Background
Osteoarthritis (OA) is a degenerative disease of the bone and joints, seriously affecting the quality of life of patients [
1]. It often occurs in middle-aged and elderly people leading to a high disability rate. Indeed, on a global scale, there were about 303.1 million prevalent cases of hip and knee OA, with an age-standardised prevalence estimate of 3754.2 per 100,000 [
2]., bringing huge economic burdens to families of affected patients and society.
OA is characterized by degeneration of the articular cartilage and secondary bone hyperplasia. The pathogenesis of joint diseases caused by mechanical, metabolic, inflammatory and immune factors remains unclear [
3]. OA presentation often occurs in the knee, hip, ankle and vertebral joints with clinical manifestations characterized by slow development of joint pain, stiffness, swelling, movement limitation and deformity [
4]. The incidence of cardiovascular events and all-cause mortality increase with disease progression [
5].
OA is classified into primary and secondary OA. The appearance of primary OA mostly occurs in the middle-aged and elderly, without clear induction, which is related to genetic, obesity and physical factors [
6]. The appearance of secondary OA occurs in young adults, secondary to trauma, inflammation, accumulated strain or congenital diseases, and so on [
7]. Currently, OA-specific biomarkers are still lacking in the clinical setting. Commonly used drugs for OA treatment include paracetamol, non-steroidal anti-inflammatory drugs (NSAIDs), and opioids. However, NSAID and opioids display a wide variety of marked side effects, so they are not suitable for all patients. Although drugs such as corticosteroids are also commonly used in the clinic, they usually have only short-term efficacy [
8]. Therefore, it is of great significance to explore the pathological mechanism of OA in an attempt to discover new targets for the diagnosis and treatment of OA.
Recently, studies have found that Core binding factor-β (Cbfβ) participates in chondrocyte proliferation, differentiation and osteogenesis [
9]. The core binding factors are composed of the Cbfα and Cbfβ subunits: the α-subunit is namely a Runx family (Runx) protein (including Runx1–3). Cbfβ does not directly bind DNA, but enhances the binding of Runx to DNA [
10]. Cbfβ forms a complex with Runx to regulate the transcription of downstream genes, as well as the maturation and differentiation of hematopoietic stem cells [
11]. The binding with Runx2 participates in the differentiation of chondrocytes and subchondral osteocytes, further affecting the development of the bone and joint [
12]. Johnson et al. [
13] demonstrated that target intervention to disrupt the interaction between the filament protein A and Cbfβ affects the gene transcription of Cbfβ-Runx1, regulates the process of induced differentiation of chondrocytes, and may promote the repair of degenerated cartilage seen in OA. The study suggested that taking Cbfβ and Cbfβ/Runx as putative targets might help in the elucidation of a novel pathway for the early clinical diagnosis and treatment of OA. However, most of the current studies on Cbfβ have investigated osteogenesis and chondrogenesis, and whether the abnormal expression of Cbfβ has an impact on the degenerative disease of the articular cartilage remains unclear.
On the basis of clinical investigation, this study aims to further investigation and discuss the effect of abnormal expression of Cbfβ on proliferation, differentiation and apoptosis of chondrocytes, as well as determining its association with articular cartilage degeneration, with the over-arching aim of providing newly discovered ideas for exploring novel target for the diagnosis, treatment and pathogenesis of OA.
Discussion
Joint degenerative diseases, such as osteoarthritis are closely related to the functional changes of chondrocytes in the articular cartilage tissue [
15]. Abnormal cell proliferation, differentiation and apoptosis leads to cartilage matrix degeneration and calcification, which indicates that the pathogenesis of osteoarthritis is closely related to the proliferation, differentiation and death of chondrocytes. Studies have confirmed that multiple OA-related changes in cytokines and molecular signals can induce chondrocyte abnormalities [
16‐
19], which indicates that studying the mechanism of chondrocyte degeneration and apoptotic abnormalities are promising approaches to help guide early diagnosis and treatment protocols for osteoarthritis in the clinic. At the same time, development of specific biological and drug preparations for chondrocyte proliferation, differentiation and apoptosis mechanisms is expected to effectively delay the development of the degeneration of the OA articular cartilage [
15].
Recently, it has been found that core binding factor β-subunit (Cbfβ) is closely related to chondrocyte differentiation and osteogenesis [
9]. Cbfβ controls the balance between chondrocyte proliferation and differentiation, and promotes chondrocyte proliferation and osteoblast differentiation by up-regulating Ihh expression, and inhibits PPR expression to enhance chondrocyte differentiation [
20]. Miller et al. [
21] established a transgenic model of Cbfβ mice, in which the promoter and enhancer of the Tek gene were used to express the fusion proteins of GFP and Cbfβ in the mice with Cbfβ defects. The mice died due to severe abnormal bone development after birth. Simultaneously, it has been confirmed that [
12] mice with abnormal Cbfβ expression died one day after birth. Due to abnormal development of chondrocytes, both intramembranous and endochondral osteogenesis were significantly inhibited. However, the relationship between abnormal expression of Cbfβ and degenerative diseases like OA has not been clearly clarified up to now.
In this study, biomarkers such as OA-related cartilage metabolites and inflammatory factors were detected in the cartilage. The results showed that Cbfβ was highly expressed in human OA diseased cartilage. Meanwhile, the expression of protein markers to include IL-1 β, MMP-13, comp and YKL-40 that were closely related to OA were significantly higher than that seen for normal cartilage.
Cartilage is composed of chondrocytes and extracellular matrix (ECM). Chondrocytes synthesize and secrete matrix components. In the inflammatory condition and during OA progression, significant changes in matrix degradation proteins provokes ECM remodeling. Changes in ECM will affect the chondrocyte microenvironment, which will further promote OA progression under inflammatory condition. Many studies have shown that functional changes in cartilage caused by proliferation, differentiation and apoptosis of chondrocytes are related to the degenerative joint diseases. Abnormal apoptosis often leads to degeneration and calcification of the cartilage matrix [
15]. As shown in Fig.
1, when compared with the normal diseased OA cartilage, the color was obviously dull, and the surface was not smooth with many cracks, and the cartilage was inelastic with uneven thickness, and even some joint areas were obviously degenerated and missing. Histological section staining showed that the cartilage structure level was disordered, and displayed more empty cartilage lacunae with a disordered cellular arrangement. In addition, normal cells were decreased significantly in both the surface and deep layer, the destruction of the cartilage ECM was obvious, and the expression of type II collagen in tissues was significantly decreased.
It is widely known that there is a “silent period” in the onset of osteoarthritis, in which patients may not have any clinical symptoms, nor obvious abnormalities by imaging examination. However, cartilage tissues might display extensive metabolic changes [
22]. Cartilage destruction and local inflammatory response are key pathological changes seen in OA, and thus biomarkers associated with OA are mainly associated with cartilage metabolites and inflammatory factors [
23]. In the process of cartilage degeneration, COMP, YKL-40, KS, CTX-II, PIICP and other small molecular products of cartilage matrix degradation are released gradually, and change with progression of the disease [
22]. Verma et al. [
24] confirmed that the synthesis and degradation of COMP is closely related to cartilage metabolism. YKL-40 [
25] expression levels indirectly indicate joint destruction, which can be used to evaluate the degree of joint degeneration/destruction.
Local inflammatory responses are also one of the most important pathological changes in OA development. Inflammatory mediators break the dynamic balance of cartilage matrix metabolism by affecting the metabolic activity of normal chondrocytes, which accelerates cartilage destruction [
26]. The inflammatory markers, especially interleukins [
27] (ILs) and matrix metalloproteinases [
28‐
30] (MMPs) have been widely investigated. In previous studies, IL-1β has been shown to play a key role in the development and progression of OA [
31,
32]. The results of Özler K et al. [
33] showed that MMP-13 expression in the joint fluid of patients with severe KOA was significantly related to the WOMAC score. As a specific biomarker of OA, MMP-13 could be used to determine the extent of cartilage damage to evaluate disease severity.
This is also concordant with the results of our previous detection assays on the pathological cartilage. Combined with the results of previous studies, Cbfβ was selected as the main target, as well as inflammatory related factors and cartilage metabolites including IL-1β, MMP-13, COMP and YKL-40, as the reference for chondrocyte abnormality and degeneration in the later stage of the disease. As a chaperonin of the Runx family of transcription factors, Cbfβ participates in cellular metabolic processes, such as osteogenesis and chondrogenesis, and plays an important role in chondrogenesis [
9,
12,
34]. Based on previous conclusions, it was speculated that Cbfβ might play an important role in chondrocyte apoptosis and cartilage tissue degeneration in OA pathogenesis, but the specific role remained to be elucidated.
In this study, a significant abnormal increase in Cbfβ was found in diseased cartilage tissue of OA patients and abnormal changes in various cartilage metabolites and inflammatory factors closely related to OA disease. Thus, it was speculated that abnormally increased expression of Cbfβ in the cartilage tissue of osteoarthritis might play an important regulatory role upstream of the pathogenic pathway. To further clarify the effect, after culturing normal and OA chondrocytes in vitro, we performed Cbfβ gene over-expression and inhibition treatment on OA-diseased chondrocytes by lentiviral transfection. Finally, two phenotypic chondrocytes, CBFB/pCDH (Cbfβ over-expression) and CBFB/pLKO.1 (Cbfβ expression inhibition), were obtained. The results of CCK8 cell proliferation showed that the in vitro proliferation of cells was significantly enhanced after expression of CBFB/pLKO.1 was inhibited (p < 0.01). However, after further over-expression of Cbfβ in diseased chondrocytes, the proliferation in the CBFB/pCDH group had further declined. The proliferation of normal chondrocytes was higher from that of the other three groups in CCK8 assays. However, the in vitro cell studies showed that inhibiting and regulating Cbfβ expression in OA diseased cells effectively improved the proliferative activity of chondrocytes. Annexin V/PI flow cytometry also confirmed that after the inhibition of Cbfβ gene expression in the CBFB/pLKO.1 group, the viable cell density was significantly increased from 62.82 to 88.03%, and that of early apoptosis was significantly decreased from 32.45 to 1.19%.
Our study indicated that selective up-regulation of Cbfβ expression in chondrocytes was associated with a significant increase in the total apoptotic rate, and the cell proliferative activity was also reduced. However, inhibition of Cbfβ expression could regulate and reduce the total apoptotic rate, and significantly increase the cell proliferative activity, indicating that Cbfβ might be closely related to pathological manifestations, such as abnormal chondrocyte apoptosis and cartilage tissue degeneration. Indeed, the apoptotic results of the Annexin V/PI flow cytometry assay demonstrated that after over-expression of Cbfβ in the diseased chondrocytes of the CBFB/pCDH group, the viable cell density, the total apoptotic rate and the general expression of chondrocytes in OA lesions were quite similar. While, the count of living cells and the total apoptotic rate in the CBFB/pLKO.1 group had also significantly improvement. Will it also affect the expression of OA-related inflammatory factors and cartilage metabolites in cells? To answer that question, the expression of OA-related inflammatory factors and cartilage metabolites in chondrocytes of each group was analyzed at both the gene and protein levels. The results of real-time PCR and Western blotting showed that there was also a certain expression level of Cbfβ, IL-1β, MMP-13, COMP and YKL-40 in normal chondrocytes of the control group (Fig.
4); however, there was a significant increase in Cbfβ in the OA group (
p < 0.001). Simultaneously, the expression of the above inflammatory factors and cartilage metabolites were all increased – at least to different extents, which was consistent with our findings.
In the study, we also processed OA chondrocytes by over-expression and inhibition of Cbfβ, which were then divided into the CBFB/pCDH group (Cbfβ over-expression) and the CBFB/pLKO.1 group (Cbfβ expression inhibition). The expression of COMP and YKL-40 in the CBFB/pCDH group showed slight increases in detection by real-time PCR and Western immunoblot analysis. Although there was no significant statistical difference with the OA group (
p > 0.05), the COMP and YKL-40 expression in the CBFB/pCDH and the OA group were significantly higher than was found in normal cells (
p < 0.01). Studies have shown that COMP [
24] and YKL-40 [
25] metabolites were mainly produced by chondrocytes, and were abnormally increased in chondrocyte degeneration or the inflammatory process, which can be used as diagnostic indices for articular cartilage degeneration and destruction.
The above results indicated that an abnormal increase in Cbfβ in chondrocytes significantly affected the apoptosis rate and proliferative activity of cells, and caused an abnormal increase in OA-related cartilage metabolites and inflammatory factors, which was consistent with the characteristics of Annexin V/PI flow cytometry apoptosis assay in this study. In the CBFB/pLKO.1 group, with significantly inhibited Cbfβ expression, the protein expression of disease-related inflammatory factors and cartilage metabolites was significantly decreased as compared with those of the OA group (p<0.001). The result of the Annexin V/PI flow cytometry and CCK-8 detection assays in this study indicated that in the pathological mechanism of OA development, Cbfβ played an upstream regulatory role in the inflammatory response and abnormal apoptosis signaling pathway. The abnormal expression of Cbfβ might be a key factor that induces the inflammatory response and abnormal apoptosis of chondrocytes.
Cbfβ plays a regulatory role in the dynamic balance between chondrocyte proliferation and differentiation [
20]. As a co-transcription factor of Runx2 [
35,
36], Cbfβ forms a heterodimer with the Runx2 protein, playing an important positive regulatory role in chondrocyte differentiation and osteogenesis [
37]. Thus, abnormal expression of Cbfβ is closely related to the apoptotic changes of articular chondrocytes and cartilage tissue degeneration. In the study, vector virus was used to regulate Cbfβ in human diseased chondrocytes in vitro, demonstrating a significant effect of Cbfβ regulatory changes on the proliferation, differentiation and apoptosis of chondrocytes. The inhibition of Cbfβ expression significantly reduced OA-related biomarkers to include IL-1β, MMP-13, COMP, and YKL-40. Moreover, the abnormal proliferation and apoptosis of OA-diseased chondrocytes was improved. Thus, the inhibition of Cbfβ expression might play a role in delaying cartilage degeneration, which was expected to provide a novel pathway for the exploration of new targets for the diagnosis, treatment, and pathogenesis of OA.
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