在人骨关节炎软骨细胞中鉴定与人类基质金属蛋白酶13近端启动子中的新型调控位点AGRE结合的蛋白质。

Identification in human osteoarthritic chondrocytes of proteins binding to the novel regulatory site AGRE in the human matrix metalloprotease 13 proximal promoter.

作者信息

Fan Zhiyong, Tardif Ginette, Boileau Christelle, Bidwell Joseph P, Geng Changshan, Hum David, Watson Alexander, Pelletier Jean-Pierre, Lavigne Martin, Martel-Pelletier Johanne

机构信息

Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montreal, Quebec, Canada.

出版信息

Arthritis Rheum. 2006 Aug;54(8):2471-80. doi: 10.1002/art.21961.

DOI:10.1002/art.21961

PMID:16868967

Abstract

OBJECTIVE

Matrix metalloprotease 13 (MMP-13) plays a major role in osteoarthritic (OA) processes. We previously identified the AG-rich element (AGRE) regulatory site (GAAAAGAAAAAG) in the proximal promoter of this gene. Electrophoretic mobility shift assays (EMSAs) done with nuclear extracts from OA chondrocytes showed the presence of 2 AGRE protein-binding complexes, the formation of which depended on the pathophysiologic state (high or low) of the cells; the low OA (L-OA) chondrocytes have low MMP-13 basal levels and high interleukin-1beta (IL-1beta) inducibility, and the high OA (H-OA) chondrocytes have high MMP-13 basal levels and low IL-1beta inducibility. In this study, we sought to determine the importance of individual AGRE bases in promoter activity and to identify AGRE binding proteins from L-OA and H-OA chondrocyte complexes.

METHODS

Promoter activity was determined following transient transfection into human OA chondrocytes. AGRE binding proteins were identified by mass spectroscopy.

RESULTS

Individual mutations of the AGRE site differentially modulated promoter activity, indicating that the intact AGRE site is required for optimal MMP-13 expression. Damage-specific DNA binding protein 1 (DDB-1) was identified in the L-OA chondrocyte-binding complex. EMSA experiments performed with the mutation of the left AGRE site (GTGCTGAAAAAG) and nuclear extracts of L-OA chondrocytes reproduced the pattern seen in the H-OA chondrocytes. Mass spectroscopy identified p130cas as one of the proteins in this complex. Supershift experiments showed the presence of p130cas and nuclear matrix transcription factor 4 (NMP-4) in the wild-type AGRE/H-OA chondrocyte complex.

CONCLUSION

These data suggest that the binding of p130(cas) and NMP-4 to the AGRE site regulates MMP-13 expression and may trigger the change in human chondrocytes from the L-OA state to the H-OA state.

摘要

目的

基质金属蛋白酶13（MMP - 13）在骨关节炎（OA）进程中起主要作用。我们之前在该基因的近端启动子中鉴定出富含AG的元件（AGRE）调控位点（GAAAAGAAAAAG）。用OA软骨细胞核提取物进行的电泳迁移率变动分析（EMSA）显示存在2种AGRE蛋白结合复合物，其形成取决于细胞的病理生理状态（高或低）；低OA（L - OA）软骨细胞具有低MMP - 13基础水平和高白细胞介素 - 1β（IL - 1β）诱导性，而高OA（H - OA）软骨细胞具有高MMP - 13基础水平和低IL - 1β诱导性。在本研究中，我们试图确定AGRE单个碱基在启动子活性中的重要性，并从L - OA和H - OA软骨细胞复合物中鉴定AGRE结合蛋白。

方法

将其瞬时转染到人OA软骨细胞后测定启动子活性。通过质谱鉴定AGRE结合蛋白。

结果

AGRE位点的单个突变对启动子活性有不同的调节作用，表明完整的AGRE位点是MMP - 13最佳表达所必需的。在L - OA软骨细胞结合复合物中鉴定出损伤特异性DNA结合蛋白1（DDB - 1）。用左AGRE位点（GTGCTGAAAAAG）突变体和L - OA软骨细胞核提取物进行的EMSA实验重现了在H - OA软骨细胞中观察到的模式。质谱鉴定出p130cas是该复合物中的一种蛋白质。超迁移实验表明在野生型AGRE/H - OA软骨细胞复合物中存在p130cas和核基质转录因子4（NMP - 4）。