Purification of guinea pig YKL40 and modulation of its secretion by cultured articular chondrocytes.
The aim of this study was to purify, characterize, and study the regulation at the chondrocyte level of the guinea pig (gp) homologue of human (R) YKL40, a putative marker of arthritic disorders. Studying YKL40 in guinea pigs is of particular interest, as age-related osteoarthritis develops in this species spontaneously. Both N-terminal sequencing and total amino acid composition of gpYKL40 purified from the secretion medium of cultured articular chondrocytes indicate a high degree of identity with hYKL40. gpYKL40 was found to contain complex N-linked carbohydrate, as demonstrated by N-glycosidase F and endoglycosidase F digestion. Isoelectric focusing demonstrated the presence of a major band at pI 6.7. The secretion of gpYKL40 by confluent articular chondrocytes in the extracellular medium was studied by immunoblotting. gpYKL40 was released by chondrocytes continuously over a 7 day period and did not appear to be degraded by proteinases, as its signal intensity in cell-free medium at 37 degrees C did not decrease with time. Thus, gpYKL40 displays high stability and accumulates in extracellular medium without reaching a steady-state level. Among the main factors known to regulate cartilage metabolism, IL-1beta, TNF-alpha, bFGF, or 1,25(OH)2D3 did not alter the basal level of gpYKL40, and retinoic acid had a slight inhibitory effect; TGF-beta and IGF-I and -II dose-dependently and inversely modulated this basal level. TGF-beta at 5 ng/ml decreased extracellular gpYKL40 2.9-fold, whereas IGF-I and IGF-II at 50 ng/ml increased extracellular gpYKL40 3.6- and 3.4-fold, respectively. The present biochemical and biological findings give new insights for studying the function of YKL40 in cartilage.
De Ceuninck
F
F
Institut de Recherches Servier, Division de Rhumatologie, Suresnes, France.
Pastoureau
P
P
Bouet
F
F
Bonnet
J
J
Vanhoutte
P M
PM
D054392:Adipokines; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism*; D002478:Cells, Cultured; D000071451:Chitinase-3-Like Protein 1; D019902:Chondrocytes / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology; D006023:Glycoproteins; D006031:Glycosylation; D006168:Guinea Pigs; D007526:Isoelectric Point; D007700:Kinetics; D037102:Lectins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017421:Sequence Analysis
D054392:Adipokines; D000596:Amino Acids; C084533:CHI3L1 protein, human; D000071451:Chitinase-3-Like Protein 1; D016207:Cytokines; D006023:Glycoproteins; D037102:Lectins; D011506:Proteins