Gene Expression Profile of the Synovium and Cartilage in a Chronic Arthritis Rat Model

Abstract

Abstract: Background: Primary osteoarthritis (OA) is a polygenic disease. To investigate the gene expression profile of cartilage and synovium from osteoarthritis and healthy rats using cDNA microarray is beneficial to recognize the pathogenesis of osteoarthritis and provide evidence for gene therapy of osteoarthritis. Objective: The present study aimed to investigate the gene expression profile of the cartilage and synovium of chronic arthritis and healthy rats through cDNA microarray assay, and identify the differentially expressed genes. This study may be helpful for understanding the role of differentially expressed genes in osteoarthritis and the gene polymorphism of osteoarthritis. Methods: A total of 24 male Wistar rats were randomly divided into control group and osteoarthritis group (n = 12 per group). The synovial and cartilage were obtained and total RNA was extracted. cDNA microarray assay was performed to identify the differentially expressed genes, and cluster analysis was conducted. Results and Conclusion: A total of 82 differentially expressed genes were identified, among which 27 were up-regulated and 55 down-regulated. Gene microarray assay is effective to identify differentially expressed genes and may find out novel osteoarthritis associated genes. Multiple genes are involved in the pathogenesis of osteoarthritis. The differentially expressed genes provide important information for further studies on the pathogenesis of osteoarthritis and gene therapy of osteoarthritis.

Keywords::

• osteoarthritis
• gene expression
• cDNA microarray
• antibody
• monoclone
• cartilage tissue engineering

INTRODUCTION

It is well known that osteoarthritis (OA) is a gene-related disease [1–3] and identifying susceptibility genes related to OA is very important. It is helpful for elucidating the potential pathogenesis of OA; through genetic research, we can deeply recognize the molecular biological pathogenesis of OA and find out novel therapeutic strategies for OA including gene therapy. It is also critical for prevention of OA in susceptible population: through screening susceptibility genes, we can identify the subjects with high risk for OA and provide genetical evidence of OA, which may be beneficial for early intervention and early prevention. Koshizuka et al. [4] investigated the gene polymorphism in OA rats. Their results showed cystatin (CST) 10 was related to the pathogenesis of OA and implicated in endochondral ossification. The present study aimed to investigate the changes in the quantity and types of genes during the occurrence and development of OA, which may be helpful to find out novel biological markers for the diagnosis, treatment, and drug development and provide evidence for the diagnosis and gene therapy of OA.

MATERIALS AND METHODS

Design

A randomized controlled animal experiment was carried out.

Setting

The experiment was performed from October 2007 to October 2008 in the central laboratory of Shenzhen Second Hospital. The level of biosafety containment was graded as 2 (Biosafety Level 2, BSL-2).

Materials

A total of 24 Wistar rats (specific pathogen-free) from the same litter weighing 130 ± 20g were purchased from the Animal Center of Southern Medical University (production license number: 2007A072) and the male to female ratio was 1. These rats were randomly assigned into control group and OA group. Furthermore, all conformed to the Guidance Suggestions for the Care and Use of Laboratory Animals developed by Ministry of Science and Technology of the People's Republic of China (2006) [5].

Reagents

Kits for Atlas™ rat cDNA expression array were purchased from Clontech (Cat No.634554; USA) and the cDNA microarray includes 588 genes.

Experimental Methods

Establishment of the OA Model. Both forelimbs and tails of rats in OA group were immobilized with plaster and then these rats were given ad libitum access to food and water [6], but rats in the control group were not immobilized. Twelve weeks later, animals were sacrificed and the synovium of hip joint and cartilage of bilateral femoral heads were obtained.

Sample Collection and Preparation. After 12 weeks of immobilization, the synovium of hip joint and cartilage of bilateral femoral heads were isolated and immediately frozen in liquid nitrogen. The samples were then taken out of liquid nitrogen under an aseptic condition and crushed for use.

Total RNA Extraction. The samples were mixed with Trizol and total RNA was extracted according to the manufacturer's instruction. The quality of extracted RNA was determined by gel electrophoresis, and the opacity density (OD) was detected at 260 nm. The RNA concentration was calculated and DNA contamination was determined. Reverse transcription was then followed when the quality of extracted RNA met the requirement.

Probes. Then 60 μg of RNA were applied for reverse trascription in the presence of reverse transcriptase, and cDNA was labelled followed by purification. Fluorescence conjugated dUTP was added when the first strand was synthesized. cy3-dUTP was used to label the RNA of the control group, and cy5-dUTP label those of the OA group and control group followed by microarray asay.

Hybridization. Biostar H - 40 scDNA microarray (Beijing Bios Bio-chip, Co., Ltd.) was used for assay. Probes were dissolved in 20 μL of hybridization solution containing 5 × SSC and 0.2% SDS. The microarray and hybridization solution were kept at 95 C for denaturation for 5 min, and immediately the probes were added into microarray followed by sealing with coverlips. Then, hybridization was performed at 60 for 15∼17 h in a sealed chamber. The coverlips were removed and the microarray was rinsed with SSC and SDS solution for 10 min and then was being air-dried. The cDNA undergoing labelling was hybridized with the nylon membrane of blank group to detect the non-specific hybridization.

Microarray Scanning. Scanarray Express 4000/5000 dual-channel laser scanner (Perkin Elmer USA) was used to scan the microarray. The hybridization area was determined and background noise then filtered. The fluorescence signal intensity was detected followed by comparisons of signal intensity between the control group and OA group. When the ratio of signal intensity of a gene in the OA group to that in the control group was greater than 2 (up-regulation) or lower than 0.5 (down-regulation), these genes were subsequently identified.

Main Outcome Measures. Gross observation: At the end of the experiment, the synovium of hip joint and cartilage of bilateral femoral heads were obtained and the general presentations were recorded. Analysis of gene expression profile: Based on the OMIM Gene Bank, the differentially expressed genes were determined.

RESULTS AND DISCUSSION

Number of Rats

All the rats were used for analysis.

General Morphology of Cartilage and Synovium

At the end of the experiment, the cartilage and synovium had the characteristics of arthritis: no luster, sallowish, and erosion and ulceration at load-bearing sites. The cartilage became thin and fragmented, and subchondral bone was partially exposed. The synovium had congestion and edema. No obvious abnormalities were noted in the cartilage and synovium of the control group.

Gene Expression Profiles of Both Groups

A total of 82 differentially expressed genes were identified, among which 27 were up-regulated (as seen in Table 1) and 55 down-regulated (as seen in Table 2).

Table 1. A fraction of genes with up-regulation.

Type	Name	No. in Gene Bank	Ratio
Growth factor	matrix metalloproteinase-11	U0046034	9.77
	growth response factor-3	NM004430	4.23
Immune gene	interleukin Iβ	NM000265	14.67
	tumor necrosis factor α	X0066539	22.51
Apoptosis gene	Ras-induced senescence 1	AF438313	2.94
Growth- and development-related gene	BTC gene family 2	NM006763	5.30
	RNA helicase /DEAD/H polypeptide 3	NM001356	3.51
Disease-related gene	Fragile X syndrome related gene	NM005087	2.42
Energy-related gene	F1 complex, O subunit gene	NM001697	2.82
	Regulator of G-protein signaling 2	NM002923	2.77
Other genes	urinary vasopressin 2	NM021995	6.12
	platelet-derived growth factor	Z0014117	20.05

Table 2. Genes with down-regulation.

Type	Name	No. in Gene Bank	Ratio
Growth factor	transforming growth factor β1	NM001964	0.12
Growth- and development-related gene	heterogeneous nuclear ribonucleoprotein	NM001533	0.36
Energy-related gene	Nucleus VCP-like gene	NM002533	0.39
Other genes	K(+) channel subfamily K gene	NM022358	0.46

Relationships Between OA and Differentially Expressed Genes

Anti-inflammatory Factors and Matrix Metalloproteinase (MMP). Recently, studies showed in the progression of OA that the expressions of interleukin 1, tumor necrosis factor α, and MMPs were abnormal and played critical roles in the pathogenesis of OA. In the present study, the expressions of MMP 11 (U0046034) and interleukin 1β (NM000265) were significantly up-regulated, which suggested that both proteins played important roles in the molecular biological pathogenesis of OA and might become targets in the gene therapy of OA [7–8].

MMPs can be secreted by fibroblasts, epithelial cells, inflammatory cells, endothelial cells, etc., and are the main degrading enzymes of the matrix [9]. In OA, MMP was activated and the endogenous MMP inhibitor in the cartilage could not regulate the activity of MMP. Therefore, the extracellular matrix in the cartilage was degraded. Under the action of mechanical load and inflammatory factors, the ability of cartilage to resist stress was compromised. Subsequently, chondrocytes became apoptotic, and extracellular matrix was exposed from the cartilage. Finally, the cartilage was injured and degenerated, resulting in progressive damage to joints [10–12].

The interleukin 1 (IL-1) family includes IL-1α, IL-1β and endogenous IL 1 receptor antagonist (IL-1Ra). The genes encoding these proteins are located on chromosome 2ql3∼24 and have high homology. These genes constitute IL 1 gene cluster. A majority of IL-1s secreted by the cartilage and synovium are IL-1β, which plays a more crucial role in the inflammation of OA than other members [13]. IL-1 is one of the important mediators leading to cartilage degeneration in OA, and a main initiator resulting in pathophysiological imbalance of articular cartilage [8]. IL-1 mainly suppresses the synthesis of the cartilage matrix leading to degradation of the cartilage matrix. In addition, IL-1 is a potent mediator resulting in inflammation and pain in OA [14].

Genes Promoting Expressions of Growth Factors. Growth factors are a series of polypeptides that can affect the cell activity through intercellular signal transmission. A lot of growth factors in the cartilage are secreted by chondrocytes and stored in the cartilage matrix. These growth factors can influence the proliferation and differentiation of chondrocytes and metabolism of cartilage matrix through autocrine and paracrine, playing an important regulatory role in the chondrosis. Among the growth factors, insulin-like growth factor 1, transforming growth factor β1, and bone morphogenetic protein contribute to the repair of cartilage degeneration [15].

Our results showed changes in the expressions of some growth factors such as early growth response gene 2, early growth factor gene 1 (NM001964), and selective connexin gene (NM003100). Early growth response protein 2 can regulate the growth and differentiation, early growth factor 1 can activate transforming growth factor β1, and selective connexin can suppress the epidermal growth factor receptor (EGFR) and is involved in the degradation of EGFR in the lysosome.

The transforming growth factor family includes more than 40 polypeptides and each member has 7 cysteine residues. These polypeptides can bind specific receptors on the cell membrane and activate regulatory proteins [16]. The regulatory proteins assemble in the nucleus and form a complex which can initiate translation as a transcription factor. Transforming growth factor β is a multi-functional factor exerting stimulatory or suppressive effects on cells. Through alterating the phenotype of receptors on chondrocytes or signal transduction, transforming growth factor β can enhance the sensitivity of chondrocytes and plays a dominant role in the cartilage repair in OA [17–19].

OA and Apoptosis Gene. Currently, evidence suggests that the imbalance between anti-apoptosis and pro-apoptosis leads to abnormal apoptosis of chondrocytes involved in the occurrence of OA. Studies have shown abnormal expressions of pro-apoptotic proteins (Bax and Fax), anti-apoptotic proteins (Bcl-2), and caspases, which were associated with the apoptosis of chondrocytes [20]. At least two independent pathways are involved in the apoptosis of chondrocytes in OA. One pathway is not related to synovitis and mediated by nitric oxide (NO). The other pathway is associated with synovitis and mediated by Fas. FAS is also called APO-1 or CD95 and the type I transmembrance protein. It belongs to the tumor necrosis factor and nerve growth factor (NGF) receptor family and Fas plays an important role in the chondrocyte apoptosis in OA [21–23]. Our results indicated up-regulation of Ras-induced senescence 1 (AF438313), suggesting Fas-induced chondrocyte apoptosis promoted the occurrence of OA.

OA and Energy Related Genes. OA has been found to be related to decreased energy metabolism (Na⁺ -K⁺ -ATPase). ATP is often called the “molecular unit of currency” of intracellular energy transfer [23]. Studies found abnormal expressions of some genes related to ATP metabolism, such as similar VCP nuclear-like gene (NM002533) (a member of ATP-binding cassettes), gene of F1 complex H+ transporting ATP synthase (NM001697) (a member of mitochondrial respiratory chain), and gene of regulators of G-protein signaling 2 (RGS2) (a protein activating GTPase).

OA and Growth- and Development-related Genes. A lot of growth and development related genes are involved in the cell differentiation and nucleic acid metabolism [24]. Our results showed, in OA, the expressions of some growth- and development-related genes were abnormal and these genes included heterogeneous nuclear ribonucleoprotein (a gene related to the synthesis and maturation of mRNA), RNA helicase /DEAD/H polypeptide 3 (NM001356), and BTC gene family 2 (NM006763) (a gene involved in cell cycle regulation).

OA and Disease-related Genes. Our study showed abnormal expressions of Fragile X syndrome-related genes (NM005087). Abnormal expressions of these genes should be observed in specific diseases, but were also noted in OA. These results implied that the expression of disease genes might be affected by physique, age, and environment, and these factors also influence the pleiotropy of genes. The same gene may control different phenotypes and different genes may control the same phenotype. These results also suggested the uncertainty of gene therapy of OA [25].

CONCLUSIONS

Studies on the susceptibility gene of OA have been conducted at tissue and cell levels. The materials are obtained from the knee joint and hip joint, and include bone, cartilage, synovium, and peripheral blood. Some types of cells are also studied and include primary chondrocytes, primary synovial cells, and the chondrosarcoma cell line. Especially, the relationships between OA and differentially expressed genes have been investigated, and corresponding results show that the gene expression profile of the synovium and cartilage in the chronic arthritis rat model provides important information for further studies on the pathogenesis of osteoarthritis and gene therapy of OA.

Currently, studies have not effectively identified the pathogenic genes or susceptibility genes of OA. In respect to the pathogenesis of OA, the characteristic of joints is an important factor. Almost all joint tissues (bone, cartilage, synovium, ligament, muscle) can be affected by load-bearing. When the compliance of some tissues is compromised, the load borne by articular cartilage is increased. On the contrary, when the tissues are intact, they can bear certain loads and provide support for articular cartilage. Therefore, the types of tissues for assay can be increased. In addition, OA has a lot of risk factors, especially age, gender, and race, significantly affecting the incidence of OA. Therefore, the susceptibility genes of OA in the Chinese population have their own characteristics and further studies are needed. With the development of a microarray technique, the complex gene network of OA will be clarified, and identifying susceptibility genes will be beneficial for the early diagnosis of OA and provide a basis to reverse OA progression or even cure OA.

Declaration of interest: The authors report no conflicts of interest. The author alone are responsible for the content and writing of the paper.