Abstract
ObjectivesThis paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4).Materials and MethodsMurine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz) by a Flexcell® FX-5000™ Tension System. Fluorescence microscope and western blot were used to observe subcellular location of HDAC4. The gene expression was analyzed by real-time RT-PCR. The concentration of Glycosaminoglycans in culture medium was quantified by bimethylmethylene blue dye; Collagen II protein was evaluated by western blot. Cells phenotype was identified by immunohistochemistry. Cell viability was evaluated by live-dead cell detect kit. Okadaic acid, an inhibitor of HDAC4 nuclear relocation, was used to further validate whether HDAC4 nuclear relocation plays a role in gene expression in response to tension stimulation.Results87.5% of HDAC4 was located in the cytoplasm in chondrocytes under no loading condition, but it was relocated to the nucleus after CTS. RT-PCR analysis showed that levels of mRNA for aggrecan, collagen II, LK1 and SOX9 were all increased in chondrocytes subjected to CTS as compared to no loading control chondrocytes; in contrast, the levels of type X collagen, MMP-13, IHH and Runx2 gene expression were decreased in the chondrocytes subjected to CTS as compared to control chondrocytes. Meanwhile, CTS contributed to elevation of glycosaminoglycans and collagen II protein, but did not change collagen I production. When Okadaic acid blocked HDAC4 relocation from the cytoplasm to nucleus, the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS.ConclusionsCTS is able to induce HDAC4 relocation from cytoplasm to nucleus. Thus, CTS alters chondrocytes gene expression in association with the relocation of HDAC4 induced by CTS.
Highlights
Chondrocytes are only cells in the articular cartilage to maintain the integrity of extracellular matrix, mainly including collagen and proteoglycans [1]
cyclic equibiaxial tensile strain (CTS) alters chondrocytes gene expression in association with the relocation of histone deacetylase 4 (HDAC4) induced by CTS
In this study we demonstrated that cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz) could induce HDAC4 nuclear import, which resulted in increase of the gene expression of the anabolism and proliferation, and repression of hypertrophic gene expression
Summary
Chondrocytes are only cells in the articular cartilage to maintain the integrity of extracellular matrix, mainly including collagen and proteoglycans [1]. 18 HDACs have been discovered so far, which are divided into three major classes. Class IIa HDACs plays a significant role in tissue-specific growth and development[13,14,15]. HDAC4, a pivotal ingredient of the class IIa HDACs, is expressed in the brain, heart, skeletal muscle and cartilage [13,16]. Studies have shown that HDAC4 subcellular translocation plays a pivotal role in regulation of neuronal cell death [18], muscle cell differentiation [19], and growth plate chondrocyte differentiation [16]. Whether biomechanics can regulate HDAC4 translocation between the cytoplasm and nucleus in chondrocytes remain unknown. We hypothesize that the biomechanics can regulate the gene expression of the chondrocytes via promoting HDAC4 relocation from cytoplasm to nucleus
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