title: Purification of guinea pig YKL40 and modulation of its secretion by cultured articular chondrocytes.
abstract: 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.
authors.affiliation: Institut de Recherches Servier, Division de Rhumatologie, Suresnes, France.
authors.forename: F
authors.initials: F
authors.lastname: De Ceuninck
authors.forename: P
authors.initials: P
authors.lastname: Pastoureau
authors.forename: F
authors.initials: F
authors.lastname: Bouet
authors.forename: J
authors.initials: J
authors.lastname: Bonnet
authors.forename: P M
authors.initials: PM
authors.lastname: Vanhoutte
mesh_terms: 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
chemical_list: D054392:Adipokines; D000596:Amino Acids; C084533:CHI3L1 protein, human; D000071451:Chitinase-3-Like Protein 1; D016207:Cytokines; D006023:Glycoproteins; D037102:Lectins; D011506:Proteins