Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Role of Raf Kinase Inhibitor Protein in Cytokines, Matrix Metalloproteinases Regulation and Invasiveness of Rheumatoid Fibroblast-Like Synobiocytes.

Ahn1,  Joong Kyong, Lee2,  You Sun, Park3,  Eun-Jung, Hwang3,  Ji-Won, Oh3,  Ji-Min, Lee3,  Jaejoon, Jeon4,  Chan-Hong

Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine
Masan Samsung Hospital, Sungkyunkwan University School of Medicine
Samsung Medical Center, Sungkyunkwan University School of Medicine
Soonchunhyang University College of Medicine

Objective:

Raf kinase inhibitor protein (RKIP) is a protein that directly interacts with the kinase domain of Raf-1, and has been shown to modulate many processes, including Alzheimer's disease, diabetes and cancer. RKIP negatively regulates the Raf/MEK/ERK pathway by interfering with the activity of Raf-1. Also, RKIP-1 interferes with the activity of the IKK complex and prevents the movement of NF-kB to the nucleus. The role of RKIP in rheumatoid fibroblast-like synoviocytes (FLS) is not known. The present study was performed to investigate the expression and function of RKIP in rheumatoid arthritis (RA) FLS.

Methods:

RKIP expression was measured in synovial tissue (ST) and FLS by Western blot analysis. Plasmid containing RKIP or control vector, RKIP small interfering RNA (siRNA), or control siRNA were transfected into FLS using the Amaxa system. Expression of cytokines and matrix metalloproteinases (MMPs) mRNA were examined by quantitative real-time PCR. Phosphorylated MAP kinases were measured in RA FLS by Western blot analysis. NF-kB EMSA was performed after TNFa stimulation in RKIP-overexpressed FLS. In vitro cell invasion assay was performed to measure the invasiveness of RA FLS according to RKIP silencing.

Results:

RKIP protein was detected in RA ST and FLS, which was similar to osteoarthritis ST and FLS (n=4 each). RKIP overexpression decreased IL-6, IL-8, MMP-1, and MMP-3 mRNA expression (40.76±24.38%, 32.16±18.93%, 31.25±25.09%, and 30.39±14.16% inhibition, respectively) in TNF-a-stimulated RA FLS (n=4), but these results did not reach statistical significance except the result of IL-6 mRNA (p=0.001). RKIP silencing by siRNA resulted in significantly increased MMP-1 and MMP-3 mRNA expression (1.8±0.4 and 2.3±0.8 fold increase, respectively) in TNF-a-stimulated RA FLS (n=3, p=0.01 for each). RKIP silencing also increased IL-6 and IL-8 mRNA expression (1.4±0.3 and 2.1±0.8 fold increased, respectively) in TNF-a-stimulated RA FLS (n=3), but it's not statistical significant. RKIP overexpression suppressed TNF-a-induced ERK phosphorylation. However, p38 and JNK phosphorylation by TNF-a were not affected by RKIP overexpression. RKIP overexpression also suppressed TNF-a-induced NF-kB activation measured by EMSA. RKIP silencing resulted in significantly higher invasion index in TNF-a-stimulated RA FLS compared to controls (10.33±1.45 vs. 4.00±0.58, n=3, p=0.02).

Conclusion:

RKIP plays an important role in inflammatory cytokine and MMP production by regulating ERK and NF-kB activation in RA FLS. These results suggest that RKIP might be a potential therapeutic target for RA.

To cite this abstract, please use the following information:
Ahn, Joong Kyong, Lee, You Sun, Park, Eun-Jung, Hwang, Ji-Won, Oh, Ji-Min, Lee, Jaejoon, et al; Role of Raf Kinase Inhibitor Protein in Cytokines, Matrix Metalloproteinases Regulation and Invasiveness of Rheumatoid Fibroblast-Like Synobiocytes. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :366
DOI: 10.1002/art.28135

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