Abstract
A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.
Highlights
The number of type I1 procollagen mRNA molecules/ (Ninomiya et al, 1986; Reginato et al, 1986; Wu and Eyre, cell was found to be about 2300 in vertebralchondro- 1984)
There wereabout 630 copies of type I pro- of total collagen synthesis, typeIf and type I collagen synthecollagen mRNAswith analia2 ratio of 1.6in vertebral chondrocytes compared with 5100 copies and an al/a2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells
Chondrocytes which undergo endochondral bone replacement and chondrocytes which remain as hyaline cartilage have previously been found to differ in that short chain type
Summary
Ment of chondrocyte media was carried out by resuspending 500,000 Probe back hybridization was determined by measuring the cpm or cpm of nondialyzable labeled protein in 5 ml of 5% (v/v) acetic acid relative densitometric areas of six DNA concentrations from 20 to and digesting the protein with 10 pg/ml pepsin at 4 "C for 24h. The ligation and transformation were prepared from pCs2 (Young et al, 1984); a 380-base pair Bum-Sal carried out asdescribed above This plasmid contains 220 nucleotides fragment for al(1) prepared from pMFlA8 (Finer et al, 198%) and of 5"flanking sequence and 93 nucleotides of exon 1. A 364-base pair Aua-Sau3A fragment for a2(I)prepared from pMF2l Labeled antisense RNA probes were prepared by digestion of SP64/. Primer hybridization and cDNA synthesis were carried outas described by Tate et al (1983)
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