Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: rapid induction of type X collagen in culture.
Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15-20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Keefe
R J
RJ
Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, New York.
Puzas
J E
JE
Loveys
L
L
Hicks
D G
DG
Rosier
R N
RN
D000818:Animals; D001345:Autoradiography; D015152:Blotting, Northern; D002458:Cell Fractionation; D002478:Cells, Cultured; D002645:Chickens; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006132:Growth Plate / Q000166:cytology / Q000378:metabolism*; D017403:In Situ Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000378:metabolism
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D003094:Collagen