OBJECTIVE:To examine the ability of a broad-spectrum histone deacetylase (HDAC) inhibitor to protect cartilage in vivo, and to explore the effects of class-selective HDAC inhibitors and small interfering RNA (siRNA)-induced knockdown of HDACs on metalloproteinase expression and cartilage degradation in vitro.METHODS:A destabilization of the medial meniscus (DMM) model was used to assess the in vivo activity of the HDAC inhibitor trichostatin A (TSA). Human articular chondrocytes (HACs) and SW-1353 chondrosarcoma cells were treated with cytokines and TSA, valproic acid, MS-275, or siRNA, and quantitative reverse transcription-polymerase chain reaction was performed to determine the effect of treatment on metalloproteinase expression. HDAC inhibitor activity was detected by Western blotting. A bovine nasal cartilage (BNC) explant assay was performed to measure cartilage resorption in vitro.RESULTS:Systemically administered TSA protected cartilage in the DMM model. TSA, valproic acid, and MS-275 repressed cytokine-induced MMP1 and MMP13 expression in HACs. Knockdown of each class I HDAC diminished interleukin-1-induced MMP13 expression. All of the HDAC inhibitors prevented degradation of BNC, in which TSA and MS-275 repressed cytokine-induced MMP expression.CONCLUSION:Inhibition of class I HDACs (HDAC-1, HDAC-2, HDAC-3) by MS-275 or by specific depletion of HDACs is capable of repressing cytokine-induced metalloproteinase expression in cartilage cells and BNC explants, resulting in inhibition of cartilage resorption. These observations indicate that specific inhibition of class I HDACs is a possible therapeutic strategy in the arthritides.

Presentationstransfections).These results were also validated using primary human OA chondrocytes.Results: Sequencing of a 10kb fragment upstream of human Pitx1 promoter allowed us to identify a SNP in a potential E2F respond element located in -3900/-3800 region of Pitx1 gene promoter.However, no association was found after validation of this SNP in a larger OA patient cohort (n=150).Nevertheless, using a 30bp probe, corresponding to the response element, and a DNA pull-down approach followed by mass spectrometry analysis (MS/MS), we discovered Prohibitin (PHB1) as the protein that interact with this response element.We confirmed this result with DNA pull-down followed by a Western blot against PHB1 and by chromatin immunoprecipitation (ChIP) assay.We have also found that in human OA chondrocytes, PHB1 is mostly accumulated in nucleus whereas in control subject it is mostly localized in cytoplasm.Overexpression of PHB1 in chondrocytes clearly demonstrated that PHB1 represses Pitx1 at mRNA and protein level.We also confirmed this repression using a gene reporter approach and point out that PHB1 use the -3900/-3800 response element discovered serendipitously.It has been shown that PHB1 represses the genes that are normally regulated by E2Fs transcription factor.Using a Luciferase reporter gene assay, we demonstrated that E2F1 is a transcriptional activator of Pitx1 gene and PHB1 over-expression block E2F1 effect on Pitx1 gene expression.Conclusions: In healthy subjects, Pitx1 is normally regulated by E2F1, but in OA condition, accumulation of PHB1 in chondrocyte nucleus leads to Pitx1 gene repression.Further work will be undertaken to determine the mechanism that lead to nuclear PHB1 accumulation in OA chondrocytes.