Expression of cartilage intermediate layer protein/nucleotide pyrophosphohydrolase parallels the production of extracellular inorganic pyrophosphate in response to growth factors and with aging.
OBJECTIVE
To evaluate the role of the extracellular inorganic pyrophosphate (ePPi)-generating ectoenzyme cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) in chondrocyte PPi elaboration, we studied CILP/NTPPH expression in response to growth factors during aging.


METHODS
Porcine chondrocytes from adult (3-4-year-old) and young (2-week-old) animals were stimulated with transforming growth factor beta1 (TGFbeta1), which enhances ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which diminishes ePPi elaboration. Measurements of ePPi, NTPPH enzyme activity, Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis were performed.


RESULTS
Elaboration of ePPi into conditioned media from adult chondrocytes was significantly increased by TGFbeta1 and significantly inhibited by IGF-1, but no significant differences were observed in young chondrocytes. The protein levels of CILP/NTPPH by Western analysis in the media from adult and young porcine chondrocytes were increased by TGFbeta1. RT-PCR and Northern analysis showed that CILP/NTPPH messenger RNA (mRNA) expression in both adult and young chondrocytes was increased by TGFbeta1 and decreased by IGF-1, but these changes were less significant in the young chondrocytes. Basal and TGFbeta1-up-regulated levels of CILP/NTPPH expression were higher in adult chondrocytes than in young chondrocytes.


CONCLUSIONS
These results provide evidence that CILP/NTPPH expression and ePPi elaboration are concomitantly stimulated by TGFbeta1 and down-regulated by IGF-1, especially in adult chondrocytes, implicating CILP/NTPPH as a functional participant in ePPi elaboration. Increased CILP/NTPPH mRNA expression in chondrocytes derived from aged animals compared with young animals might promote the formation of calcium pyrophosphate dihydrate crystals in aged cartilage.
Hirose
J
J
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226, USA.
Masuda
I
I
Ryan
L M
LM
D000375:Aging / Q000187:drug effects*; D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D011756:Diphosphates / Q000378:metabolism*; D005110:Extracellular Space / Q000737:chemistry; D006133:Growth Substances / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism*; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine
D017077:Culture Media, Conditioned; D011756:Diphosphates; D006133:Growth Substances; D010710:Phosphates; D012333:RNA, Messenger; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase