Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling

PLOS ONE, Dec 2019

Objectives This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4). Materials and Methods Murine 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. Results 87.5% 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. Conclusions CTS 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.

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Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling

May Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling Chongwei Chen 0 1 Xiaochun Wei 0 1 Zhi Lv 0 1 Xiaojuan Sun 0 1 Shaowei Wang 0 1 Yang Zhang 0 1 Qiang Jiao 0 1 Xiaohu Wang 0 1 Yongping Li 0 1 Lei Wei 0 1 0 Department of Orthopaedics, the Second Hospital of Shanxi Medical University; Shanxi Key Lab of Bone and Soft Tissue Injury Repair , Taiyuan, Shanxi , China , 2 Department of Orthopaedics, Warren Alpert Medical School of Brown University/Rhode Island Hosptal , Providence, Rhode Island , United States of America 1 Editor: Dmitry I Nurminsky, University of Maryland School of Medicine , UNITED STATES This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4). Murine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz) by a Flexcell1 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. Data Availability Statement; All relevant data are within the paper - OPEN ACCESS Funding: The project was supported by Grant R01AR059142 from National Institute of Arthritis and Musculoskeletal and Skin Diseases (US), P20GM104937 from National Institute of General Medical Sciences (US), and 81572098 from National Natural Science Foundation of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Objectives Results 87.5% 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, Competing Interests: The authors have declared that no competing interests exist. the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS. Conclusions CTS 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. Introduction Chondrocytes are only cells in the articular cartilage to maintain the integrity of extracellular matrix, mainly including collagen and proteoglycans [ 1 ]. As the joint bearing loading, the chondrocytes are exposed to a combination of mechanical stress, in which tensile strain plays a critical role [ 2,3 ]. A large number of studies demonstrated that low-magnitude-and-frequency tensile strain has an anti-inflammatory function and promotes cartilaginous gene expression and matrix synthesis [ 3–6 ]; in contrast, high-magnitude-and-frequency tensile strain inhibites anabolism and induces catabolism as well as expression of inflammatory factor in chondrocytes [ 3,7,8 ]. However, the mechanisms by which the biomechanics, such as tension, regulates metabolism in cartilage are still not well understood. Epigenetic evidence indicates that dynamic control of histone acetylation is an important physiological event of reversible post-translational modifiation in regulating gene expression [ 9 ]. Histone deacetylation by Histone deacetylase (HDACs) promotes chromatin condensation; however, histone acetylation by Histone acetylase (HATs) relaxes the structure of nucleosomes, which consequently alters interaction between histone and DNA, and controls gene transcription [ 10–12 ]. In mammalian cells, 18 HDACs have been discovered so far, which are divided into three major classes. Class I (HDAC1, 2, 3 and 8) and class III HDACs (consisting of a large family of sirtuins) are ubiquitously expressed; conversely, class II has a tissue-specific pattern of expression, which can be further divided into the following two subgroups: class IIa (HDAC4, 5, 7 and 9) and class IIb (HDAC6 and 10) [ 13 ]. Class IIa HDACs plays a significant role i (...truncated)


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Chongwei Chen, Xiaochun Wei, Zhi Lv, Xiaojuan Sun, Shaowei Wang, Yang Zhang, Qiang Jiao, Xiaohu Wang, Yongping Li, Lei Wei. Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling, PLOS ONE, 2016, Volume 11, Issue 5, DOI: 10.1371/journal.pone.0154951