CDK9Inhibition Attenuates Cartilage Inflammatory Responses In Vitro And Preventspost-traumatic Osteoarthritis In Short-term In Vivo

Osteoarthritis (OA) affects more than half of the people over age65in the United States. OA is a degenerative disease of the articular joints characterized by slow but progressive lost of cartilage. The main protein component of articular cartilage is a fibrillar network of type II collagen (Col2a), which provides tensile strength to the cartilage. The compressive stiffness of the cartilage is provided by the proteoglycan components, through their attraction of water molecules. Although the etiology of OA remains incompletely understood, various inflammatory conditions that cause damage to the collagens and proteoglycans in cartilage are suspected of initiating OA.Pro-inflammatory cytokines, such as interleukin1beta (IL-1β) and tumor necrosis factor alpha (TNFα), can induce the catabolic destruction of the cartilage matrix. These cytokines elicit a cascade of events that activate inflammatory mediator genes and apoptosis in chondrocytes. Pro-inflammatory cytokines induce the expression of proteinases that degrade cartilage matrix, including matrix metallopeptidases (MMPs), aggrecanases, and cathepsins. Pro-inflammatory cytokines are induced by a variety of stress conditions in cartilage, including joint overloading and physical damage such as occurs in sports-related injuries. Strikingly, a disproportionally large number of people with joint injuries developed OA. Since injury activates pro-inflammatory cytokines expression, we hypothesize that injury-related activation of the inflammatory response in chondrocytes ultimately contributes to cartilage destruction and the development of OA. Therefore, a strategy to suppress the inflammatory response in chondrocytes after acute tissue stress or injury may help prevent the onset of OA.Acute tissue stress and inflammatory signaling activate primary response genes (PRG) that do not require de novo protein synthesis. Recent advances demonstrate that despite their initiation by diverse signaling pathways, the transcriptional activation of most, if not all, PRG is similarly controlled by a general transcription factor, namely the cyclin-dependent kinase9(CDK9). CDK9is ubiquitously and abundantly expressed in all cell types, but unlike the other more well-known CDKs, it is not directly involved in regulating cell cycle progression. It was believed for many years that the rate-limiting step in transcriptional activation of PRG is the recruitment of transcription factors and RNA Polymerase II (Pol II) complex to the promoters. However, recent studies show that in order for these PRG to be activated rapidly, in their basal and unstimulated states, the Pol II complex is already pre-assembled and producing short mRNA transcripts. However, the Pol II is then paused～40base-pair downstream of the transcription start site. Promoter-proximal pausing of Pol II is accomplished by the concerted efforts of the negative elongation factor (NELF) and the DRB-sensitive inducible factor (DSIF). The rapid activation of PRG is the result of signal-induced recruitment of CDK9to the promoters, where it phosphorylates NELF and DISF to negate their repression. CDK9also phosphorylates Pol II at the Serine2residues of the C-terminal domain heptapeptide repeats. Phosphorylation by CDK9induces a conformational change that allows Pol II to enter possessive elongation to efficiently transcribe full-length mRNAs. The recruitment of CDK9to activated PRG promoters is accomplished by the bromo-domain containing protein4(Brd4), through its binding to acetylated histones. Thus, CDK9regulation represents a unique mechanism of activating PRG transcription and has a broad impact on many aspects of biological functions.In this study, based on cell culture, tissue culture and mouse knee injury model, we investigated:1) if CDK9inhibition protect chondrocyte from catabolic effects induced by pro-inflammatory cytokines,2) if CDK9inhibition protect cartilage from mechanical injury, and3) if CDK9inhibition prevent or delay the onset of osteoarthritis in mouse knee model. ObjectiveThe objective of this study is to determine whether CDK9inhibition effectively suppress the inflammatory response in chondrocytes and protect cartilage from the catabolic effects of pro-inflammatory cytokines in vitro and in vivo.Methods1. Human chondrocytes were challenged with pro-inflammatory cytokines like IL-1β, TNFaand LPS, cartilage explants harvested from bovine stifle knee joints suffered from single mechanical loading at30%strain. All the samples were continued to be cultured in the presence or absence of300nM Flavopiridol. The anterior cruciate ligaments of mouse knee joints were ruptured by a custom made device, with or without intra peritoneal injection of7.5mg/kg Flavopiridol, knee samples were also harvested at various time points.2. Ssamples were smashed in liquid nitrogen with mortar and pestle. RNA was immediately extracted. Absorbance measurements were measured at260nm and280nm to determine the concentration of RNA extracted from the plug and the purity of the extract. Revers transcription of equal quantities of RNA (2.5ug) from each sample was performed.For the determination of individual mRNA expression,4.5ul of cDNA was used for quantitative RT-PCR (in a final volume of10ul) performed in triplicate. Results were normalized to18s rRNA and calculated as fold-change in mRNA expression relative to untreated control, using the2-△△CT method.3. Chondrocytes were seeded in96-well plates. Cytotoxicity was assessed using the Vybrant Cytotoxicity Assay kit. For determining long-term effects of Flavopiridol on the viability of chondrocytes in cartilage, bovine cartilage explants (6-mm disk) were cultured in6-well plates in DMEM and10%FBS, in the presence or absence of300nM Flavopiridol for6days, with media changed every other day. The live and dead cells were stained using the LIVE/DEAD Viability/Cytotoxicity kit (Invitrogen, cat#L3224) according to the manufacturer’s protocol. The percentages of live and dead cells were determined by counting the cell numbers at three random fields of the cross-section images of explants captured using a fluorescence microscope. A total of3different donors were used.4. Western blot analysis-Chondrocytes grown in12-well plates were harvested and lysed with sample loading buffer. Lysates were resolved by4-12%SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (BioRad). The membranes were blocked with3%skim milk in TBST, followed by incubation with rabbit anti-CDK9(0.6ug/ml)mouse anti-MMP13(1:500dilution), or mouse anti-GAPDH at4oC overnight. Blots were then probed with horseradish peroxidase-conjugated secondary antibody, and reactive protein bands were visualized with Western Lightning Plus-ECL (Perkin Elmer) on radiographic films.5. Assessment of cartilage degradation-Human cartilage explants (-3mm cubes) were treated with1ng/ml IL-1βfor6days, in the presence or absence of6or300nM Flavopiridol (with media change on day3). The amount of glycosaminoglycan (GAG) released into the media was determined by the colorimetric dimethylmethylene blue dye-assay, with chondroitin sulfate as standard. The release of Col2a degradation products into the media was determined by measuring the amount of cleaved Col2a peptides with the C2C ELISA kit (IBEX Pharmaceuticals) according to the manufacturer’s protocol.6. Samples fixed with4%paraformaldehyde were transferred to75%ethanol after24hours. H&E staining was then performed on5um-thick sections using standard protocol. Slides were observed under optical microscope, and photo was taken under same magnification.7. Statistical analysis-Values of all measurements were expressed as the mean+standard deviation. Data were analysed by Student’s t test for two samples comparison and analysis of variance (ANOVA) for multiple varies comparison. A p<0.05was considered significant.Results1. CDK9inhibition protects chondrocyte from deleterious effects of pro-inflammatory cytokines. CDK9inhibition by Flavopiridol effectively suppressed iNOS mRNA induction by all three pro-inflammatory stimuli. Results from NFkB-targets PCR array showed that Flavopiridol suppressed the induction of a broad range of inflammatory mediator genes (59out of67tested) by IL-1β. Flavopiridol also inhibited the induction of catabolic genes MMP1,3,9,13, and ADAMTS4,5; but did not affect the basal expression of anabolic genes such as Col2a, aggrecan, and COMP, and housekeeping genes. Flavopiridol had no apparent short-term cytotoxicity as assessed by glucose-6-phosphate dehydrogenase activity. Finally, in IL-1β-treated cartilage explants, Flavopiridol reduced the release of matrix degradation products GAG and cleaved Col2a peptides, but did not affect long-term chondrocyte viability.2. CDK9inhibition protect cartilage explants from single impact mechanical injury2.1CDK9inhibition suppresses the expression of catabolic genes in cartilage explants injury modelSingle mechanical over-loading increased those selected catabolic genes, in which IL-6, ADAMTS4and MMP1showed a time dependent manner, shortly after injury. As expected, those catabolic genes expression were remarkably suppressed by co-cultured with Flavopiridol in each time point. On the other hand, we also tested two anabolic genes (Aggreacan and Collagen2a) expression, and results showed neither of them was impacted by single injury or Flavopiridol treatment in same24hours frame.2.2CDK9inhibition saves chondrocytes via prevents chondrocytes from apoptosis caused by mechanical injuryChondrocytes viability assay showed injury killed30%chondrocytes when compared with uninjured control, and Flavopiridol rescue chondrocytes, although the result is not significant. This result supports that Flavopiridol saves chondrocytes from mechanical injury.2.3CDK9inhibition prevents GAG degradationCartilage explants were continuously cultured for5day in the presence or absence of300nM Flavopiridol after injury; medium was collected and processed for DMMB staining for measuring the release of GAG. GAG releases into the medium was increased after injury, however, the concentration of GAG was significantly reduced and return to baseline level by treatment of300nM Flavopiridol. This result showed that the CDK9inhibition keeps cartilage matrix intact after mechanical injury.3. CDK9inhibition prevents the very early stage onset of OA after mouse knee joint injury model3.1CDK9inhibition suppresses the expression of pro-inflammatory cytokines and down-stream effect genes in vivo.In the PTOA mouse model, the expression of IL-1β and IL-6mRNA increased rapidly2hrs after knee injury and peaked at4hrs, then returned gradually to baseline after3-7days. However, their induction was greatly reduced by Flavopiridol\. Similar results were seen in other catabolic genes such as MMP13and ADAMTS4. In contrast, the expression of the anabolic genes, Col2al and aggrecan, were neither impacted by knee injury nor by Flavopiridol.3.2CDK9inhibitoin prevent sub-chondral bone loss in short term after joint injuryMicroCT demonstrated a significant subchondral bone loss at the femoral epiphysis of the injured knees3days after injury, but was prevented by CDK9inhibition. BV/TV ratio of Flavopiridol treated injury group was almost as same as control group.3.3CDK9inhibiton prevent inflammation infiltration after knee injuryHistological analysis of3-days post-injured knees showed heavy signs of inflammation, synovial hyperplasia, pennas formation, and leukocyte infiltration. However, these clinical presentations were reduced by Flavopirdol.Conclusion1. CDK9inhibition effectively suppresses the induction of inflammatory mediators and catabolic genes to protect chondrocyte from the deleterious effects of pro-inflammatory cytokines, without impacting cell viability and functions.2. CDK9inhibition in the suppression of pro-inflammatory cytokine induced by mechanical injury and prevention of chondrocyte apoptosis in cartilage explants. In addition, our data strongly indicate that Flavopiridol is an effective agent to prevent cartilage degradation and save cartilage mechanical properties.3. CDK9inhibition markedly attenuates the pro-inflammatory cytokines expression and followed catabolic gene activation, sub-chondral bone resorption and remodeling, and inflammation infiltraion induced by anterior cruciate ligament rupture in mouse knee injury model.