FKBP51 is a cochaperone that regulates several biological processes in the cell, through protein-protein interaction. Very recently, we have found that FKBP51 is hyperexpressed in malignant melanoma and plays a crucial role in its chemo- and radio-resistance. Downmodulation of FKBP51, by gene silencing, produces an increase of melanoma apoptosis induced by several cytotoxic stimuli. To investigate the mechanism of the increased apoptosis sensitivity of FKBP51-silenced melanoma, we performed a study of protein expression profiles in melanoma cells that were depleted or not of FKBP51, using ionizing radiation (IR) as apoptotic stimulus. Among the multiple signaling pathways that were found modulated in FKBP51-silenced cells, a marked upregulation of TRAIL receptors was found. In particular, the death receptors were significantly increased in FKBP51-depleted cells, while the decoy receptors, that do not transduce death signal, were decreased in the same cells. Among TRAIL receptors, TRAIL R2 (DR5) transduces most efficiently the death signal. Aim of the present study was to validate the increase of DR5 at mRNA and protein level in FKBP51-silenced melanoma cells and investigate the effect of FKBP51 silencing on TRAIL-induced apoptosis. Summary of key findings FKBP51-silencing increased DR5 transcript by 6,1 folds (range 2,8/14,7 folds), in melanoma cells, measured by Real-time PCR. Flow cytometry confirmed the increased expression of DR5 (by more than 100%) in FKBP51 silenced cells, compared with non silenced melanoma cells. The study of apoptosis was in accordance with the expression of death receptors. Indeed, FKBP51 silencing increased TRAIL-induced apoptosis, as suggested by both propidium iodide incorporation and annexin V staining of melanoma cells. Low levels of apoptosis were measured in melanoma cells stimulated with 100ng/ml TRAIL (18 + 6%). By contrast, 39 + 14% of apoptosis was measured in FKBP51-depleted melanoma cells, following TRAIL stimulation (P= 0.01). FKBP51 regulated the acetylation pattern of melanoma as suggested by the reduced amount af acetylated protein in FKBP51 silenced-melanoma cells (D), most interestingly, reduced levels of acetylated Yin Yang 1 transcription factor (YY1) involved in control of DR5 tanscription, were found in FKBP51-silenced melanoma, as suggested by western blot assay. Conclusions FKBP51 gene silencing produces a marked increase in TRAIL sensitivity of melanoma, due to upregulation of TRAIL death receptors. FKBP51 decreases the acetylation pattern of melanoma, and in particular of the DR5 transcriptional repressor YY1, suggesting that epigenetic modification of DNA could be responsible for reduced TRAIL-receptor transcription and resistance to apoptosis of melanoma.
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