Inhibition of CDK9 prevents mechanical injury-induced inflammation, apoptosis and matrix degradation in cartilage explants

Abstract

Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acute injury responses to trauma induce production of pro-inflammatory cytokines and catabolic enzymes, which promote chondrocyte apoptosis and degrade cartilage to potentiate PTOA development. Recent studies show that the rate-limiting step for transcriptional activation of injury response genes is controlled by cyclin-dependent kinase 9 (CDK9), and thus it is an attractive target for limiting the injury response. Here, we determined the effects of CDK9 inhibition in suppressing the injury response in mechanically-injured cartilage explants. Bovine cartilage explants were injured by a single compressive load of 30 % strain at 100 %/s, and then treated with the CDK9 inhibitor Flavopiridol. To assess acute injury responses, we measured the mRNA expression of pro-inflammatory cytokines, catabolic enzymes, and apoptotic genes by RT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. For long-term outcome, cartilage matrix degradation was assessed by soluble glycosaminoglycan release, and by determining the mechanical properties with instantaneous and relaxation moduli. Our data showed CDK9 inhibitor markedly reduced injury-induced inflammatory cytokine and catabolic gene expression. CDK9 inhibitor also attenuated chondrocyte apoptosis and reduced cartilage matrix degradation. Lastly, the mechanical properties of the injured explants were preserved by CDK9 inhibitor. Our results provide a temporal profile connecting the chain of events from mechanical impact, acute injury responses, to the subsequent induction of chondrocyte apoptosis and cartilage matrix deterioration. Thus, CDK9 is a potential disease-modifying agent for injury response after knee trauma to prevent or delay PTOA development.

Fig. 1

CDK9 inhibition prevents injury-induced upregulation of pro-inflammatory cytokine and catabolic genes. A) Schematic representation of the cartilage explant injury and drug treatment, and the subsequent tests. B–F) Suppression of pro-inflammatory cytokine and catabolic enzyme genes by Flavopiridol in cartilage explants (C = control uninjured, I = injured) within 24 h post-injury, G–H) Flavopiridol does not affect the expression of the anabolic genes aggrecan and collagen 2A. All values were the mean ± standard deviation obtained from n = 6 individual donors (* p < 0.05).

Fig. 2

CDK9 inhibition reduces mRNA expression of apoptotic mediators. Flavopiridol suppresses mRNA expression of the pro-apoptotic genes p53, Bcl-2, and PTEN. All values were the mean ± standard deviation obtained from n = 6 individual donors (* p < 0.05).

Fig. 3

CDK9 inhibition rescues chondrocyte from injury-induced apoptosis. A) Flavopiridol treatment prevents chondrocyte apoptosis. At the indicated times, cartilage explants were processed for histological examination and TUNEL staining. Injury increased apoptosis in cartilage explants but Flavopiridol reduced the apoptotic cell population. Results were the mean ± standard deviation obtained from n = 3 individual donors (* p < 0.05). B) Flavopiridol enhances chondrocyte survival. At 5-days post-injury, cartilage explants were stained with LIVE/DEAD viability stain. Injury decreased cell viability but the cells were rescued by Flavopiridol. Results were the mean ± standard deviation obtained from n = 6 individual donors (* p < 0.05).

Fig. 4

Protection of cartilage from injury-induced matrix degradation. Mechanically injured cartilage explants were treated for 5 d with Flavopiridol. The amount of GAG released into the medium was measured by dimethylmethylene blue dye binding assay. Results were normalised to the wet weight of the explants. Injury caused cartilage degradation, as indicated by increased GAG release. In the presence of 300 nM Flavopiridol, levels of GAG release returned to baseline. Results were the mean ± standard deviation obtained from n = 6 individual donors (* p < 0.05).

Fig. 5

CDK9 inhibition preserves the mechanical properties of injured cartilage. Cartilage explants were cultured for 4 weeks post-injury. The cartilage compressive properties were assessed by stress-relaxation testing in unconfined compression. The 10 % relaxation and instantaneous moduli, and coefficient of viscosity are shown. Flavopiridol treatment enhanced the compressive properties of both the injured and uninjured explants. Results were the mean ± standard deviation obtained from n = 6 individual donors. Within each chart, means that do not share a letter are significantly different from each other (p < 0.05).