. Methods: B6 females carrying a transgene encoding for a chimeric protein with the iSH2 domain of the p85 subunit of PI3K fused to the N-terminus of p110 (PIK3ca*) were crossed to B6 males carrying the Min allele of Apc (ApcMin/+). The resulting B6 PIK3ca* ApcMin/+ mice were then crossed to mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3ca* ApcMin/+ progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon, as well as having lost one allele of Apc. An additional novel model system was developed to induce mutations in the cells of the mid to distal colon using a minimally invasive technique to instil adenovirus expressing the Cre recombinase. These tumors could then be monitored over time with colonoscopy with biopsies taken for histological and molecular analyses. Results: FC PIK3ca* ApcMin/+ mice develop multiple small intestinal and colon tumors, including invasive adenocarcinomas with lymphatic and mesenteric metastases. Tumor multiplicity and size were increased. Moreover, the tumors appeared less differentiated and were more invasive as compared to those in controls. Advanced adenomas and adenocarcinomas were easily detectible with a novel near-infrared phospholipid ether analog, CLR1502, that selectively accumulates in tumors. In the proximal colon, mucinous adenocarcinomas that were initiated by the dominant active PI3K rarely exhibited nuclear β-catenin and when observed, this localization was limited to select areas. This indicates that in these tumors loss of APC in these cells was a late event and can be a source of clonal outgrowth. The tumors that form in this model are microsatellite stable. Interestingly this synergy appears to be independent of PI3K mediated regulation of GSK-3β and β-catenin signaling. Conclusion: Expression of a dominant active PI3K synergizes with loss of APC activity resulting in dramatic changes in tumor multiplicity, size, morphology, and invasiveness. Citation Format: Dustin Deming, Alyssa Leystra, Laura Nettekoven, Chelsie Kohns, Linda Clipson, Dawn Albrecht, Jeff Bacher, Mary Kay Washington, Christopher Pazoles, Jamey Weichert, Richard Halberg. PIK3CA and APC mutations are synergistic in the development of intestinal cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2738. doi:10.1158/1538-7445.AM2013-2738

Lineage Targeted MHC-II Transgenic Mice Demonstrate the Role of Dendritic Cells in Bacterial-driven Colitis

Inflammatory bowel disease (IBD) pathogenesis involves an inadequately controlled immune reaction to intestinal microbiota, and CD4(+) T cells, dependent on MHC class II (MHC-II) processing and presentation by antigen-presenting cells (APC), play important roles. The role of professional APC (macrophages and dendritic cells [DCs]) and nonprofessional APC (intestinal epithelial cells [IECs]) in microbial-driven intestinal inflammation remains controversial. We generated transgenic animals on an MHC-II(-/-) genetic background in which MHC-II is expressed on 1) DC via the CD11c promoter (CD11cTg) or 2) IEC via the fatty acid binding protein (liver) promoter (EpithTg). These mice were crossed with Rag2(-/-) mice to eliminate T and B cells (CD11cTg/Rag2(-/-) and EpithTg/Rag2(-/-)). Helicobacter bilis (Hb) infection and adoptive transfer (AT) of naïve CD4 T cells were used to trigger IBD. CD11cTg/Rag2(-/-) mice infected with Hb+AT developed severe colitis within 3 weeks post-AT, similar to disease in positive control Rag2(-/-) mice infected with Hb+AT. CD11cTg/Rag2(-/-) mice given AT alone or Hb alone had significantly less severe colitis. In contrast, EpithTg/Rag2(-/-) mice infected with Hb+AT developed mild colitis by 3 weeks and even after 16 weeks post-AT had only mild lesions. MHC-II expression restricted to DCs is sufficient to induce severe colitis in the presence of T cells and a microorganism such as Hb within 3 weeks of AT. Expression of MHC-II solely on IEC in the presence of a microbial trigger and T cells was insufficient to trigger severe colitis.

Abstract 1341: Mice expressing activated PI3K develop advanced colon cancer

Abstract Background: Mutations in PIK3CA are a common feature of human cancers and multiple new PI3K inhibitors are in clinical development. These mutations are present in 20-30% of colorectal cancers in humans; however, the effects of such mutations on the intestinal mucosa have not been previously studied in animal models. Here we demonstrate that the expression of a dominant active form of the PI3K protein in the mouse intestine results not only in hyperplasia but also advanced neoplasia. Methods: Mice carrying a transgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC) were crossed to mice carrying a transgene encoding a chimeric protein with the iSH2 domain of the p85 subunit fused to the N-terminus of p110 (PIK3ca*). Progeny carrying both transgenes (FC PIK3ca*) express this constitutively active form of p110 in epithelial cells of the distal small bowel and colon. Results: Invasive mucinous adenocarcinomas develop in the proximal colon by only 50 days of age. These tumors are very aggressive, spreading into the mesentery and adjacent organs, and their histological characteristics are strikingly similar to those of invasive colon cancers in humans. Neoplastic cells exhibit a high rate of cell proliferation as seen by increased Ki67 immunohistochemical staining and phosphorylation of AKT, pS6, and p4E-BP1 by western blot demonstrating downstream signaling of PI3K. Interestingly, these cells did not exhibit perturbations in ERK and WNT signaling. Consistent with the lack of aberrant WNT signaling, no benign polypoid intermediaries were identified. Together, the results indicate that tumors in this model form via a non-canonical mechanism. A cohort of FC PIK3ca* mice were imaged by FDG dual hybrid PET/CT colonography and mice with similar sized tumors were divided into control and experimental arms. The experimental arm was treated with rapamycin (6mg/kg/day) for 14 days. Both groups were re-imaged 14 days later. The full results of the rapamycin treatment will be presented. Conclusion: This unique model of advanced colonic invasive adenocarcinomas has the potential to further our understanding of human disease and facilitate the development of therapeutics, especially inhibitors targeting downstream of PI3K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1341. doi:1538-7445.AM2012-1341

Netrin-1 overexpression in kidney proximal tubular epithelium ameliorates cisplatin nephrotoxicity

Netrin-1, a multifunctional laminin-related protein is widely expressed in various tissues, including kidney. The pathophysiological roles of netrin-1 in toxic acute kidney injury are unknown. To determine the role of netrin-1 in cisplatin-induced nephrotoxicity, we used netrin-1 transgenic mice that overexpress netrin-1 in the proximal tubular epithelium using the fatty acid binding protein promoter. Administration of cisplatin caused severe renal injury in WT mice but not in netrin-1 transgenic mice. Functional improvement was associated with better preservation of morphology, reduced cytokine expression and oxidative stress in the kidney, and reduced serum and urine cytokine and chemokine levels of transgenic mice as compared with WT mice. Cisplatin induced an increase in neutrophil infiltration into the kidney of WT mice, which was not significantly reduced in netrin-1 transgenic mice. Interestingly, ischemia reperfusion induced a large increase in apoptosis in WT mice but not in netrin-1 transgenic mice (215±40 vs 94±20 cells/5 HPF ( × 400), P<0.0001), which was associated with reduced caspase-3 and p53 activation in the transgenic kidney. These results suggest that netrin-1 protects renal tubular epithelial cells against cisplatin-induced kidney injury by suppressing apoptosis and inflammation.

Constitutive Activation of IKKβ in Adipose Tissue Prevents Diet-Induced Obesity in Mice

The IκB kinase β (IKKβ) is a master kinase involved in obesity-related inflammation and insulin resistance through nuclear factor κB dependent and independent pathways. However, the effect of IKKβ activation in adipose tissue, the organ critical for storage of excessive energy and initiation of inflammatory responses in the context of obesity, on systemic insulin sensitivity and metabolism, has not been investigated. In our study, we found that mice overexpressing the constitutively active IKKβ in adipose tissue under the control of murine adipocyte fatty acid binding protein (aP2) promoter were protected from age-related and diet-induced body weight gains, despite increased food intake. The aP2-IKKβ SE mice have significantly reduced weights in all white adipose tissue depots and reduced triglyceride contents in adipose tissue, liver, and muscle. Despite increased systemic and tissue inflammation, aP2-IKKβ SE mice displayed decreased blood glucose levels, improved glucose, and insulin tolerance. This may be at least partially attributable to increased energy expenditure. Histological analysis revealed presence of many small adipocytes in white adipose tissue of aP2-IKKβ SE mice fed on high-fat diet. Furthermore, transgenic expression of IKKβ in adipose tissue improved high-fat diet-induced hepatosteatosis. Collectively, increased energy expenditure and reduced plasma free fatty acid levels may contribute to enhanced systemic insulin sensitivity in aP2-IKKβ SE mice. Our study demonstrates that presence of inflammation in adipose tissue before the development of obesity has beneficial effect on metabolism.

Optimization of hCFTR Lung Expression in Mice Using DNA Nanoparticles

Efficient and prolonged human cystic fibrosis transmembrane conductance regulator (hCFTR) expression is a major goal for cystic fibrosis (CF) lung therapy. A hCFTR expression plasmid was optimized as a payload for compacted DNA nanoparticles formulated with polyethylene glycol (PEG)-substituted 30-mer lysine peptides. A codon-optimized and CpG-reduced hCFTR synthetic gene (CO-CFTR) was placed in a polyubiquitin C expression plasmid. Compared to hCFTR complementary DNA (cDNA), CO-CFTR produced a ninefold increased level of hCFTR protein in transfected HEK293 cells and, when compacted as DNA nanoparticles, produced a similar improvement in lung mRNA expression in Balb/c and fatty acid binding protein promoter (FABP) CF mice, although expression duration was transient. Various vector modifications were tested to extend duration of CO-CFTR expression. A novel prolonged expression (PE) element derived from the bovine growth hormone (BGH) gene 3' flanking sequence produced prolonged expression of CO-CFTR mRNA at biologically relevant levels. A time course study in the mouse lung revealed that CO-CFTR mRNA did not change significantly, with CO-CFTR/mCFTR geometric mean ratios of 94% on day 2, 71% on day 14, 53% on day 30, and 14% on day 59. Prolonged CO-CFTR expression is dependent on the orientation of the PE element and its transcription, is not specific to the UbC promoter, and is less dependent on other vector backbone elements.

Regulated release of nitric oxide by fibroblastic reticular cells controls the frequency and pathogenicity of autoreactive T cells in a model of intestinal tolerance (44.37)

Abstract Fibroblastic reticular cells (FRCs) are non-hematopoietic stromal cells of lymphoid organs wielding significant immunological impact. They exert important effects on the migration and homeostasis of naïve T cells however, their influence on activated T cells is undescribed. Here we report that nitric oxide (NO) release by FRCs profoundly inhibits T cell proliferation without altering early activation signals. Mechanistically, the expression of nitric oxide synthase 2 and production of NO paralleled the activation of T cells and required a tripartite synergism between IFNgamma, TNFalpha, and direct contact with activated CD8 T cells. We have also assesed the role of NOS2 in the iFABP-tOVA transgenic mouse model in which a truncated, cytosolic form of ovalbumin (tOVA136-336) is expressed as a self-antigen by mature intestinal epithelial cells under the control of the intestinal fatty acid binding protein (iFABP) promoter. In this model, where FRCs play a major role in the induction of CD8 T cell tolerance, FRC-derived NO controlled the magnitude of the response at the level of T cell proliferation. The percentage and absolute number of self-reactive CD8 T cells was increased in NOS2-/- iFABP mice, resulting in increased weight loss and more severe ileocolitis. Our study illuminates a hitherto unrecognized role of FRCs in regulating T cell responses that may have important implications for inflammatory bowel diseases.

Cystic fibrosis transmembrane conductance regulator with a shortened R domain rescues the intestinal phenotype of CFTR −/− mice

Gene transfer could provide a novel therapeutic approach for cystic fibrosis (CF), and adeno-associated virus (AAV) is a promising vector. However, the packaging capacity of AAV limits inclusion of the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA together with other regulatory and structural elements. To overcome AAV size constraints, we recently developed a shortened CFTR missing the N-terminal portion of the R domain (residues 708-759, CFTRΔR) and found that it retained regulated anion channel activity in vitro. To test the hypothesis that CFTRΔR could correct in vivo defects, we generated CFTR(-/-) mice bearing a transgene with a fatty acid binding protein promoter driving expression of human CFTRΔR in the intestine (CFTR(-/-);TgΔR). We found that intestinal crypts of CFTR(-/-);TgΔR mice expressed CFTRΔR and the intestine appeared histologically similar to that of WT mice. Moreover, like full-length CFTR transgene, the CFTRΔR transgene produced CFTR Cl(-) currents and rescued the CFTR(-/-) intestinal phenotype. These results indicate that the N-terminal part of the CFTR R domain is dispensable for in vivo intestinal physiology. Thus, CFTRΔR may have utility for AAV-mediated gene transfer in CF.

Transgenic Expression of Walleye Dermal Sarcoma Virus rv-cyclin Gene in Zebrafish and Its Suppressive Effect on Liver Tumor Development After Carcinogen Treatment

A retrovirus homologue gene of cellular cyclin D₁, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors.

Ectopic expression of P-cadherin correlates with promoter hypomethylation early in colorectal carcinogenesis and enhanced intestinal crypt fission in vivo.

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential "field effect" of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.

Over‐expression of monoacylglycerol lipase (MGL) in mouse small intestine results in an obese phenotype

We recently demonstrated that, in addition to the well known anabolic metabolism of intestinal MG, MG hydrolytic activity and expression of the MG lipase (MGL) gene were present in rodent small intestine. The function of small intestinal MGL is unknown. We therefore generated transgenic mice (iMGL mice) that over‐expressed MGL specifically in small intestine using the intestinal fatty acid binding protein promoter. Marked induction of transgenic MGL mRNA was found in transgenic animals and intestinal specific expression of the transgene was confirmed. Western analysis and MGL activity measurements were also performed. Following 3 weeks of high fat feeding (40% Kcal), body weight gain and total fat pad weight were significantly increased in iMGL mice compared to non‐transgenic littermates. DXA measurements showed that body fat mass was significantly higher in the iMGL mice whereas lean mass was not altered. Triacylglycerol (TG) content of intestinal mucosa and liver were also increased in iMGL mice. Plasma TG levels in iMGL mice were high but free fatty acid and total cholesterol levels were not changed. The metabolism of 14C oleate and 3H monoolein in small intestinal mucosa were not substantially altered, although less labeled MG remained following in vivo incubation, likely due to the action of the MGL transgene. Food intake data showed that the iMGL mice were hyperphagic. Based on this phenotype, we hypothesize a role for intestinal MGL in whole body energy balance, possibly via regulation of food intake.

Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.

Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice

Adipocytes express the rate-limiting enzymes required for glycogen metabolism and increase glycogen synthesis in response to insulin. However, the physiological function of adipocytic glycogen in vivo is unclear, due in part to the low absolute levels and the apparent biophysical constraints of adipocyte morphology on glycogen accumulation. To further study the regulation of glycogen metabolism in adipose tissue, transgenic mice were generated that overexpressed the protein phosphatase-1 (PP1) glycogen-targeting subunit (PTG) driven by the adipocyte fatty acid binding protein (aP2) promoter. Exogenous PTG was detected in gonadal, perirenal, and brown fat depots, but it was not detected in any other tissue examined. PTG overexpression resulted in a modest redistribution of PP1 to glycogen particles, corresponding to a threefold increase in the glycogen synthase activity ratio. Glycogen synthase protein levels were also increased twofold, resulting in a combined greater than sixfold enhancement of basal glycogen synthase specific activity. Adipocytic glycogen levels were increased 200- to 400-fold in transgenic animals, and this increase was maintained to 1 yr of age. In contrast, lipid metabolism in transgenic adipose tissue was not significantly altered, as assessed by lipogenic rates, weight gain on normal or high-fat diets, or circulating free fatty acid levels after a fast. However, circulating and adipocytic leptin levels were doubled in transgenic animals, whereas adiponectin expression was unchanged. Cumulatively, these data indicate that murine adipocytes are capable of storing far higher levels of glycogen than previously reported. Furthermore, these results were obtained by overexpression of an endogenous adipocytic protein, suggesting that mechanisms may exist in vivo to maintain adipocytic glycogen storage at a physiological set point.

Pharmacological Suppression of Premature Stop Mutations that Cause Genetic Diseases

Aminoglycoside antibiotics have long been used as antibacterial agents due to their ability to inhibit bacterial translation. However, aminoglycosides also stimulate translation errors in mammalian cells. Aminoglycosides bind to a pocket formed in a domain of the ribosomal RNA (rRNA) of the small ribosomal subunit that constitutes the decoding site in both prokaryotes and eukaryotes. Normally, accurate base pairing takes place between each successive codon and its cognate aminoacyl-tRNA within this region of the ribosome. When aminoglycosides bind to the decoding site, a conformational change decreases discrimination between cognate and near-cognate tRNAs, leading to errors in the decoding process. The ability of aminoglycosides to bind to the decoding site and induce translational misreading in eukaryotic cells is less efficient than in prokaryotic cells due to subtle differences in the sequence of the decoding site rRNA. The observation that aminoglycosides induce low levels of misreading in eukaryotic cells has inspired many investigations to determine whether aminoglycosides can suppress nonsense mutations that cause human diseases. Disease models in which aminoglycosides have been shown to efficiently suppress nonsense mutations include cystic fibrosis, Duchenne muscular dystrophy, Hurler syndrome, infantile neuronal lipfuscinosis, cystinosis, x-linked nephrogenic insipidus, spinal muscular atrophy, and cancer. However, if aminoglycosides are to be used clinically for suppression therapy, their efficacy must be improved and their toxicity reduced. The co-administration of other compounds that reduce aminoglycoside toxicity or the development of new compounds that suppress stop mutations may allow the realization of suppression therapy as a clinical treatment to suppress disease-causing stop mutations. Keywords: aminoglycosides, bacterial 70S ribosome, cognate tRNAs, Cystic fibrosis (CF), fatty acid binding protein promoter (FABP), Duchenne muscular dystrophy (DMD)