The effects of PPARγ/TGFβ cross-talk on CD36 gene expression in the intestinal epithelium

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Maintenance of the intestinal epithelium requires both constant renewal and strict control over the processes of cell proliferation, migration, differentiation and death. Peroxisome-proliferator activated receptor gamma (PPARγ) and transforming growth factor beta (TGFβ) are signaling molecules expressed in the intestinal epithelium that are thought to play a role in its maintenance. Additionally, aberrant PPARγ/TGFβ signaling within the intestinal epithelium has been linked to inflammation, formation of pre-neoplastic lesions, and cancer. In this study, CD36 was chosen as a representative of a set of genes that exhibit potential PPARγ/TGFβ cross-talk. CD36 is a scavenger receptor that has been implicated in diverse functions including fatty acid transport, cellular adhesion, angiogenesis and cell signaling, all of which are critical for intestinal epithelium homeostasis. Quantitative PCR was used to determine the expression of CD36 in mouse colon and in a rat intestinal epithelial cell line stably transfected with PPARγ. In mice, CD36 was differentially expressed along the length of the colon and that its expression was upregulated in response to the synthetic PPARγ ligand RS5444. In cell culture, CD36 was also upregulated in response to RS5444. Importantly, expression was unaffected by treatment with TGFβ alone but the PPARγ mediated induction was inhibited by simultaneous treatment with RS5444 and TGFβ. We hypothesize that TGFβ has a direct effect on PPARγ or its co-activators/co-repressors thereby interfering with transcription complex formation at the CD36 promoter. To test this hypothesis, the rat CD36 promoter has been cloned. Reporter gene and oligonucleotide binding assays are being utilized to characterize potential PPARγ response elements (PPRE). The sequence of the cloned promoter fragment revealed that two DR-1 elements previously shown to bind PPARγ in human and mouse are conserved to rat. The reporter gene response to RS5444 and TGFβ was similar to the changes in RNA abundance observed for the endogenous gene. Furthermore, deletion or mutation of the 5′ DR-1 had a profound effect on PPARγ mediated induction while deletion or mutation of the 3′ DR-1 had little to no effect. A double stranded biotinylated oligonucleotide containing the sequence of the 5′ DR-1 element was used in an oligonucleotide binding assay. The results indicated that the sequence specifically binds PPARγ, that the binding occurs in the absence of PPARγ ligand, and that the PPARγ binding affinity is significantly lower than the binding affinity to the known HMG Co A PPRE. Current experiments are designed to determine the EC50 for binding and to test the effect of TGFβ on the EC50. Completion of these experiments will lead to a greater understanding of the mechanism by which TGFβ inhibits PPARγ mediated induction of a subset of PPARγ target genes. Supported in part by NRSA F32 DK071436-01 and by a grant from Sankyo Corporation, LTD.