Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis of E157D PPARÎ³ transcriptional activity in NIH-3T3 fibroblasts

ABSTRACT: The E157D PPARÎ³ mutation presents as an atypical case of familial partial lipodystrophy and diabetes in a human cohort and has a novel molecular mechanism. The mutation is located in the DNA-binding domain, has no dominant negative activity but disrupts transcription activation after the protein has bound the DNA. The mutation causes a transcriptional activation defect on many PPARÎ³ target genes, while some genes are induced normally. A small number of non-PPARÎ³ target genes are induced by the E157D mutant, possibly leading to the atypical features in this cohort. mRNA was amplified from total RNA collected from NIH-3T3 cells expressing either wild-type or E157D PPARÎ³ through lentiviral infection. Cells were treated with DMSO or 20ÂµM rosiglitazone for 24 hours prior to RNA isolation in three independent experiments.

ORGANISM(S): Mus musculus

SUBMITTER: Philippe Campeau

PROVIDER: E-GEOD-37990 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Studies in mice have shown that PPARÎ± is an important regulator of lipid metabolism in liver and a key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARÎ± in human liver. Here we set out to study the function of PPARÎ± in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARÎ± agonist Wy14643. Results: Quantitative PCR indicated that PPARÎ± is well expressed in human liver and human liver slices and that the classical PPARÎ± targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARÎ± activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARÎ± activation (q value<0.05). Many genes induced by PPARÎ± activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARÎ± activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon Î³-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARÎ± is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARÎ± activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARÎ± were identified that were commonly induced by PPARÎ± activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L. Conclusion: Our manuscript demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARÎ± in human liver. Our data underscore the major role of PPARÎ± in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARÎ± in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease. Precision-cut liver slices, prepared from liver biopsies obtained from obese subjects undergoing bariatric surgery, were incubated with the peroxisome proliferator-activated receptor alpha (PPARÎ±) agonist Wy14643 or vehicle for 24hrs, after which gene expression was profiled by array.

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Genome-wide analysis of E157D PPARÎ³ transcriptional activity in NIH-3T3 fibroblasts

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