Probing the role of PPARγ in the regulation of late-onset Alzheimer’s disease-associated genes

<div><p>Peroxisome proliferator-activated receptor-γ (PPARγ), is a transcription factor that governs pathways, such as lipid metabolism and immune response, that have been implicated in the etiology of LOAD. Previously, we established HepG2-derived cell-lines with stable knockdown of PPARγ gene, and showed an increase in mRNA levels of genes mapped in the <i>APOE</i> linkage disequilibrium (LD) region on chromosome 19q13.32, with the greatest effect observed for <i>APOE</i>-mRNA. Here, we extended the analysis using our <i>PPARγ</i> knockdown model system and investigated the broader effect on expression changes of genes implicated in LOAD via genome wide association studies (GWAS). We applied the nCounter gene expression assay (NanoString) using a panel of twenty-four LOAD-associated genes inferred by proximity to the top significantly associated SNPs. Two independent <i>PPARγ</i> knockdown cell-lines showed changes in mRNA levels of a total of seven genes compared to a control HepG2 cell-line; six of which, <i>ABCA7</i>, <i>APOE</i>, <i>CASS4</i>, <i>CELF1</i>, <i>PTK2B</i>, and <i>ZCWPW1</i>, were upregulated and one, <i>DSG2</i>, was downregulated upon <i>PPARγ</i> knockdown. Our results propose that <i>PPARγ</i> may act as a master regulator of the transcription of several genes involved in LOAD pathogenesis. Our study provided the premise for further analyses including a larger set of genes positioned within a wider range of linkage disequilibrium (LD) regions tagged by all LOAD significantly associated SNPs.</p></div

LOAD-risk genes located ± 100 kb of the top SNP.

<p>LOAD-risk genes located ± 100 kb of the top SNP.</p

<i>PPARγ</i> knockdown effects on mRNA levels of LOAD-genes.

<p><i>PPARγ</i> knockdown effects on mRNA levels of LOAD-genes.</p

Validation of the reduction in <i>PPARγ</i>-mRNA expression in HepG2-derived <i>PPARγ</i>-KD1 and -KD2 cells.

<p>RNA was extracted from four HepG2 derived cell-lines: PPARγ-KD1, PPARγ-KD2, GFP, and untransduced (U). For each cell-line, RNA samples from four biological replicates were pooled. The levels of <i>PPARγ</i>-mRNA relative to the geometric mean of <i>GAPDH</i>, <i>B2M</i>, and <i>LDHA</i> -mRNAs was assessed by nCounter NanoString technology. The different HepG2 derived cell-lines are indicated on the X-axis, and the fold change of mRNA (log2 transformed) is indicated on the Y-axis. <i>PPARγ</i>-mRNA were decreased in both PPARγ-KD cell-lines compared to the GFP cells (by ~4–5 fold), and compared to ‘U’ cells (~3–4 fold).</p

A20 expression in IECs restricts colon tumorigenesis.

<p>(A) immunoblot analysis of isolated IECs indicating efficient deletion of A20 from the small bowel (SB) and colon (C) of villin-Cre A20^FL/FL APC^min/+ mice (fl/fl) compared to control villin-Cre A20^+/+ APC^min/+ mice (+/+) mice. GAPDH is shown as a loading control. (B) Tumor number (left panel) and aggregate tumor size (right panel) in colons of A20 (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. (C) Tumor numbers in small intestines of A20 (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. (D) Colon and small intestine lengths from (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. Each point represents one mouse. Lines indicate mean values. (f) Hematoxylin and eosin staining (upper panels) and Ki-67 and cleaved caspase-3 immunostaining (lower panels) of colonic sections from villin-Cre A20^FL/FL APC^min/+ mice (fl/fl) and control villin-Cre A20^+/+ APC^min/+ mice. 40X magnification shown.</p

A20 supports β-catenin ubiquitination and degradation through an interaction with the destruction box.

<p>(A) Luciferase assay showing transcriptional activity of a β-catenin dependent TCF/LEF4 reporter in RKO cells. Cells were treated with A20 specific or control siRNA and recombinant human wnt3a (rhwnt3a) as indicated. Relative luciferase units (RLU) are shown. **indicates p<0.01. (B) Co-precipitation of A20 with Axin. RKO cells transfected with the indicated expression plasmids were lysed, immunoprecipiated (IP) for the indicated epitope tag, and immunoblotted (IB) for the indicated proteins. Cells were stimulated with rhwnt3a or control as indicated for four hours. Input levels of MYC, FLAG, and GAPDH are shown as controls below. (C) Co-precipitation of partial A20 proteins with Axin. Co-transfection experiments as in (B). Input levels of MYC, FLAG, and GAPDH shown below. (D) A20 suppresses wnt3a stimulated induction of β-catenin expression. Immunoblot analyses of active and total β-catenin expression in RKO cells treated with A20 specific or control siRNA. A20 and GAPDH levels shown below as loading control. (E) A20 supports wnt3a stimulated β-catenin ubiquitination. RKO cells were treated with A20 specific or control siRNAs and wnt3a for the indicated times. Lysates were immunoprecipitated for β-catenin followed by immunoblotting for ubiquitin. Input amounts of beta-catenin, A20, and GAPDH proteins shown below as controls. All data are representative of three or more independent experiments.</p

A20 Restricts Wnt Signaling in Intestinal Epithelial Cells and Suppresses Colon Carcinogenesis

<div><p>Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Here, we have studied the role of A20 (also known as TNFAIP3), a ubiquitin-editing enzyme that restricts NFκB and cell death signaling, in intestinal homeostasis and tumorigenesis. We have found that A20 expression is consistently reduced in human colonic adenomas than in normal colonic tissues. To further investigate A20’s potential roles in regulating colon carcinogenesis, we have generated mice lacking A20 specifically in intestinal epithelial cells and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APC^min). While A20^FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20^FL/FL villin-Cre APC^min/+ mice contain far greater numbers and larger colonic polyps than control APC^min mice. We find that A20 binds to the β-catenin destruction complex and restricts canonical wnt signaling by supporting ubiquitination and degradation of β-catenin in intestinal epithelial cells. Moreover, acute deletion of A20 from intestinal epithelial cells in vivo leads to enhanced expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer. Taken together, these findings demonstrate new roles for A20 in restricting β-catenin signaling and preventing colon tumorigenesis.</p> </div

Human colonic adenomas express less A20 than normal colonic mucosa.

<p>Expression of A20 (top panel), cyclin D1 (middle panel) and c-myc (bottom panel) mRNAs in normal colonic mucosa and colonic adenomas, as quantitated by the Genome Expression Omnibus (GDS2947). Relative expression levels are shown. **indicates p<0.01.</p

Acute deletion of A20 from IECs leads to increased levels of Cyclin D1 and MYC mRNA in vivo.

<p>Villin-ER/Cre A20^FL/FL (fl/fl) and control Villin-ER/Cre A20^+/+ (+/+) were injected with 1 mg of tamoxifen daily for 5 days. IECs were then isolated and studied for expression of A20 (upper panel), Cyclin D1 (middle panel), and MYC (lower panel) mRNAs by qPCR. Each point represents one mouse. *indicates p<0.05; **indicates p<0.01.</p