Effect of adenovirus-mediated overexpression of bovine ADAMTS-4 and human ADAMTS-5 in primary bovine articular chondrocyte pellet culture system.
BACKGROUND
Articular cartilage matrix synthesis and degradation are dynamic processes that must be balanced for proper maintenance of the tissue. In osteoarthritis (OA), this balance is skewed toward degradation and ultimate loss of matrix. The transcriptional and/or activity levels of hundreds of genes are dysregulated in chondrocytes from osteoarthritic cartilage, and a subset of these genes may represent pivotal factors that could be modulated if their specific role in the disease process could be identified.


OBJECTIVE
To investigate the role of ADAMTS-4 and ADAMTS-5 in cartilage matrix degradation by developing a chondrocyte pellet culture assay in combination with adenoviral gene expression, and to demonstrate the utility of this assay by assessing the specific functional contribution of these genes to cartilage matrix metabolism.


METHODS
A full-length cDNA for bovine ADAMTS-4 (bADAMTS-4) was isolated, and used to evaluate the expression, regulation, and activity of this gene in bovine cartilage. Adenoviruses expressing bADAMTS-4, human ADAMTS-5 (hADAMTS-5) or human bone morphogenetic protein 2 (BMP-2) were used to infect primary chondrocytes, and their effect on extracellular matrix metabolism was assessed by monitoring the accumulation and release of glycosaminoglycans (GAG) in three-dimensional chondrocyte pellet cultures.


RESULTS
Analysis of bADAMTS-4 transcriptional regulation in chondrocytes revealed that interleukin-1alpha (IL-1alpha) was the most potent inducer of bADAMTS-4 mRNA and subsequent aggrecan degradation in cartilage explant cultures of those cytokines tested. bADAMTS-4 mRNA induction by IL-1alpha was greater in nasal cartilage than in articular cartilage. Chondrocytes infected with adenovirus expressing either bADAMTS-4 or hADAMTS-5 genes showed increased aggrecan degradation in newly synthesized matrix by pellet cultures while chondrocytes overexpressing BMP-2 showed increased aggrecan synthesis.


CONCLUSIONS
Adenoviral delivery of genes to primary bovine chondrocytes, followed by culture in three-dimensional pellet format and evaluation of extracellular matrix protein metabolism, is a useful functional assay for assessing the role of genes on cartilage matrix synthesis and degradation.
Arai
Maya
M
Department of Discovery Medicine, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA. mxarai@wyeth.com
Anderson
Dina
D
Kurdi
Yahya
Y
Annis-Freeman
Bethany
B
Shields
Kathleen
K
Collins-Racie
Lisa A
LA
Corcoran
Christopher
C
DiBlasio-Smith
Elizabeth
E
Pittman
Debra D
DD
Dorner
Andrew J
AJ
Morris
Elisabeth
E
LaVallie
Edward R
ER
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002358:Cartilage, Articular / Q000166:cytology / Q000201:enzymology*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011348:Procollagen N-Endopeptidase; D012333:RNA, Messenger / Q000235:genetics; D013045:Species Specificity
D012333:RNA, Messenger; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human