Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes

Abstract

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 ␤ (TGF␤)-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 TGF␤ (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).Conclusion. 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.Articular cartilage chondrocytes have the unique ability to constitutively elaborate relatively large amounts of extracellular inorganic pyrophosphate (PPi), and cartilage is the major source of free PPi in joints (1-3). Moreover, in aging and osteoarthritic (OA) articular cartilage, a dysregulated increase in PPi elaboration in chondrocytes and other changes in the chondrocyte differentiation and matrix composition promote calcium Ms Moffa

Pyrophosphate: a key inhibitor of mineralisation

“…To maintain joint health and integrity, cartilage calcification needs to be tightly controlled and restricted to specific regions. Chondrocytes, the resident cell type in cartilage, release ATP constitutively [70], display high levels of NPP1 activity and can produce large amounts of extracellular pyrophosphate [71,72]. In the degenerative joint disease osteoarthritis (OA), aberrant articular cartilage calcification may occur and damage the surrounding tissue [73].…”

Extracellular nucleotide metabolism and signaling in the pathophysiology of articular cartilage

“…The enzyme-inactive mutant of TNAP does not upregulate NTPPH, indicating that ePi production is required. Conversely, NPP1 overexpression in articular chondrocytes does not influence TNAP expression (48). These data suggest that TNAP activity may regulate ePPi production, but not vice versa.…”

“…CILP/NTPPH expression is 6-fold and 3-fold higher in OA with and without CPPD crystals, respectively, than it is in normal articular chondrocytes (116). However, mRNA levels for NPP1 and NPP3 are not significantly higher in OA than in normal cartilage (48). In contrast, OA enhances NPP1 and CILP/NTPPH expression in human knee menisci (36).…”

“…Articular chondrocytes constitutively elaborate large amounts of ePPi (48,49) and possess the highest known NTPPH activity (50). Several tissues that synthesize collagenous extracellular matrices express NPP1, NPP2␣, and NPP3 (36,41,48,51).…”

“…Several tissues that synthesize collagenous extracellular matrices express NPP1, NPP2␣, and NPP3 (36,41,48,51). All 3 ectoenzymes have been immunolocalized in human knee menisci (36), whereas human articular chondrocytes transcribe mRNA for NPP1 and NPP3 (48). NPP1 is located in the plasma membrane and MVs, while NPP3 is restricted to the pre-Golgi (48) (Figure 4).…”