The Role And Mechanism Of HDAC4 Nuclear Relocation In Regulation Of Gene Expression Of Chondrocytes By Compression

Back ground:Cartilage covers the surfaces of articulating joints,inside which chondrocytes are only cells.Load-bearing is the fundamental function of cartilage and results in direct compression of articular cartilage.Mechanics have been reported to be able to modulate chondrocytes gene expression.Normal biomechanical loading increases cartilaginous gene expression and matrix protein production.Alternatively,abnormal mechanical loading(excessive or insufficient loading)can promote the onset of cartilage degeneration,and lead to osteoarthritis.Nevertheless,the mechanisms that chondrocytes sense and respond to mechanical stimulation remain largely unknown.HDAC4 has a surprising ability to translocate between the cytoplasm and nucleus.HDAC4 subcellular relocation plays a critical role in cell differentiation and proliferating.Studies have shown that HDAC4 subcellular relocation plays a prominent role in muscle cell differentiation,neuronal cell death.Our previous studies have shown that Subcellular relocation of histone deacetylase 4regulates growth plate chondrocyte differentiation.But it is unclear whether mechanical loading can inducd HDAC4 subcellular translocation.In this study,we aim to investigate the effect of compression on HDAC4 subcellular localization,and the role that HDAC4 subcellular localization play in regulation of gene expression in chondrocytes.Objective:(1)To investigate the effect of compression on HDAC4 subcellular localization in chondrocytes by using a Flexcell? FX-5000? Compression system and culturing chondrocytes in alginate;(2)To analyze the effect of the compression on gene expression of chondrocytes,and to discusse the role of HDAC4 subcellular localization in metabolism,proliferation and differentiation of chondrocytes;(3)To analyze the mechanisms by which compression elicits HDAC4 subcellular localization,and the mechanisms that compression regulates gene expression of chondrocytes through HDAC4 subcellular localization.Methods:The topic included the following five studies:(1)Cultivation of cell in alginate and choice of compression protocol:(1)Murine chondrocytes were isolated from the ventral parts of the rib cages of 6-d-old mice(C57Bl/6),and sufferred primary culture;(2)Adenovirus vector expressing HDAC4 was constructed in vitro and transfected murine chondrocyte;(3)The transfected murine chondrocyte encapsulated in 2% alginate disks and sufferred 3D alginate culture.(4)To choose the duration of the compression protocol necessary to promote most high gene expression in chondrocytes,a pilot study was performed to observed the time-dependent changes in gene expression levels of aggrecan and type II collagen following compression.(2)Observation of the effect of compression on HDAC4 subcellular localization in chondrocytes:(1)cell/alginate constructs pre-cultured for 7 days were subjected to 3 h compression at 0.5 Hz and 20 kPa and the intracellular localization of GFP-HDAC4 was observed by confocal laser scanning microscope.(2)To further verify that translocation of HDAC4 from the cytoplasm to nucleus occurred in response to compression,immunoprecipitation was performed with an antibody against HDAC4.(3)Observation of the effect of the compression on proliferation and differentiation of chondrocytes:(1)Observation of the effect of compression on gene expression of aggrecan,type II collagen,LK1,SOX9,CDKN1 A,Type X collagen,MMP-13,Ihh and Runx2 by real-time PCR;(2)Analysis of the effect of compression on production of matrix protein by chondrocytes,using Safranin-O staining and western blot analysis of type II collagen protein;(3)Observation of the effect of compression on the proliferation of chondrocytes by using the method of an EdU-based cell proliferation assay;(4)Observation of the effect of compression on dedifferentiation of chondrocytes by immunohistochemistry analyses for type II collagen and type I collagen.(4)Exploration of the mechanism by which compression elicits HDAC4 subcellularlocalization.and compression regulates the gene expression of chondrocytes through HDAC4 subcellular localization.(1)We first investigated the effect of compression on phosphorylation of HDAC4 in chondrocytes by western blot analysis with antiphosphoserine antibody after immunoprecipitating;(2)We further investigated the effect of compression on the activity of PP2 A by using PP2 A immunoprecipitation phosphatase assay kit.(5)Inhibition experiment of Okadaic acid(OA)was performed to investigate the relationship between HDAC4 relocation and HDAC4 dephosphorylation by PP2 A.(1)The confocal laser scanning microscope and immunoprecipitating were used to investigate whether the PP2 A inhibitor,OA,inhibits HDAC4 nuclear import;(2)Using antiphosphoserine antibody,western blot analysis was carried through following immunoprecipitating to investigate whether OA blocks the dephosphorylation of HDAC4 by compression;(3)A PP2 A immunoprecipitation phosphatase assay kit was used to investigate whether OA blocks activity of PP2A;(4)Real-time PCR was performed to observe whether OA abrogates the effect of compression on m RNA expression;(5)We used Hoechst 33342 / PI Double Stain Apoptosis Detection Kit to investigate whether OA elicits chondrocyte death.Results:(1)Cultivation of cell in alginate and compression protocol.(1)We used a Flexcell?FX-5000? Compression system,and chose a loading regime applied at 0–20 kPa strains with a sinusoidal waveform at a frequency of 0.5 Hz(0.5 Hz,20kPa).(2)The confocal microscopy demonstrated that the transfection efficiency of HDAC4 was around 89.8%(86.9% to 92.6%)after 1 week culture in 2% alginate.(3)The pilot study indicated that gene expression of aggrecan and type II collagen was significantly upregulated after 2 and3 h of compression,but decreased after 4 h of compression(P < 0.05 versus unloaded group).Therefore,we performed all subsequent experiments with a protocol of compression of 3 h duration.(4)The experiments of cell viability indicated that there were no detectable dead cells in the loaded groups.This confirms that the chondrocytes in 2%alginate were highly viable after 3 h of compression at 0.5 Hz and 20 kPa.(2)Compression induced HDAC4 nuclear import.(1)Confocal laser scanning microscope showed that HDAC4 was mainly located in the cytoplasm of cells in the unloaded cell/alginate constructs,while most of the HDAC4 was relocated in the nuclei of cells that had undergone the 3-h compression protocol.The percentage of cells that had HDAC4 located in their nuclei was higher in the compression group than in the unloaded group(P=0.009).(2)Immunoprecipitation experiment further conformed that HDAC4 level in the cytoplasmic fraction was far higher in the unloaded vs.compressed samples.In contrast,the HDAC4 level in the nuclear fraction was less in the unloaded group than in the compression group.This result indicates that compression induces HDAC4 relocation from cytoplasm to nucleus.(3)Compression regulated gene expression of chondrocytes.(1)Levels of mRNA for aggrecan,type II collagen,LK1 and SOX9 were increased in chondrocytes that were subjected to compression as compared with chondrocytes that did not undergo loading(P< 0.05);CDKN1A,Type X collagen,MMP-13,Ihh and Runx2 showed the opposite pattern.The level of expression of these genes was lower in chondrocytes subjected to compression as compared with unloaded chondrocytes(P<0.05).(2)Safranin-O staining showed a strong red-staining around the chondrocytes subjected to compression compared with those that were not loaded.Western blot analysis showed that levels of type II collagen protein were higher in the compressed cell/alginate constructs than that in the unloaded group.(3)EdU cell proliferation staining(red)showed that compression significantly promoted chondrocyte proliferation,P=0.0047 versus the unloaded control group.(4)Immunohistochemistry analyses showed a positive stain for type II collagen,but not for type I collagen.(4)Compression induced HDAC4 nuclear import by dephosphorylation of HDAC4 in a PP2A-dependent manner.(1)western blot and immunoprecipitating analysis showed that compression decreased the level of phosphorylated-HDAC4(Ps-HDAC4)in chondrocytes;(2)PP2A immunoprecipitation phosphatase assay revealed that compression increases PP2 A activity.(5)OA,PP2 A inhibitor,impaired the HDAC4 nuclear import.(1)OA abrogates the compression-induced increase of HDAC4 in the nuclei.HDAC4 was mainly located in the nuclei of cells subjected to compression without OA,while HDAC4 was mainly located inthe cytoplasm of cells subjected to compression with OA;(2)Compression decreased the level of Ps-HDAC4 in chondrocytes,while compression with OA was unable to decrease the level of Ps-HDAC4 of chondrocytes;(3)PP2A immunoprecipitation phosphatase assay shows that blocking of PP2 A by its inhibitor reduced the induction of PP2 A activity by compression.(4)The levels of mRNA for aggrecan,type II collagen and LK1 were all decreased in the chondrocytes subjected to compression with OA inhibitor when compared with compression alone;Likewise,the mRNA expression of CDKN1 A,type X collagen,MMP-13,Ihh and Runx2 were all increased in the group subjected to compression with OA compared with compression only.(5)Hoechst 33342 / PI Double Stain apoptosis detection show that compression with OA(1nM)did not induce chondrocyte death.Conclusions:This study demonstrates that compression induce HDAC4 relocation from cytoplasm to nuclus.Compressive stimulation regulates chondrocytes gene expression through HDAC4 relocation from cytoplasm to the nucleus via PP2A-depended HDAC4 dephosphorylation.HDAC4 is located in the cytoplasm in chondrocytes under no loading condition and relocates to the nucleus in the chondrocytes subjected to compression.Compression stimulates PP2 A activity which lead to dephosphorylation of HDAC4.When HDAC4 is dephosphorylated,it detaches from 14-3-3 chaperone proteins and relocates to the nucleus to achieve transcriptional repression of RunX2 and regulation of chondrocytes gene expression.