Endocrine Abstracts (2004) 7 P58

Targeted RNA interference to selectively modulate expression of PPARgamma2 and 1 isoforms

E Schoenmakers1, DH Hadaschick2, S O'Rahilly1,2 & VKK Chatterjee1

1Department of Medicine, University of Cambridge, Cambridge, UK; 2Department of Clinical Biochemistry, University of Cambridge, Cambridge, UK.

The PPARgamma is a ligand activated transcription factor that plays an important role in various biological processes including adipocyte differentiation and glucose homeostasis. Alternate promoter usage generates two receptor isoforms: PPARgamma2, containing 28 additional aminoterminal residues, is selectively expressed in adipose tissue whereas PPARgamma1 is more ubiquitously distributed. PPARgamma2 is strongly up regulated during adipogenesis, suggesting a specific role for this isoform in fat cell differentiation. However, studies of the ability of each isoform to stimulate adipogenesis have yielded ambiguous results, in part because PPARgamma stimulate its own expression.

We have used RNA interference (RNAi) to analyse the role of each receptor isoform. RNAi uses 21-nucleotide RNA duplexes, called small interfering RNAs (siRNA), which are exogenously added to eukaryotic cells resulting in a targeted degradation of mRNA. The siRNAs are thought to incorporate into a protein-RNA complex, the RNA-induced silencing complex, where they serve to guide a nuclease to the target mRNA. siRNA oligonucleotides, directed against either PPARgamma2 or PPARgamma1/2 isoforms were generated. When transfected into HEK-293 cells, each siRNA oligo was capable of selectively blocking transcriptional activation mediated by coexpressed PPARgamma2 or 1 isoforms. These effects were specific, and the siRNA oligos had no effect on signalling by other PPARs (alpha, delta) or unrelated (LXR) nuclear receptors. Cotransfection of siRNA oligos with EGFP-PPARgamma fusions visualised receptor downregulation and western blotting confirmed selective reduction in gamma2 or gamma1protein expression. In functional assays, the siRNA oligos were able to inhibit the differentiation of murine 3T3-L1 preadipocytes and studies to assess their effect on primary human preadipocyte differentiation are being undertaken.

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