The nucleoside triphosphate pyrophosphohydrolase isozyme PC-1 directly promotes cartilage calcification through chondrocyte apoptosis and increased calcium precipitation by mineralizing vesicles.
OBJECTIVE
Aging associated elevations of cartilage extracellular inorganic pyrophosphate (PPi) and PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) are linked with degenerative arthritis in chondrocalcinosis. Increased chondrocyte apoptosis and expression of annexin V occur at sites of matrix calcification in degenerative arthritis, and membrane limited chondrocyte apoptotic bodies containing NTPPPH may promote chondrocalcinosis by acting as mineralizing matrix vesicles (MV). Because chondrocytes express 3 related NTPPPH isozymes [PC-1, autotaxin (ATX), and B10/PDNP3], we evaluated the effects on apoptosis and MV mediated calcium precipitation of direct expression of NTPPPH isozymes.


METHODS
To achieve "gain of function" of NTPPPH isozymes, we expressed the isozymes in cultured chondrocytic cells.


RESULTS
Plasmid cDNA transfection of PC-1, but not ATX or B10/PDNP3, markedly increased apoptosis of cultured chondrocytic knee meniscal cells and increased calcium precipitation by MV fractions. The capacity of PC-1 to increase chondrocyte and meniscal cell apoptosis, and calcium precipitation by MV, further analyzed using adenoviral gene transfer in cultured meniscal cells and articular chondrocytes, was shown to be dependent on integrity of the PC-I NTPPPH catalytic site. The MV-containing fraction released from meniscal cells and chondrocytes that overexpressed wild-type PC-1 had increased annexin V. Use of antibodies to annexin V and PC-1 revealed that both annexin V and PC-1 directly mediated the elevated calcium-precipitating capacity of MV. The increased ability of MV to precipitate calcium from PC-1-overexpressing cells did not require exogenous ATP.


CONCLUSIONS
Upregulated expression of enzymatically active PC-1 directly promotes apoptosis, increased MV annexin V, and an increased capacity of meniscal cell and articular chondrocyte MV to precipitate calcium. These results suggest a direct link between increased PC-1 expression and the pathogenesis of chondrocalcinosis.
Johnson
K
K
VA Medical Center University of California San Diego, 92161, USA.
Pritzker
K
K
Goding
J
J
Terkeltaub
R
R
D017209:Apoptosis; D002358:Cartilage, Articular / Q000166:cytology / Q000201:enzymology / Q000473:pathology; D020170:Caspase 1 / Q000378:metabolism; D002478:Cells, Cultured; D002805:Chondrocalcinosis / Q000201:enzymology* / Q000473:pathology; D019902:Chondrocytes / Q000201:enzymology* / Q000473:pathology; D005109:Extracellular Matrix / Q000201:enzymology*; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes; D007719:Knee Joint; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D014162:Transfection; D015854:Up-Regulation; D000099189:Lysophospholipase D
D020170:Caspase 1; D005956:Glucose-6-Phosphate Isomerase; D006023:Glycoproteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D000099189:Lysophospholipase D; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; C021895:nucleoside triphosphate pyrophosphatase