Analysis of cartilage maturation using micromass cultures of primary chondrocytes.
A micromass culture (MM-C) system of primary immature chondrocytes for functional analysis of soluble factors involved in the maturation step of cartilage was previously developed. Ectopically expressed BMP-2 was shown to induce the expression of the Ihh and Noggin genes. Here it is demonstrated that, upon longer culture, secreted bone morphogenetic protein-2 (BMP-2) further promotes the maturation step as judged by the induction of type X collagen and BMP-6 expression, which are known to be detectable in the later phase of cartilage maturation. Induction of all of these genes by secreted BMP-2 was not inhibited by ectopic expression of parathyroid hormone-related peptide (PTHrP) induced by retrovirus vector infection, although the same virus vector showed strong inhibitory effects on the expression of type X collagen gene or alkaline phosphatase activity in mature chondrocytes. These results suggest that the maturation-promoting activity exhibited by BMP-2 is dominant over the suppressive effect of PTHrP in immature chondrocytes. When the BMP-6 gene was introduced into the same virus vector as that used for BMP-2, it induced the same sets of genes (Ihh, Noggin, type X collagen and endogenous BMP-6) as BMP-2 did. These results also suggest that BMP-6 would autonomously maintain and/or promote a later stage of chondrocytic maturation.
Kameda
T
T
Department of Gene Regulation, Institute of Medical Science, University of Tokyo, Japan.
Koike
C
C
Saitoh
K
K
Kuroiwa
A
A
Iba
H
H
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins; D002356:Cartilage / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D018929:Cell Culture Techniques; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D053823:Hedgehog Proteins; D017403:In Situ Hybridization; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein
D055396:Bone Morphogenetic Protein 2; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D002352:Carrier Proteins; D003094:Collagen; D004279:DNA, Viral; D053823:Hedgehog Proteins; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D000099176:Noggin Protein