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)