Sen Malini, Reifert Jack, Lauterbach Kevin, Wolf Vladimir, Rubin Jeffrey S, Corr Maripat, Carson Dennis A
Department of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663, USA.
Arthritis Rheum. 2002 Nov;46(11):2867-77. doi: 10.1002/art.10593.
The enhanced release of extracellular matrix proteins by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients is suggestive of joint remodeling. Because Wnt proteins play a critical role in joint development, we investigated whether up-regulated Wnt signaling plays a role in the enhanced synthesis of extracellular matrix proteins. The purpose of the present experiments was to determine the role of Wnt-1-like molecules in the expression of matrix proteins by RA FLS and to ascertain the effects of Wnt antagonists on RA FLS function and survival.
Transfection with a reporter plasmid (TOPflash) was performed to assess whether Wnt signaling is active in RA FLS. Wnt signaling was up-regulated in normal FLS by transfection with a Wnt-1 expression plasmid and was down-regulated in RA FLS by transfection with dominant-negative lymphoid enhancer factor 1 (LEF-1)/T cell factor 4 (TCF-4) and secreted Frizzled receptor protein 1 (sFRP-1) expression plasmids. Recombinant sFRP-1 and anti-Wnt-1 antibody were also administered to RA FLS to block Wnt signaling.
RA FLS had constitutive activation of the canonical Wnt signaling pathway. Transfection of normal FLS with a Wnt-1 expression vector enhanced not only fibronectin, but also pro-matrix metalloproteinase 3 (proMMP-3) expression. In a complementary manner, interference with Wnt signaling using anti-Wnt-1 antibody, the Wnt antagonist sFRP-1, or dominant-negative vectors that inhibited the transcription factors TCF-4/LEF-1 blocked the expression of fibronectin by RA FLS and reduced cell survival. Anti-Wnt-1 antibody and sFRP-1 also blocked the expression of proMMP-3.
Our results indicate that the canonical Wnt pathway regulates fibronectin and metalloproteinase expression in RA FLS.
类风湿关节炎（RA）患者的成纤维样滑膜细胞（FLS）细胞外基质蛋白释放增强提示关节重塑。由于Wnt蛋白在关节发育中起关键作用，我们研究了Wnt信号上调是否在细胞外基质蛋白合成增强中起作用。本实验的目的是确定Wnt-1样分子在RA FLS基质蛋白表达中的作用，并确定Wnt拮抗剂对RA FLS功能和存活的影响。
进行报告质粒（TOPflash）转染以评估Wnt信号在RA FLS中是否活跃。通过用Wnt-1表达质粒转染使正常FLS中的Wnt信号上调，通过用显性负性淋巴增强因子1（LEF-1）/T细胞因子4（TCF-4）和分泌型卷曲受体蛋白1（sFRP-1）表达质粒转染使RA FLS中的Wnt信号下调。还将重组sFRP-1和抗Wnt-1抗体施用于RA FLS以阻断Wnt信号。
RA FLS具有经典Wnt信号通路的组成性激活。用Wnt-1表达载体转染正常FLS不仅增强了纤连蛋白的表达，还增强了前基质金属蛋白酶3（proMMP-3）的表达。以互补的方式，使用抗Wnt-1抗体、Wnt拮抗剂sFRP-1或抑制转录因子TCF-4/LEF-1的显性负性载体干扰Wnt信号，可阻断RA FLS中纤连蛋白的表达并降低细胞存活率。抗Wnt-1抗体和sFRP-1也阻断了proMMP-3的表达。
我们的结果表明经典Wnt通路调节RA FLS中纤连蛋白和金属蛋白酶的表达。
