Different levels of regulation accomplish the switch from type II to type I collagen gene expression in 5-bromo-2'-deoxyuridine-treated chondrocytes.
The shift of chick embryo chondrocytes to a fibroblastic phenotype by 5-bromo-2'-deoxyuridine (BrdUrd) has been used to examine the molecular basis of the switch from type II to type I collagen gene expression. Transcription rates of each of these three collagen genes before and after this shift were measured in nuclear run-on transcription assays with double-stranded 3'-cDNA probes specific for each of these three mRNAs. Degradation rates of each of these RNAs were calculated from the rate of decrease in the concentration of each RNA after the inhibition of synthesis with actinomycin D. The shut-off of the expression of the type II collagen gene during this shift was shown to occur at the transcriptional level, since the transcription rate of this gene decreased dramatically. The decay rate of the type II mRNA (half-life of approximately 15 h) is not significantly faster in BrdUrd-treated cells. The alpha 1(I) gene is transcribed at similar rates in untreated and shifted chondrocytes, but the steady state level of alpha 1(I) RNA in chondrocytes is only 1.5% of that in shifted cells. Although the measured degradation rate of the total alpha 1(I) RNA from untreated chondrocyte cultures is approximately the same as in shifted cells (half-life of approximately 12 h), indirect evidence suggests that this alpha 1(I) RNA is derived from a low level of fibroblast contamination of these chondrocyte cultures. The alpha 1(I) RNA synthesized by untreated chondrocytes is assumed therefore to be broken down very rapidly in the nucleus. The alpha 2(I) gene is also transcribed in untreated chondrocytes at rates similar to shifted cells but, unlike alpha 1(I) RNA, its steady state level in untreated chondrocytes is approximately 30% of its level in shifted chondrocytes. The increased level of alpha 2(I) RNA in shifted cells may be regulated in part by an increase in stability of the alpha 2(I) mRNA, which has half-lives of 5.2 and 10.4 h, respectively, in untreated and shifted chondrocytes. The alpha 2(I) RNA in the untreated chondrocytes was found to have a different 5' end from that present in the BrdUrd-shifted chondrocytes or in chick embryo fibroblasts. The presence of this altered RNA in untreated chondrocytes explains the absence of synthesis of the fibroblastic alpha 2(I) collagen polypeptide chains in these chondrocytes, despite the presence of the alpha 2(I) RNA as measured with 3' probes.
Askew
G R
GR
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459.
Wang
S
S
Lukens
L N
LN
D000818:Animals; D001973:Bromodeoxyuridine / Q000494:pharmacology*; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D007700:Kinetics; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic
D012333:RNA, Messenger; D003094:Collagen; D012260:Ribonucleases; D001973:Bromodeoxyuridine