류마티스 섬유모세포양 활막세포에서 저산소증 및 사이토카인에 의한 Aggrecanase-1과 Aggrecanase-2 발현 조절
Regulation of aggrecanase-1 and aggrecanase-2 expressions by hypoxia and cytokines in rheumatoid fibroblast-like synoviocytes
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목적 : 류마티스 관절염 FLS에서 저산소증에 의한 aggrecanase-1, -2 발현의 변화를 알아보고, 각종 사이토카인이 aggrecanase의 발현에 미치는 영향을 살펴 보고자 하였다. 방법 : Aggrecanase-1, -2의 mRNA 발현을 관찰하기 위하여 hypoxic chamber에서 FLS를 24시간 동안 배양시킨 세포로부터 분리한 RNA로 cDNA를 합성하고, Real Time PCR을 시행하여 그 결과를 분석하였다. 또한 FLS에 IL-1β, IL-6, IL-8, EGF, TNF-α, TGF-β를 각각 처리하고 24시간 동안 배양하였다. 배양 후 RNA를 분리하여 합성한 cDNA로 real-time PCR을 시행하여 그 결과를 분석하였다. 결과 : 저산소 상태에서 aggrecanse-1 mRNA 발현은 정상 산소 상태와 비교하여 8시간 이후부터 2배 이상 증가하였다. Aggrecanase-2 mRNA 발현은 30분 후부터 증가하기 시작하여 1시간째에 이르러 약 2배까지 증가하였으나 그 이후부터는 감소하는 경향을 보였다. Aggrecanase-1 mRNA 발현은 IL-1β에 의해 1.29±0.41배, IL-6에 의해 2.1±0.13배, EGF에 의해 2.65±1.49배, TNF-α에 의해 1.62±0.45배 그리고 TGF-β에 의해 5.0±1.79배 증가하였다. 그러나 IL-8은 aggrecanase-1 유전자 발현을 의미있게 증가시키지 못하였다. Aggrecanase-2 mRNA 발현은 IL-6에 의해 2.28±1.21배 증가하였고, 그 외 TGF-β에 의해 1.88±0.75배, EGF에 의해 1.96±0.62배 증가한 소견을 보였다. IL-1β와 IL-8은 aggrecanase-2 유전자 발현의 증가에 의미 있는 영향을 미치지 못하였으며 TNF-α는 오히려 발현을 감소시킨 것으로 나타났다.
Abstract
Background : While aggrecanases (aggrecanase-1 and aggrecanase-2) are substantially responsible for cartilage aggrecan breakdown in rheumatoid arthritis (RA), not much information is available on the regulation or expression of the two key aggrecanases in rheumatoid fibroblast-like synoviocytes (FLS). The aim of this study is to determine the effect of hypoxia and several cytokines on the expression of the aggrecanases in rheumatoid FLS. Methods : FLS obtained from RA patients were cultured under hypoxic condition for 24 hours. Quantitative polymerase chain reaction assays for mRNA expression of aggrecanase-1 and aggrecanase-2 were performed on FLS cultured under hypoxia. Additionally, to see the effect of various cytokines, same experiments were conducted after treating FLS with IL-1β, IL-6, EGF, TGF-β and TNF-α, compared with control. Results : Hypoxia significantly increased both aggrecanase-1 and -2 mRNA expression in rheumatoid FLS compared with normoxia. IL-1β, IL-6, EGF, TGF-β and TNF-α upregulated the mRNA expression of aggrecanase-1. Both EGF and TGF-β upregulated the mRNA expression of aggrecanase-2, but TNF-α significantly downregulated the mRNA expression of aggrecanase-2. Conclusions : These results showed upregulation of aggrecanase-1 and -2 by hypoxia and differential regulation of aggrecanase-1 and -2 by various cytokines in rheumatoid FLS. It suggests that hypoxia and cytokines enhance the aggrecanase activity of RA FLS and contribute to joint destruction.(Korean J Med 71:182-188, 2006)
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