Characterization of the promoter in ADAMTS‐5 (aggrecanase‐2)
Authors: Heming M.R. , Wainwright S.D. , Caterson B. , Hughes C.E. 
Published: September 2009

Introduction  Loss of aggrecan metabolites from the cartilage matrix into the surrounding synovial fluid have allowed for identification of two major cleavage sites within aggrecan: Asn341–Phe342 and Glu373–Ala374 peptide bonds. Proteolysis at the Glu373–Ala374 bond has been specifically attributed to ADAMTS‐4 and ADAMTS‐5. Genetic reporters are commonly used in cell biology to study mechanisms regulating gene expression. Firefly luciferase is widely used as such a reporter for the immediate availability of reporter activity, the sensitivity of the assay, and quickness and ease of use. By making time‐controlled cuts in the 5′‐noncoding region of ADAMTS‐4 and ‐5 and inserting the deletions into the genetic reporters, which are then transfected into a suitable cell line, activity can be measured. The project aim was to identify the cis and trans acting factors in the 5′‐noncoding regions that regulate ADAMTS‐5 expression using the Luciferase Assay System (Promega, Madison, WI, USA).Methods  A 3‐kb fragment containing the 5′‐flanking region of ADAMTS‐5 was subcloned into pGL3‐Basic (Promega), a luciferase reporter vector lacking a eukaryotic promoter. Truncations have also been made to the full‐length construct using Erase‐a‐Base System (Promega) at restriction enzyme sites SacI at 11 bp from the origin and NheI at 21 bp from the origin, resulting in 200–2300 bp deletions on the TS‐5 vector. Adherent kidney embryonic fibroblast‐like cells (PEAK) were grown in RPMI 1640 media containing supplements (Gibco Invitrogen Corporation) at 37 °C in 5% CO2. Cells were transfected using Fugene‐6 Reagent (Roche) as recommended by the manufacturer. 1.5 × 105 cells were plated out in 12‐well plates and grown overnight. The next day, serum media was removed and RPMI 1640 media containing only glutamate was added. DNA complexes were prepared by mixing 97 µl of serum‐free medium, 3 µl of Fugene‐6, and 2 µg of DNA from Control, Basic, or TS‐5 full constructs and selected truncations and incubated for 30 min at RT. Hundred microlitres of the complex was pipetted directly into each well. After a 4–5‐h incubation, 100 µl of FBS was added to each well and left overnight at 37 °C in 5% CO2. The next morning, the cells were harvested and washed twice in PBS, and resuspended in ×1 Passive Lysis Buffer. About 20 µl of the cell lysate was assayed for luciferase activity as described in the manufacturer's protocol (Promega).Results and discussion  Full‐length and seven deletion fragments (numbering downstream from the ATG start codon) spanning the 5′‐noncoding region were selected from a library of 41 deletions prepared by the Erase‐a‐Base System: 3b.4 (−2322 bp), 4a.5 (−2110 bp), 5a.2 (1810 bp), 4a.2 (1427 bp), t3a.3 (−718 bp), 2a.1 (−433 bp) and 4a.1 (−278 bp). Firstly, an increase in activity occurs with 256 base pairs deleted from the full‐length vector (3b.4). With control considered 100% of activity, the full‐length vector produces greater than that amount indicating TS5 vector as having double the activity. A greater increase in activity (×6 that of the control) between the full length and 3b.4 suggests the presence of a suppressor somewhere in the first 256 base pairs of the 5′‐noncoding region. Activity then drops between 3b.4 and 4a.5. However, activity is still greater than that produced by the full length. A levelling‐off occurs at 5a.2, which continues until a sudden decrease at t3a.3 that is maintained in 2a.1 and 4a.1. Loss of activity between 4a.2 and t3a.3 is linked to loss of a TATA box at 1753–59 bp. There is a total of seven motifs existing in the first few hundred base pairs that could be possible suppressors including an H1 conserved site at 248–254 base pairs. By following the same methods using selected deletion fragments within −2565 to −2322, it will be possible to identify which motif is responsible for suppressing activity in this cell line. Similar experiments have been carried out in isolated articular chondrocytes.