Ospelt Caroline, Mertens Joachim C, Jüngel Astrid, Brentano Fabia, Maciejewska-Rodriguez Hanna, Huber Lars C, Hemmatazad Hossein, Wüest Thomas, Knuth Alexander, Gay Renate E, Michel Beat A, Gay Steffen, Renner Christoph, Bauer Stefan
University Hospital, Zurich, Switzerland.
Arthritis Rheum. 2010 May;62(5):1224-35. doi: 10.1002/art.27395.
Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP-4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage.
Expression of FAP and DPP-4/CD26 by RASFs was analyzed using fluorescence-activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L-glutamyl L-boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real-time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell-derived factor 1 (SDF-1 [CXCL12]), MMP-1, and MMP-3 protein levels were measured using enzyme-linked immunosorbent assay. The impact of FAP and DPP-4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry.
Inhibition of serine protease activity of FAP and DPP-4/CD26 in vitro led to increased levels of SDF-1 in concert with MMP-1 and MMP-3, which are downstream effectors of SDF-1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP-expressing RASFs and marked by a significantly higher accumulation of MMP-1 and MMP-3, when compared with controls.
Our results indicate a central role for the serine protease activity of FAP and DPP-4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.
由于类风湿关节炎（RA）患者滑膜中的成纤维细胞表达丝氨酸蛋白酶成纤维细胞活化蛋白（FAP）和二肽基肽酶4（DPP-4）/CD26，我们开展了本研究以确定这两种酶在RA滑膜成纤维细胞（RASF）侵入关节软骨中的功能作用。
使用荧光激活细胞分选和免疫细胞化学分析RASF中FAP和DPP-4/CD26的表达。通过荧光底物的裂解测量丝氨酸蛋白酶活性，并用L-谷氨酰-L-硼脯氨酸处理后抑制其活性。使用实时聚合酶链反应检测RASF中基质金属蛋白酶（MMP）和金属蛋白酶组织抑制剂（TIMP）的诱导和表达。使用免疫印迹对MMP进行光密度测量，在信使RNA水平上证实了我们的发现。使用酶联免疫吸附测定法测量基质细胞衍生因子1（SDF-1 [CXCL12]）、MMP-1和MMP-3蛋白水平。在RA的SCID小鼠共植入模型中，使用免疫组织化学分析FAP和DPP-4/CD26抑制对RASF侵袭性的影响。
体外抑制FAP和DPP-4/CD26的丝氨酸蛋白酶活性导致SDF-1水平升高，同时MMP-1和MMP-3水平也升高，MMP-1和MMP-3是SDF-1信号传导的下游效应物。使用SCID小鼠共植入模型，体内酶活性抑制显著促进异种移植的RASF侵入共移植的人软骨。与对照相比，软骨吸收区域被表达FAP的RASF浸润，并且MMP-1和MMP-3的积累明显更高。
我们的结果表明FAP和DPP-4/CD26的丝氨酸蛋白酶活性在保护关节软骨免受RA滑膜成纤维细胞侵袭方面起着核心作用。
