ABSTRACT
OBJECTIVE: Extracellular inorganic pyrophosphate (ePPi) plays a key role in the regulation of normal and pathologic mineralization. The purpose of this work was to evaluate the role of P1 and P2 purine receptors in modulating ePPi production by articular chondrocytes. METHODS: Porcine cartilage explants and chondrocyte monolayers were cultured in the presence of P1 agonists, or a P2 agonist or antagonist and inhibitors of P2 signaling. Ambient media ePPi concentrations were measured after 48-96h. RESULTS: The P1 agonists NECA and CGS 21680 significantly decreased ePPi concentrations surrounding chondrocytes and cartilage explants. The P2 agonist, ADP, increased ePPi levels, and the P2 antagonist, suramin, decreased ePPi concentrations. Thapsigargin and 1,2 bis-(2-aminophenoxy)ethane-N,N,N'N'-tetra acetic acid (BAPTA), which dampen Ca(2+)-related P2 signaling, suppressed the response to ADP. CONCLUSIONS: Purine receptors are important regulators of ePPi production by chondrocytes. P1 receptor stimulation diminishes and P2 receptor stimulation enhances ePPi production. Alterations in receptor signaling or aberrations of extracellular purine nucleotide metabolism resulting in abnormal quantities or proportions of P1 and P2 receptor ligands could foster changes in ePPi production that in turn affect mineralization. We propose a homeostatic role for extracellular purine nucleotides and purine receptors in stabilizing ePPi concentrations.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Diphosphates/metabolism , Purinergic P1 Receptor Agonists/pharmacology , Purinergic P2 Receptor Agonists/pharmacology , Receptors, Purinergic/drug effects , Adenosine Diphosphate/pharmacology , Animals , Cells, Cultured , Cyclic AMP/metabolism , Purinergic P2 Receptor Antagonists/pharmacology , RNA, Messenger/metabolism , Suramin/pharmacology , SwineABSTRACT
Whitlockite crystals have been observed in both degenerating and normal articular cartilages. To determine their potential for inducing cartilage degeneration, we studied their ability to induce mitogenesis and synthesis and secretion of metalloproteases in vitro. Whitlockite crystals were found to stimulate cell proliferation and to stimulate synthesis and secretion of stromelysin and collagenase. However, they were less stimulatory than crystals that contained calcium (Ca) and phosphate without magnesium substitution for Ca. Whitlockite crystals elicit biologic cellular responses that suggest potential pathogenicity in arthritis, but are less potent than Ca phosphate crystals without magnesium.
Subject(s)
Calcium Phosphates/pharmacology , Metalloendopeptidases/metabolism , Cell Division , Cells, Cultured , Collagenases/metabolism , Fibroblasts/drug effects , Fibroblasts/physiology , Humans , Immunoblotting , Matrix Metalloproteinase 3/metabolismABSTRACT
OBJECTIVE: To investigate cellular signaling mechanisms that influence chondrocyte production of inorganic pyrophosphate (PPi), which promotes calcium pyrophosphate dihydrate (CPPD) crystal deposition. METHODS: Articular chondrocyte and cartilage cultures were stimulated with protein kinase C (PKC) activator and adenyl cyclase activator. Generation of extracellular PPi was measured. RESULTS: Adenyl cyclase activation resulted in diminished pyrophosphate generation. PKC activation stimulated pyrophosphate elaboration. CONCLUSION: Two signaling pathways, cAMP and PKC, modulate generation of extracellular pyrophosphate by cartilage and chondrocytes. They are novel targets for potentially diminishing extracellular pyrophosphate elaboration that leads to CPPD crystal deposition.
Subject(s)
Adenylyl Cyclases/metabolism , Calcium Pyrophosphate/metabolism , Chondrocalcinosis/metabolism , Chondrocytes/enzymology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Alprostadil/metabolism , Animals , Carcinogens/pharmacology , Cartilage/enzymology , Cartilage/pathology , Chondrocalcinosis/drug therapy , Chondrocytes/drug effects , Cyclic AMP/metabolism , Enzyme Inhibitors/pharmacology , Protein Kinase C/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Swine , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Articular cartilage vesicles (ACV), isolated by differential centrifugation of adult hyaline articular cartilage collagenase digests, mineralized in the presence of calcium and ATP. Mineral analysis by microscopy, chemical analysis, energy-dispersive analysis, and infrared spectroscopy revealed crystals resembling calcium pyrophosphate dihydrate (CPPD). Adult articular cartilage also underwent ATP-dependent mineralization, supporting the contention that vesicles in situ fostered adult articular cartilage mineralization. Phosphocitrate (PC) is a recognized in vitro inhibitor of hydroxyapatite and calcium oxalate monohydrate crystal formation, but it is not known whether PC can similarly restrict CPPD crystal development. In the present study we examine the effect of PC, citrate, and n-sulfo-2-amino-tricarballylate (SAT, a PC analogue) on the ATP-induced CPPD crystal formation in both ACV and articular cartilage models. Only PC (10-1000 microM) blocked both the ATP-dependent and -independent mineralization in ACV in a dose-dependent fashion. At 1 mM, SAT and citrate blocked the ATP-independent mineralization. Similarly, only PC blocked both the ATP- and non-ATP-dependent mineralization in native articular cartilage slices. PC, SAT, and citrate had no effect on ACV nucleoside triphosphate pyrophosphohydrolase activity, suggesting that none of these agents blocked mineralization through the inhibition of nucleoside triphosphate pyrophosphohydrolase activity, which generates inorganic pyrophosphate from ATP.
Subject(s)
Calcium Pyrophosphate/metabolism , Cartilage, Articular/metabolism , Cartilage/metabolism , Citrates/pharmacology , Adenosine Triphosphate/metabolism , Adult , Calcification, Physiologic , Cartilage/drug effects , Cartilage, Articular/drug effects , Crystallization , Humans , In Vitro Techniques , Tricarboxylic Acids/pharmacologyABSTRACT
Our objective was to examine the effect of insulin-like growth factor-1 (IGF-1) on extracellular pyrophosphate (ePPi) elaboration by porcine cartilage. These studies further define the factors influencing ePPi accrual, a key step in calcium pyrophosphate dihydrate (CPPD) crystal formation. ePPi was measured in adult porcine organ and monolayer culture media in the presence of IGF-1, transforming growth factor beta-1 (TGFbeta-1), IGF-1 antibody and synovial fluid (SF). As previously shown, TGFbeta-1 stimulated ePPi elaboration by cartilage and chondrocytes. IGF-1 significantly inhibited the stimulatory effect of TGFbeta-1 on ePPi elaboration by both cartilage explants and chondrocytes. Anti-IGF-1 antibody blocked this inhibition. Anti-IGF-1 antibody also decreased the inhibitory effect of SF on ePPi elaboration, suggesting the presence of active IGF-1. These results support an important regulatory role for IGF-1 in cartilage ePPi elaboration. IGF-1 inhibited the effects of the ePPi-stimulatory factor TGFbeta-1 and thus may protect normal joints from excess accumulation of ePPi and subsequent CPPD crystal formation.
Subject(s)
Cartilage, Articular/drug effects , Chondrocytes/drug effects , Diphosphates/antagonists & inhibitors , Insulin-Like Growth Factor I/pharmacology , Transforming Growth Factor beta/antagonists & inhibitors , Animals , Cartilage, Articular/cytology , Cartilage, Articular/metabolism , Cells, Cultured/drug effects , Chondrocytes/metabolism , Sulfates/metabolism , Swine , Synovial Fluid/physiologyABSTRACT
OBJECTIVE: To determine whether adult articular cartilage mineralizes in the presence of ATP. METHODS: Intact adult porcine articular cartilage and monolayers of chondrocytes were cultured in physiologic media containing ATP, and mineralization was measured as retention of 45Ca. Cartilage was analyzed by electron microscopy. RESULTS: Articular cartilage sequestered 45Ca when incubated with 100 microM ATP: Use of the ATP analog alpha, beta-methylene ATP did not promote mineralization and addition of pyrophosphatase inhibited mineralization, indicating that hydrolysis of ATP to AMP and inorganic pyrophosphate is necessary for the process to occur. Mineral was concentrated in articular cartilage vesicles in the perichondral area. CONCLUSION: Adult articular cartilage mineralizes in the presence of ATP, in a manner similar to that found with isolated matrix or articular cartilage vesicles. This supports the notion that these structures have a role in chondrocalcinosis.
