Background/Purpose: Although current inflammation-targeted therapy improved the outcome of patients with rheumatoid arthritis (RA), the achievement of complete disease remission was still challenging. Since synovial fibroblasts (SFs) in RA are epigenetically altered, acquiring invasiveness, and excessive cytokine-producing and proliferating capacity, SFs-targeted therapy was expected to be alternative or complementally therapeutic strategy. This study was designed for discerning the underlying mechanisms involved in cyclin-dependent kinase (CDK) 4/6-mediated regulation of inflammatory mediators, including matrix metalloproteinases.

Methods: CDK4/6 activity in RASFs was inhibited or enhanced using a small-molecule CDK4/6 inhibitor (CDKI) or gene transduction. The gene and protein expressions were evaluated with quantitative PCR and ELISA under the combination treatment with IL-1β and TNFα (0.2 ng/ml, respectively) as a stimulation in the presence (10%) or absence (0.5%) of fetal bovine serum (FBS). The nuclear protein binding to the DNA sequence was assessed with an electrophoresis mobility shift assay. Protein expression and ubiquitination were assessed with Western blotting using specific antibodies. Gene knockdown was performed using RNA interfering. RNA-Seq was performed to identify genes affected by CDKI treatment in cytokine stimulation using RASFs from 5 individuals.

Results: In RASFs, cytokine productions induced with the combination of IL-1β and TNFα were enhanced in the presence of FBS. Among them, CDKI suppressed the production of MMP-1 and MMP-3, but not MMP-2, CXCL8 and IL-6. MMP-1 and MMP-3 shared AP-1 binding motif in their promoter region. CDKI impaired the binding of AP-1 components to DNA. CDK4/6 protected JUN, one of the AP-1 components, from proteasome-dependent degradation by inhibiting ubiquitination, indicating that CDK4/6 inhibition would result in the repression of a set of genes regulated by AP-1. The RNA-Seq analysis confirmed the hypothesis, namely, the AP-1 motif was enriched in a set of genes suppressed by CDKI treatment. Interestingly, according to the KEGG pathway classification, these CDKI-repressing inflammatory genes were also enriched in RA associated genes (Q value 0.03), Cytokine-cytokine receptor interaction (Q value 0.07), and IL-17 signaling (Q value 0.07).

Conclusion: The active CDK4/6 enhanced transcriptional activity of AP-1 via JUN stabilization, indicating that CDK4/6 would determine the cytokine hyper-responsiveness of RASFs. We have revealed that inhibition of CDK 4/6 prevented joint destruction in animal models of arthritis by inhibiting synovial cell proliferation and by synergizing with TNF inhibition or IL-6 inhibition. Since the pharmacologic inhibition of CDK4/6 was established as tolerable in the cancer treatment, it would exert anti-arthritic effects to attenuate pathogenic characteristics of RASFs in addition to suppressing synovial hyperplasia.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cyclin-dependent-kinase-4-and-6-determine-the-cytokine-responsiveness-by-stabilizing-jun-in-rheumatoid-arthritis-synovial-fibroblasts/