Clock gene Bmal1 influences the cell cycle of chondrocytes in osteoarthritis

doi:10.21203/rs.3.rs-4961150/v1

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Clock gene Bmal1 influences the cell cycle of chondrocytes in osteoarthritis

https://doi.org/10.21203/rs.3.rs-4961150/v1

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Osteoarthritis (OA) is a degenerative disease caused by multiple factors. This study used in vitro and animal models to investigate the connection between the biological clock and cell cycle in osteoarthritic cartilage. The results indicate that the transcription levels of the circadian clock gene Bmal1 exhibit a negative correlation with Mmp13 and positive correlation with Wee1. Thus, increased expression of Bmal1 and Wee1 may be a potential protective factor in osteoarthritis, while high expression of Per1, Cdk1, Ccnb1, and Mmp13 may be a risk factor. By controlling the cell cycle and contributing to the pathophysiology of OA, the biological clock may impact the apoptosis of chondrocytes.

Biological clock

Cell cycle

Osteoarthritis

Cartilage

Apoptosis

Osteoarthritis (OA) is a long-term, disabling joint disease that severely impacts physical and mental health. It affects the articular cartilage, subchondral bone, ligaments, capsules, and synovium. According to statistics, 14.8% of the global population over the age of 30 suffered from some form of OA in 2020, which is equivalent to more than one billion people worldwide [1]. In addition to increasing the burden on individuals, the socioeconomic costs of OA are considerable. For example, osteoarthritis caused about $80 billion in medical expenses in 2016 in the United States alone [2]. However, there are few effective treatments for OA, and the disease's complex pathogenesis is attributed to a variety of potential causes—among which we will concentrate on the biological clock and cell cycle.

The circadian rhythm has a length of approximately 1 day, and there is a coupling relationship between it and the cell cycle, whereby both can affect each other [6–8]. The biological clock is an adaptive mechanism that synchronizes individual physiological functions and behaviors, so that most organisms can better adapt to changes inside the body and in the surrounding environment. Studies have shown that synchronous expression of cyclic genes is more conducive to early cartilage formation. Circadian clock genes can drive the rhythmic expression of key genes such as Matrix Metallopeptidase 13 (Mmp13), Collagen Type II Alpha 1 Chain (Col2a1), and Indian Hedgehog Signaling Molecule (Ihh), which are related cartilage homeostasis. In addition, they can directly or indirectly control the expression of multiple cell cycle genes, such as WEE1 G2 Checkpoint Kinase (Wee1), Cyclin-Dependent Kinase Inhibitor 1A (P21), Cyclin-E, and Cyclin-B [9–12]. Similar to the circadian rhythm of the biological clock, the cell cycle also has a strictly regulated rhythm, regulated by cyclins, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CKIs). The abnormal expression of these cell cycle regulatory proteins can affect the normal process of the cell cycle and lead to diseases. For example, the abnormal expression of Cyclin-D1 (CCND1), Cyclin Dependent Kinase 1 (CDK1), Cyclin-B1 (CCNB1), as well as WEE1, and P21 proteins, can affect the normal division, proliferation, and differentiation of chondrocytes, cause apoptosis, and promote the pathogenesis of osteoarthritis [13–15]. However, the intrinsic balance between the biological clock and the cell cycle in articular cartilage, as well as how the biological clock regulates the cell cycle and co-initiates the mechanism of OA, remains poorly understood. This study combined the analysis of rat OA samples with the ATDC5 chondrocyte cell line Bmal1 overexpression model to demonstrate the regulation of cell cycle by the clock gene Bmal1 in articular cartilage tissue, as well as its relationship with the occurrence and development of OA.

2.1. Animals

All animal experiments were authorized by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval No. A2310-22) and performed in accordance with institutional, national, and international ethical standards. Male Sprague–Dawley (SD) rats (2 months old, n = 40) were obtained from the Animal Center of Xinjiang Medical University (Urumqi, China). The number of rats in each group was chosen based on the calculation formula reported by Xinlian and Hartmann [16]. After 10 days of adaptation, 20 rats underwent surgery for the anterior cruciate ligament transection to generate an OA model, while the remaining 20 rats were the normal control group. Rats in the OA group were subjected to forced activity for 1 hour a day 1 week after surgery, and all rats were euthanized 4 weeks later.

2.2. Cell culture

Mouse cartilage cell line ATDC5 (Jiangsu, China) was cultured in DMEM/F12 medium (Gibco, USA) containing 10% fetal bovine serum (BioInd, Israel). When confluence reached 30–40%, the complete medium was replaced with an induction medium containing insulin-transferrin-selenium (ITS, USA, Sigma) for chondrogenic induction of ATDC5 cells. After successful induction, the ITS induction medium was replaced with a complete medium containing IL-1β ( PeproTech Inc, USA ). The concentration of IL-1β was 10 ng / ml, and the induction culture was 24 hours to become OA cartilage-like ATDC5 cells. In the process of chondrogenic induction of ATDC5 cells, Bmal1 overexpression lentivirus ( Shanghai, China ) was used for cell transfection, and the MOI value was 40. The chondrocyte-like ATDC5 cells stably transfected with the Bmal1 overexpression vector were selected by adding puromycin to the culture medium at a final concentration of 3 mg/L. We named the normal group as chondrocyte-like ATDC5 cells, the OA group as OA chondrocyte-like ATDC5 cells, and the lentivirus Bmal1 (LV-Bmal1) group as chondrocyte-like ATDC5 cells overexpressing Bmal1.

2.3. Cell viability assessment

The cells were seeded into 96-well plates (100 µl/well) and 10 µl of CCK-8 solution was added to each well. After the cells were incubated for an hour, a microplate reader was used to measure the absorbance of each group at 450 nm.

2.4. Hematoxylin/Eosin (H&E) and Safranin O/Fast green staining

The knee joint cartilage of the normal group and the OA group was placed in the tissue fixative (Servicebio, China) for 1 day, and decalcified with EDTA (Servicebio, China) for 8 weeks. After routine dehydration, transparency, embedding and sectioning, H.E.and safranin fast green staining were performed, and the pathological changes of cartilage were observed under an inverted microscope (DM4000 B; leica Microsystems Co., Ltd.) to observe the regularity of cartilage surface, cartilage thickness and cell morphology.

2.5. RT-PCR detection

RNA was extracted using a cartilage RNA column extraction kit (Beijing TransGen Biotech Biotechnology Co., Ltd., China) and reverse transcribed into cDNA for gene amplification. The PCR reaction system comprised 10µl. The amplification conditions were as follows: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, annealing at 60°C, and extension for 30 s, for a total of 40 cycles. The chondrogenic differentiation of ATDC5 cells into cartilage-like cells was confirming by comparing the levels of Collagen Type II (Col 2) and Collagen Type X (Col 10) mRNA. We also calculated and compared the mRNA levels of Bmal1, Wee1, Per1, Cdk1, Ccnb1, and Mmp13. According to their mRNA expression levels in the OA group, correlation analysis was performed based on the Ct values.

2.6. Immunohistochemistry

The cartilage tissue was subjected to rehydration and antigen retrieval, followed by incubation with primary antibodies against BMAL1, PER1, CDK1, CCNB1, MMP13 (Cat No. 14268-1-AP, 1:300; 13463-1-A, 1:200; 10762-1- A, 1: 200; 28603-1 - A,1: 200; 18165-1 - AP, 1:100. Sanying, China, respectively) and WEE1 (ab307515, 1:300; Abcam, UK) overnight. Anti-incubation, DAB color, anti-blue, dehydration, transparent, seal, mapping. Two independent researchers used ImageJ software to measure the positive cell area for all results.

2.7. Western blot analysis

The total protein was extracted from the ATDC5 cells or cartilage tissue samples, and the protein concentration was determined using a BCA assay kit. Antibodies against BMAL1, PER1, CDK1, CCNB1, and MMP13 (Cat No. 14268-1-AP, 13463-1-A, 10762-1-APP, 28603-1, AP, 18165-1- AP, Sanying, China, 1:1000, respectively) as well as WEE1 (ab307515, AbCam, USA, 1: 1000) were used for western blotting. The next day, incubation of secondary antibody at room temperature, chemiluminescence imaging. Grayscale analysis with Image J software, protein relative expression calculation, and line visualization analysis.

2.8. Tunel staining

Rat knee joint cartilage tissue dewaxing to water, proteinase working liquid covering tissue, 37 degrees heat tank incubation, after washing and drying the tissue sections, the membrane-breaking working fluid was added dropwise. After incubation, the sections were washed and dried, buffer covering fabric, regular temperature incubations after drip mixed good working liquid, incubation after 1 hour with PBS wash 3 times, dried after dry drip with DAPI dyeing, PBS after incubating at optical room temperature washed 3 times. Cut the slice dry, with anti-fluorescence sealant. Finally, the TUNEL-stained setons were observed under a fluorescence microscope to capture images and analyze the differences in the number of apoptotic cells.

2.9. Quantification of Apoptosis by Flow Cytometry

The apoptotic rates of chondrocytes in normal, OA and LV-Bmal1 cartilage samples cells was carried out using an Annexin V-PE/7-AAD cell death testing kit (Solarbio, China). The collected cells were washed, centrifugal settling, and then re-suspended in the buffer to concentration to 1–5×10⁶/ml. Then, 100µl of cell suspension was added 5µl Annexin V / PE, mixed and incubated at room temperature in the dark, added 7AAD and PBS, immediately detected.

2.10. Statistical analysis

All data were analyzed using GraphPad Prism Version 9.0 and OriginPro 2021, with group-to-group comparisons using Student’s t-test and single-factor analysis of variance (one-way ANOVA), with a threshold for statistically significant differences of P < 0.05.

3.1. Verification of cartilage cells and animal models

The PCR technique was used to determine the mRNA expression of Col 2 and Col 10 in cartilage cells. Following induction by ITS, the mRNA expression of Col 2 significantly increased on the 7th day and peaked after 14 days, indicating early stage of chondrogenic differentiation in ATDC5 cells. The mRNA expression of Col 10 increased gradually after induction and peaked at 21 days, indicating the late phase of chondrogenic differentiation in ATDC5 cells (Fig. 1A). With an MOI of 40, the transfection rate was approximately 30% after 24 hours and more than 70% after 72 hours, indicating that the Bmal1 expression vector exhibited slow cell transfection (Fig. 1B). The surface of cartilage from the normal group was smooth and complete, the thickness was normal, and the base was uniform, while the OA group the surface was rough and the cartilage was thin (Fig. 1C,D). Safranin O/Fast green staining showed that the thickness of the cartilage matrix in the normal group was unchanged and uniformly red. In the OA group, the cartilage matrix was destroyed, the proteoglycan was significantly lost, and the cross-section was unevenly red. The thickness of the cartilage was reduced, and the substructure was eroded under the cartilage (Fig. 1E,F). In the OA group of post-modeling cartilaginous cells and cartilage tissue, the mRNA expression of the Mmp13 gene, closely related to cartilage degradation, and the MMP13 protein expression levels were significantly higher than in the normal group. These results confirmed that ITS induced chondrogenic differentiation of ATDC5 cells after 14 days, when mature cartilage cells were formed, OA cartilage-like ATDC5 cells and OA animal modeling are successful.

3.2. Cell viability and apoptosis

After inflammatory induction of cartilaginous ATDC5 cells into the OA phenotype, cell viability increased, while that of the LV-Bmal1 cell group decreased compared to the normal and OA groups (Fig. 2A). The TUNEL staining results of the tissue specimens indicated that the positive apoptotic cells were dispersed unequally among normal cells, and there was a significant increase in the number of apoptotic cells in the OA group compared to the normal group (Fig. 2B, C). In addition, the flow cytometry analysis also revealed a significant increase in the proportion of early and late apoptotic cells in the OA group compared to the normal group, while there was a significant decrease in the LV-Bmal1 group compared to the normal group (Fig. 2D, E; P < 0.05).

3.3. Relationship and regulation of the biological clock and cell cycle

There were significant differences in the mRNA expression of Bmal1, Per1, Wee1, Cdk1, Ccnb1, and Mmp13. Compared with the Normal group, the relative mRNA expression of Bmal1 and Wee1 decreased in the OA group, while the relative mRNA expression of Per1, Cdk1, Ccnb1, and Mmp13 increased significantly. After the relative expression of Bmal1 mRNA in the LV-Bmal1 group increased due to transfection, the relative expression of Wee1 mRNA also increased, while that of Per1, Cdk1, Ccnb1 and Mmp13 mRNA decreased compared with the normal group (Fig. 3A, B). Correlation analysis showed that there was a positive correlation between the mRNA expression levels of Bmal1 and Wee1 in OA chondrocytes and knee cartilage. The mRNA expression of Bmal1 and Wee1 was negatively correlated with that of Per1, Cdk1, Ccnb1, and Mmp13, while there was a positive correlation among the latter four (Fig. 3C). These results indicated that Bmal1 may have a positive regulatory effect on Wee1 while having a negative regulatory effect on Per1, Cdk1, Ccnb1 and Mmp13. It has been reported that OA is accompanied by circadian clock disorder, which in turn may influence the cell cycle. Based on this, high expression of Bmal1 and Wee1 may be a protective factor in OA, while high levels of Per1, Cdk1, Ccnb1 and Mmp13 may be risk factors for OA.

The results of western blot analysis showed significant differences in the relative expression of BAML1, PER1, CDK1, CCNB1, WEE1, and MMP13 proteins in the normal, OA, and LV-Bmal1 groups, with P < 0.05 in all cases. BMAL1 and WEE1 protein levels were reduced in the OA group compared to the normal group, in both cultured cartilage cells and cartilaginous tissue, while those of PER1, CDK1, CCNB1, and MMP13 were increased. The relative expression levels of BMAL1 and WEE1 proteins in LV-Bmal1 group were increased, and the relative expression levels of PER1, CDK1, CCNB1 and MMP13 proteins were decreased(Fig. 3D,E). Research showed that BMAL1 controls the cell cycle by upregulating WEE1 as well as downregulating PER1, CDK1, CCNB1, and MMP13. Both of these factors may play a role in the development and occurrence of OA.

The immunohistochemistry results showed that there were large differences in the amounts of BAML1, WEE1, PER1, CDK1, CCNB1, and MMP13 proteins in OA cartilage tissue compared to normal cartilage tissue, with P values < 0.05 in all cases. In addition, the OA group had lower levels of BMAL1 and WEE1 compared to the normal group. However, the levels of PER1, CDK1, CCNB1, and MMP13 were much higher (Fig. 4). This adds to the evidence that the biological clock and cell cycle are dysregulated in OA, along with cartilage degradation, which may be closely linked to the development of OA.

Osteoarthritis is mainly characterized by damage to the joint cartilage, but it can also involve the subchondral bone, synovial membrane, and even the entire joint. Chondrocytes are the only cell type in normal cartilage. They secrete collagen and proteoglycan to form the cartilaginous matrix, which together with chondrocytes forms cartilage. Numerous internal and external factors, such as the peripheral biological clock, cell cycle regulation proteins, apoptosis, inflammatory factors, as well as nutrient metabolism inside and outside the cells, affect the division, proliferation, differentiation, and stability of chondrocytes, which can lead to local environmental imbalance and induce OA. Recent studies have increasingly recognized the importance of the peripheral biological clock in cartilage tissue and cells, whereby it has also been observed that the pain and stiffness of OA have a daily rhythm. Several studies have also found that the stability of the cartilage is regulated in the outer-circular biological clock mode [17–19]. As the core coordinator of the biological clock, Bmal1 is an important transcription factor that regulates the biorhythm and is essential for the development of hard tissue, including bone, cartilage, and teeth. It is expressed in intercellular stem cells, terminally differentiated bone cells, and cartilaginous cells [20]. Bmal1 has been widely confirmed to be associated with the occurrence and development of OA, exhibiting correlations with a lower rate of survival of cartilage cells in mouse growth plates, a decreased number of cartilaginous cells, increased apoptosis, and a significant loss of extracellular matrix [21, 22]. In addition, downregulation of Bmal1 not only impairs the survival and secretory function of cartilage cells, but also increases the expression of cartilage matrix degrading enzymes, including MMP13, which disrupts its balance, resulting in a gradual loss of cartilage [23, 24]. Among the well-known pathological changes of osteoarthritis, the progressive destruction of articular cartilage is the most significant. Matrix metalloproteinase 13 (MMP13) is the main catabolic factor involved in cartilage degradation. It has the special ability to irreversibly cleave Col2, the main component of the cartilaginous matrix, and plays a key role in the progression of OA. Bmal1 is a major contributor to OA among the biological clock genes, but the mechanism of its effects on Mmp13 in the joint cartilage is not fully understood and needs further study. In addition, a number of other biological clock genes, such as Per1 and Per2, are also associated with OA. It was found that the expression of PER1 protein showed an increasing trend in articular cartilage of OA rats and humans. Knockout of the Per2 gene in OA chondrocytes can alleviate the effects of IL-1β, hydrogen peroxide and alkaline calcium phosphate crystals on chondrocytes. It was also found that increased expression levels and ectopic expression of Per2 in chondrocytes could lead to changes in the expression of phenotypic markers, and regulate the activity of chondrocytes by affecting their phenotype [5, 25–27]. At the same time, it has also been observed that the increase of IL-1β after OA can cause the decrease of Bmal1 expression[28, 29]. Based on the finding that Bmal1 expression was decreased in OA cartilage cells, our results support the same conclusion and confirm that Per1 and Mmp13 expression also increased in OA. After expression of Bmal1 in normal cartilage cells, Per1 and Mmp13 expression decreased. The expression changes of Bmal1 influenced the expression of the cell cycle-related genes Wee1, Cdk1, and Ccnb1.

In the Cyclin-CDK-CKI cell cycle molecular network, cyclins form the regulatory core. At different times, CDKs combine with different cyclins that activate CDK functions and keep the cell cycles moving along the G1-S-G2-M phase transitions. At the same time, different CKIs can also bind corresponding CDKs or Cyclin/CDK complexes to stop CDK activity and phase transitions during the cell cycle. WEE1 is a G2 checkpoint enzyme-cell cycle regulator that protects the nucleus from the effects of the CDK1-CyclinB1 complex. It does this by mediating the phosphorylation of CDK1 on Thr14 and Tyr-15 as well as controlling the entry into mitosis (G2-M). CDK1 belongs to the family of CDKs, which can facilitate the transition of cell cycle phase G2 to phase M, as well as adjust the progression of phases G1 and S. CCNB1 is a cyclin that is mainly involved in the regulation of mitosis. Transcription begins in the S phase, peaks in the late G2 phase, and it is ubiquitinated and degraded in the late mitosis, thus exiting the M phase. CDK1 and CCNB1 are important regulators of cell proliferation, division, differentiation and apoptosis by regulating the transition from G2 to M phase of cell cycle and controlling cell cycle progression in the form of complexes.

Studies have shown that the biological clock proteins Bmal1 and circadian locomotor output cycles kaput (CLOCK) can form a complex and directly bind the Wee1 gene promoter in tumor cells to promote its transcription, while Bmal1 can induce mRNA synthesis in the liver, promoting the expression of Wee1. Conversely, when Bmal1 is knocked out, Wee1 expression is reduced[7, 30, 31]. When DNA is damaged, WEE1 blocks cells from entering into mitosis through CDK1 phosphorylation to regulate the G2/M checkpoint, giving the cell sufficient time to repair the damaged DNA and thus promoting survival [32–34]. When WEE1 is downregulated, the function of the G2/M checkpoint disappears or is defective, causing the cell to ignore the presence of DNA damage, prematurely pass the G2/M checkpoint, and enter abnormal mitosis, resulting in a mitotic catastrophe and apoptosis [35, 36]. In the course of development, the cartilage cells eventually differentiate into the phenotype of hypertrophic chondrocytes, which is controlled by CDK1 [15]. Studies have shown that increased CDK1 activity can disrupt the antiapoptotic function of Bcl-2 family proteins, accelerating karyokinesis and partially promoting proliferation [37], while in astrocytes and microglia, downregulation of CDK1 activity can inhibit neuronal autophagy and apoptosis [38]. In addition, high activity of CCNB1 can cause cell cycle disturbances, resulting in the activation of cell death mechanisms in the event of abnormal mitosis [39]. The results of this study showed that BMAL1 can indirectly lower the expression of CDK1 and CCNB1 while raising the activity of WEE1 in chondrocytes. When BMAL1 levels drop, WEE1 activity goes down, the G2 checkpoint stops working or malfunctions, but CDK1 and CCNB1 stay very active, increasing cell proliferation. At this point, cells cannot normally exit the S, G2, and M phases, DNA replication and mitosis times are shortened, which speeds up the cell cycle so that there isn't enough time to fix DNA damage cells, which leads to apoptosis. This offers a plausible mechanism through which the biological clock may affect the cell cycle, thus playing a role in the occurrence and development of OA.

The biological clock is a genetic oscillator that is closely linked to the cell cycle. This study confirmed that the expression of Bmal1 and Wee1 is positively correlated, while being negatively correlated with that of Per1, Cdk1, Ccnb1, and Mmp13. Bmal1 can positively control the expression of Wee1 as well as negatively control the expression of Per1, CDK1, CCnb1, and Mmp13. The amounts of BMAL1 and WEE1 proteins in OA chondrocytes decreased, while those of PER1, CDK1, CCNB1, and MMP13 increased. In addition, the cell viability increased, as well as the apoptosis rate. In the LV-Bmal1 group, the protein levels of BMAL1 and WEE1 were increased, while those of PER1, CDK1, CCNB1, and MMP13 decreased. At the same time, both the cell viability and apoptotic ratio went down. Based on this, it stands to reason that BMAL1 and WEE1 may be protective factors in OA, while PER1, CDK1, CCNB1 and MMP13 may be risk factors for OA. The clock gene Bmal1 causes chondrocyte apoptosis by regulating cell cycle and affects the initiation and progression of OA, providing a new perspective for the molecular mechanism of OA.

Funding Statement

The research fund for this research is mainly supported and provided by Professor Baolan Wang.

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Clock gene Bmal1 influences the cell cycle of chondrocytes in osteoarthritis

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1. Introduction

2. Materials and Methods