Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5.
OBJECTIVE
To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity.


METHODS
Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo.


RESULTS
TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage.


CONCLUSIONS
An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.
Rogerson
Fraser M
FM
University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia. fraser.rogerson@mcri.edu.au
Stanton
Heather
H
East
Charlotte J
CJ
Golub
Suzanne B
SB
Tutolo
Leonie
L
Farmer
Pamela J
PJ
Fosang
Amanda J
AJ
D051722:ADAM Proteins / Q000172:deficiency*; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D053674:Aggrecans / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002356:Cartilage / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005270:Femur Head / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D011348:Procollagen N-Endopeptidase / Q000172:deficiency*
D053674:Aggrecans; D010450:Endopeptidases; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C498489:Adamts5 protein, mouse; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603919:Adamts4 protein, mouse; C083641:aggrecanase