PPARgamma(Peroxisome proliferator-activated receptor gamma) interacts with Sp1 transcription factor on the p21waf/cip1 promoter region to stimulate its transcription in cladosporol A-treated HT-29 cells

Dietary habits and life-style are the major etiological factors of colorectal cancer and, therefore, the use of chemopreventive agents able to interfere with the disease onset, is widely accepted. In a previous work, we demonstrated that cladosporol A, a secondary metabolite purified from Cladosporium tenuissimum, is able to inhibit the growth of colon cancer cells through the upregulation of p21waf1/cip1 gene expression mediated by an Sp1-dependent mechanism. In different cell types the increased p21waf1/cip1 transcription has been shown to depend upon a specific Sp1/PPARγ interaction. In this work we sought to investigate the functional relationship between these transcription factors in HT-29 cells treated with cladosporol A. PPARγ protein levels undergo a cyclic reduction due to a negative autoregulation mechanism (negative feedback loop) triggered by cladosporol A, supporting the notion that it is a PPARγ specific ligand and confirmed by the reciprocal effect observed after addition of GW9662, a well-recognized irreversible antagonist. The concomitant exposure of HT-29 cells to cladosporol A and GW9662, indeed, attenuates the increase of p21waf1/cip1 protein levels further proving the involvement of PPARγ in the upregulation of p21waf1/cip1 gene expression. Transient silencing of PPARγ in HT-29 cells exposed to cladosporol A reduces the upregulation of the endogenous p21waf1/cip1 gene and inhibits the transcriptional activation of an exogenous transiently transfected p21waf1/cip1 promoter. Co-immunoprecipitation assays show that PPARγ specifically interacts with Sp1, either in untreated and cladosporol A-treated cells. Finally, ChIP assays demonstrate the presence of a more intense PPARγ/Sp1 complex on the p21waf1/cip1 promoter region along with other markers of transcriptional activation. To our knowledge, this is the first report describing cladosporol A as a novel PPARγ natural ligand.