Background/Purpose:   Histone deacetylase inhibitors (HDACi) are a class of compounds that inhibits the histone deacetylase activity. MCPIP1 is a negative regulator of IL-6 expression and is expressed at low levels in human osteoarthritis (OA) cartilage. In the present study we tested the effects of class I and class II HDACi on the expression of MCPIP1 and IL-6 in OA chondrocytes.

Methods: OA chondrocytes were isolated by enzymatic digestion of cartilage samples obtained from OA patients who underwent total knee arthroplasty. Primary OA chondrocytes were treated with IL-1β in the absence or presence of SAHA-a class I & II HDACi. Gene expression and protein levels were measured by TaqMan assay and Western blot respectively. Expression of miR-9 was quantified by TaqMan assay. Secreted IL-6 levels were determined by ELISA. MCPIP1 promoter or 3’ UTR reporter vector activity was assayed using dual luciferase reporter assay system. MCPIP1 promoter deletion mutants were generated by PCR and cloned into reporter vectors. PCR based Nuclear run-on assay was employed for the analysis of ongoing transcription. Transcription factor binding to the MCPIP1 promoter was determined by ChIP assay performed with ChIP grade antibodies.

Results: Treatment of OA chondrocytes with SAHA robustly induced the expression of MCPIP1 and inhibited the expression of IL-6 mRNAs upon IL-1β stimulation and reduced the secreted IL-6 protein level in the culture medium by ~70%. Treatment of OA chondrocytes with SAHA inhibited the IL-1b-induced expression of miR-9 by ~70%. Luciferase reporter vectors containing the 3’UTR of the MCPIP1 mRNA which contains the miR-9 “seed sequence” exhibited 25% increased luciferase activity in OA chondrocytes upon SAHA treatment compared to controls treated with IL-1β alone. Nuclear run-on assays using nuclei prepared from OA chondrocytes treated with IL-1β alone or IL-1b + SAHA demonstrated increased expression of MCPIP1 (1.4 fold) in OA chondrocytes compared to IL-1β alone-stimulated OA chondrocytes. Using MCPIP1 promoter deletion mutants a 156bp fragment was identified that encompassed sequences sufficient to enhance luciferase activity in OA chondrocytes stimulated with SAHA. In-silico analyses identified CEBPa binding site in the 156 bp promoter region and expression of CEBPα was significantly increased upon SAHA treatment in OA chondrocytes. Ectopic overexpression of CEBPα was sufficient to enhance the expression of MCPIP1 and increased the luciferase activity of the reporter vectors containing either the full length MCPIP1 promoter or the 156bp promoter fragment. Recruitment of CEBPα to the MCPIP1 promoter was significantly increased upon SAHA treatment. Finally we found reduced expression of CEBPa in damaged OA cartilage compared to smooth cartilage, which suggests that low level of MCPIP1 expression could be due to the reduced expression of CEBPα in damaged OA cartilage.

Conclusion:  These data indicate that HDACi SAHA upregulates MCPIP1 expression and suppress IL-6 expression in IL-1β treated OA chondrocytes via upregulation of CEBPa and inhibition of miR-9 expression. Results of the present study propose a novel therapeutic role for HDACi SAHA for the management of OA.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-histone-deacetylase-inhibitor-saha-induce-mcpip1-expression-and-suppress-il-6-expression-by-upregulating-cebp-expression-and-downregulating-the-expression-of-mir-9-in-human-oa-chondrocytes/