Characterization of the human ADAMTS-5 (aggrecanase-2) gene promoter.
Aggrecan degradation in osteoarthritis is mediated primarily by aggrecanases (-1 and -2) that are members of the ADAMTS family of proteases. Aggrecanase-2 (ADAMTS-5) null mice are resistant to aggrecan degradation in models of experimentally-induced osteoarthritis. In order to analyze ADAMTS-5 gene expression at the transcriptional level, we have cloned a 2.6 kb promoter region (-2096 to +506) of the human ADAMTS-5 gene and generated betagal reporter constructs. Promoter functionality was tested in transient transfection assays in chondrocytic cells. Analysis of the 2.6 kb promoter sequence indicated four putative binding sites for the Runx family of transcription factors, of which one member, Runx2, plays a role in chondrocyte maturation and hypertrophy. Overexpression of Runx2 stimulated reporter gene expression approximately 7-fold over control in SW1353 human chondrosarcoma cells and approximately 5-fold over control in primary bovine articular chondrocytes, suggesting ADAMTS-5 to be a potential downstream target of Runx2. 5'-deletions in the promoter resulted in a substantial loss in responsiveness to Runx2 implicating the functional importance of the distal promoter region that harbors putative Runx binding elements for achieving maximal Runx2 effects. The promoter-reporter construct could serve as a useful tool to understand the regulation of ADAMTS-5 gene expression at the transcriptional level.
Thirunavukkarasu
Kannan
K
Musculoskeletal Research, Lilly Research Labs, Eli Lilly and Company, Indianapolis, IN, 46285, USA. kannan@lilly.com
Pei
Yong
Y
Wei
Tao
T
D051722:ADAM Proteins / Q000235:genetics*; D000071116:ADAMTS5 Protein; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017384:Sequence Deletion; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D050659:Core Binding Factor Alpha 1 Subunit; C493709:RUNX2 protein, human; D014157:Transcription Factors; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human