杉野英彦(大阪大学)2011年 欧州リウマチ学会(ロンドン)

破骨細胞はマクロファージからM-CSFとRANKLによって誘導される。
一方我々は、カルシウム結合性タンパク質であるS100A4がこの破骨細胞への分化に大きな役割を話している事をDNAチップのデータから予想していた。そこでマクロファージがM-CSFとRANKLで破骨細胞に分化する過程で、S100A4遺伝子の発現をsiRNAによって阻害した。その結果、破骨細胞は形成されなかった。一方、マクロファージも細胞死を起こすことも無かった。この結果、破骨細胞形成にS100A4は必須であると予想した。

SUPPRESSION OF INTRACELLULAR S100A4 UTILIZING SMALL-INTERFERING RNA INHIBITS OSTEOCLASTOGENESIS

○H.Sugino (杉野英彦) 1 , H.-M. Lee 1 , T. Ochi 2 , N. Nishimoto3,*
1Graduate School of Frontier Biosciences, Osaka University, Suita , 2Osaka Police Hospital, Osaka, 3Laboratory of Immune Regulation, Wakayama Medical University, Ibaraki, Japan

I confirm that my abstract complies with the rules and contains orginal data: Yes
My abstract has been or will be presented at a scientific meeting during a 12 months period prior to EULAR 2011:

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovitis andprogressive joint damage. Recently, up-regulation of S100A4 expression has been reported in the serum, synovial tissues or synovial fluid from RA patients, and its expression level correlates with the disease activity. S100A4 is a
calcium binding protein belonging to S100 family, an EF-hand super-family, and is hypothesized to regulate RANKL andM-CSF signaling pathways by modulating calcium/calcineurin, calcium/CaMKIV-CREB and NFTAC-1 inocteoclastogenesis.

Objectives: To know the function of intracellular S100A4 in osteoclastogenesis, we suppressed the intracellular expression of S100A4 in the cultured pre-osteoclast and observed the osteoclast differentiation.
Methods: Rat preosteoclast cells (mixture of monocytes and macrophages from the rat femurs) were purchased fromPrimary Cell Co., Ltd (Sapporo, Japan, http://www.primarycell.com). Preosteoclasts were once washed, resuspended with a-MEM containing 10% FCS, 1% penicillin-streptomycin, 50ng/ml of M-CSF and 50ng/ml of RANKL, and seeded into24well plates (2.0 x 10 5 cells/500ml/well). Two kinds of siRNAs against S100A4 were purchased from Ambion Inc’s, Silencer Select Pre-designed siRNA (S100a4, siRNA ID s128148 and ID s128150), and transfected into preosteoclasts by Lipofectamine RNAmax (Invitrogen) at the concentration of 5nM at day3 and examined for the capability of differentiation into mature osteoclasts. Tartrate-resistent acid phosphatase (TRAP) staining was also performed at day 5
using the TRAP staining kit (Primary Cell Co., Ltd). Total RNA was extracted from the cultured cells for rtPCR analysis for osteoclast specific genes such as TRAP, cathepsin-K, and MMP-9. MTT assay was performed to confirm the survival of
the cells.

Results: S100A4 was consistently and highly expressed during the process of osteoclastogenesis compared to other
S100 family members. Both S100A4 siRNAs (ID s128148 and ID s128150) suppressed the S100A4 expression inpreosteoclasts by 60% and 50%, respectively, compared to non-transfected or irrelevant RNA-transfected cells. Theinhibitory effect of S100A4 siRNAs (ID s128148) was stronger and thus used throughout the experiments. S100A4
siRNAs clearly suppressed the osteoclast differentiation, not only morphologically determined by microscopic analysis,but also by the several specific marker enzymes such as TRAP and cathepsin K. Most of the siRNA transfected preosteoclasts could not fuse to form multi-nucleated osteoclasts. They were negative for TRAP. Decrease in both TRAP and cathepsin-K mRNA was also confirmed. In contrast, no significant change in MMP-9 expression was observed by suppression of intracellular S100A4.MTT assay at day5 confirmed that most of the S100A4 siRNA transfected preosteoclasts were alive.
Conclusions: Suppression of intracellular expression of S100A4 in preosteoclasts using siRNA inhibited the differentiation into mature osteoclasts. Thus the intracellular S100A4 has a critical role for the osteoclastogenesis. Disclosure of Interest: None Declared

リンク先
http://www.congress.eular.org/congress_2011.cfm

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