Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions.
OBJECTIVE
To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS-4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic "unsilencing."


METHODS
Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS-4 was analyzed with methylation-sensitive restriction enzymes, followed by polymerase chain reaction amplification.


RESULTS
Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late-stage OA cartilage were immunopositive. The overall percentage of non-methylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP-13, 81% versus 47% for MMP-9, 57% versus 30% for MMP-3, and 48% versus 0% for ADAMTS-4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at -110 for MMP-13, -36 for MMP-9, -635 for MMP-3, and -753 for ADAMTS-4.


CONCLUSIONS
This study provides the first evidence that altered synthesis of cartilage-degrading enzymes by late-stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.
Roach
Helmtrud I
HI
University of Southampton, Southampton, UK. H.Roach@soton.ac.uk
Yamada
Norikazu
N
Cheung
Kelvin S C
KS
Tilley
Simon
S
Clarke
Nicholas M P
NM
Oreffo
Richard O C
RO
Kokubun
Shoichi
S
Bronner
Felix
F
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000368:Aged; D000369:Aged, 80 and over; D019902:Chondrocytes / Q000201:enzymology*; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic / Q000502:physiology; D005260:Female; D005333:Fetus; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008297:Male; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics* / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase; D011401:Promoter Regions, Genetic / Q000502:physiology
D051722:ADAM Proteins; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human