Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes.
OBJECTIVE
Increased nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity in chondrocytes is associated with cartilage matrix inorganic pyrophosphate (PPi) supersaturation in chondrocalcinosis. This study compared the roles of the transforming growth factor beta (TGFbeta)-inducible plasma cell membrane glycoprotein-1 (PC-1) and the closely related B10 NTPPPH activities in chondrocyte PPi metabolism.


METHODS
NTPPPH expression was studied using reverse transcriptase-polymerase chain reaction and Western blotting. Transmembrane PC-1 (tmPC-1), water-soluble secretory PC-1 (secPC-1), and transmembrane B10 were expressed by adenoviral gene transfer or plasmid transfection, and expression of PPi was assessed in cultured articular chondrocytes and immortalized NTPPPH-deficient costal chondrocytes (TC28 cells).


RESULTS
PC-1 and B10 messenger RNA were demonstrated in articular cartilages in situ, in untreated cultured normal articular chondrocytes, and in TC28 cells. Expression of tmPC-1 and secPC-1, but not B10, rendered the NTPPPH-deficient TC28 cells able to increase expression of extracellular PPi, with or without addition of TGFbeta (10 ng/ml) to the media. More plasma membrane NTPPPH activity was detected in cells transfected with tmPC-1 than in cells transfected with B10. Furthermore, confocal microscopy with immunofluorescent staining of articular chondrocytes confirmed preferential plasma membrane localization of PC-1, relative to B10. Finally, both PC-1 and B10 increased the levels of intracellular PPi, but PC-1 and B10 appeared to act principally in different intracellular compartments (Golgi and post-Golgi versus pre-Golgi, respectively).


CONCLUSIONS
PC-1 and B10 NTPPPH activities were not redundant in chondrocytes. Although increased PC-1 and B10 expression caused elevations in intracellular PPi, the major effects of PC-1 and B10 were exerted in distinct subcellular compartments. Moreover, PC-1 (transmembrane and secreted), but not B10, increased the levels of extracellular PPi. Differential expression of PC-1 and B10 could modulate cartilage mineralization in degenerative joint diseases.
Johnson
K
K
Department of Veterans Affairs Medical Center, University of California, San Diego 92161, USA.
Vaingankar
S
S
Chen
Y
Y
Moffa
A
A
Goldring
M B
MB
Sano
K
K
Jin-Hua
P
P
Sali
A
A
Goding
J
J
Terkeltaub
R
R
D020126:Brefeldin A / Q000494:pharmacology; D002358:Cartilage, Articular / Q000166:cytology; D002460:Cell Line; D019902:Chondrocytes / Q000737:chemistry; D011756:Diphosphates / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011755:Pyrophosphatases / Q000378:metabolism / Q000494:pharmacology
D011756:Diphosphates; D008562:Membrane Glycoproteins; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D010727:Phosphoric Diester Hydrolases; C524442:Enpp3 protein, rat; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; C021895:nucleoside triphosphate pyrophosphatase