Parallel regulation of extracellular ATP and inorganic pyrophosphate: roles of growth factors, transduction modulators, and ANK.
OBJECTIVE
Extracellular inorganic pyrophosphate (ePPi) is a key regulator of pathologic mineralization in articular cartilage. Articular chondrocytes generate ePPi by the transportation of intracellular PPi (iPPi) through transport mechanisms such as ANK or by the degradation of extracellular adenosine triphosphate (eATP) by ectoenzymes. Although numerous modulators of ePPi have been characterized, little is known about eATP elaboration in cartilage. We sought to determine (1) whether eATP is coordinately regulated with ePPi and (2) whether ANK transports ATP.


METHODS
Primary articular chondrocytes were treated with factors known to modulate ePPi levels including growth factors (TGFβ1 and IGF-1), anion channel inhibitors, and chemicals that alter adenylyl cyclase and protein kinase C activities. Additional chondrocyte monolayers were infected with adenovirus containing functional (Ad-ANK) or mutated (Ad-ANK mutant) ANK sequences. eATP levels were measured with a bioluminescent assay.


RESULTS
TGFβ1 enhanced eATP accumulation by 33%, whereas IGF-1 decreased eATP accumulation by 63% and attenuated TGFβ1-induced eATP release by 72%. Forskolin and probenecid diminished eATP accumulation by 55% and 89%. Phorbol-12-myristate-13-acetate increased eATP by 29%. Transfection of chondrocytes with Ad-ANK caused a 10-fold increase in eATP compared with control values.


CONCLUSIONS
Modulation of eATP by various factors paralleled their effects on ePPi production, suggesting a shared pathway of ePPi and eATP production and implicating ANK in eATP transport. As eATP directly contributes to pathologic mineralization in articular cartilage, understanding eATP regulation may lead to effective therapies for crystal-associated arthritis.
Costello
Jill C
JC
Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53295-1000, USA.
Rosenthal
Ann K
AK
Kurup
Indira V
IV
Masuda
Ikuko
I
Medhora
Meetha
M
Ryan
Lawrence M
LM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000256:Adenoviridae; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000502:physiology*; D002358:Cartilage, Articular / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D005576:Colforsin; D011756:Diphosphates / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D028061:Phosphate Transport Proteins; D011339:Probenecid; D011493:Protein Kinase C / Q000378:metabolism; D034421:Sus scrofa; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology
D011756:Diphosphates; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D028061:Phosphate Transport Proteins; D053773:Transforming Growth Factor beta1; C410982:ank protein, mouse; D005576:Colforsin; D007334:Insulin-Like Growth Factor I; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D011339:Probenecid