Session Type: Abstract Submissions (ACR)
Background/Purpose: Cigarette smoking is the best-known environmental risk factor for the development of rheumatoid arthritis (RA). However, the molecular mechanisms involved in the association of smoking and progression of RA are not well investigated. Sirtuins (SIRTs) are recently discovered regulators of inflammation. The aim of the current study was to investigate the impact of cigarette smoke extract (CSE) on the expression of interleukins (ILs) and matrix metalloproteinases (MMPs), and the possible function of SIRTs in the cigarette smoke induced inflammatory response in RA synovial fibroblasts (RASF).
Methods: Synovial tissues were obtained from RA patients undergoing joint replacement surgery. RASF from non smokers (n=16) were stimulated with 5% CSE or 10 ng/ml TNFα for 24 hours. Expression of SIRTs, ILs and MMPs was measured at the mRNA level by Real-time TaqMan and SYBR green PCR. Protein levels of SIRTs were detected by immunoblot in cell lysates and of ILs and MMPs in cell culture supernatants by ELISA. For silencing of SIRT6, RASF (n=10) were transfected with siRNA targeting SIRT6 or control siRNA for 48h prior to stimulation.
Results: Stimulation of RASF with CSE significantly increased protein levels of IL8 (by 1.9-fold±0.26, p=0.03) and MMP1 (by 1.8-fold±0.13, p=0.01), but did not affect the protein expression of IL6 and MMP3. Also at the mRNA level IL8 was 4.1-fold±0.7 (p=0.03) and MMP1 was 5.8-fold±1.4 (p=0.02) increased in CSE stimulated RASF, indicating that cigarette smoke regulates the expression of IL8 and MMP1 at the transcriptional level. Analysing the expression of SIRTs 1-7 revealed that CSE increases significantly the expression of SIRT6. Furthermore, SIRT6 protein levels were elevated upon TNFα stimulation. Basal expression as well as induction of SIRT6 after stimulation was successfully blocked by transfection of RASF with SIRT6-specific siRNA. Basal production of MMP1 increased significantly by 31±6% (p=0.008) after silencing of SIRT6. After stimulation with CSE, silencing of SIRT6 enhanced the levels of MMP1 protein by 35±8.8% (p=0.01) compared to control transfected, CSE stimulated cells. Also stimulation with TNFα had a significantly stronger effect on MMP1 expression in SIRT6 silenced, compared to control transfected cells (control: 4.1-fold ±1.4 induction, siSIRT6: 6.4-fold ±2.1 induction; p=0.004). Expression of IL6, IL8 and MMP3 was not affected by silencing of SIRT6 under basal as well as in stimulated conditions.
Conclusion: In the current study we found that CSE increases the expression of IL8 and MMP1 in RASF. Most interestingly, we could show that both CSE and TNFα induced production of MMP1 is specifically regulated by SIRT6. Therefore, we conclude that SIRT6 acts as a protective regulator attenuating CSE as well as TNFα induced MMP1 production in RASF. This protective function of SIRT6 must be considered in the development of therapies using pan sirtuin inhibitors that target also SIRT6 activities.
R. E. Gay,
Supported by Masterswitch-PF7 and her institution,
B. A. Michel,
Supported by his institution,
Supported by IAR and by his institution,
Supported by his institution,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/sirt6-regulates-cigarette-smoke-induced-mmp1-expression-in-rheumatoid-arthritis-synovial-fibroblasts/