Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1 beta, and okadaic acid.
The interstitial collagenase produced by the rat growth plate chondrocytes is the homologue of the human collagenase-3, or matrix metalloproteinase-13. This enzyme is responsible for the loss of collagen during hypertrophy of chondrocytes and for the degradation of transverse septa in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) had an 8-fold higher level of collagenase mRNA in the hypertrophic versus proliferative zone of growth plate cartilage. Intramuscular injection of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 1.0 micrograms/kg body weight) in rachitic rats increased collagenase mRNA another 1.5-fold in the hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)2D3 and interleukin (IL)-1 beta in cultured proliferative chondrocytes was studied by means of steady-state mRNA and half-life determination of mRNA using the transcriptional inhibitor actinomycin D, and nuclear run-on transcription analyses. Treatment of cells with 1,25-(OH)2D3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8- and 13-fold, respectively. Additionally, the collagenase mRNA half-life was increased by 1,25-(OH)2D3 and IL-1 beta. In the presence of a protein kinase C inhibitor, staurosporine, 1,25-(OH)2 D3 induction of collagenase mRNA was blocked. Here the addition of phorbol 12-myrisate 13-acetate (PMA) to activate protein kinase C increased collagenase mRNA 10-fold. However, in the presence of staurosporine (50 nM), PMA induction was blocked, whereas IL-1 beta was not. IL-1 beta is known to activate several phosphorylation pathways. Okadaic acid (500 nM), a protein phosphatase inhibitor, increased the relative collagenase mRNA abundance 10-fold. The rate of the rat collagenase gene transcription in nuclei was increased with 1,25-(OH)2D3, IL-1 beta and okadaic acid. In separate experiments, the collagenase promoter was ligated to a reporter plasmid and the plasmid was transfected into chondrocytes. The results showed that 1,25-(OH)2D3, IL-1 beta, and PMA increased reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, there are multiple nuclear and cytoplasmic mechanisms by which cartilage modulators regulate rat interstitial collagenase gene expression.
Grumbles
R M
RM
Arthritis Research Laboratory, Miami V.A. Medical Center, Florida, USA.
Shao
L
L
Jeffrey
J J
JJ
Howell
D S
DS
D000199:Actins / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D002273:Carcinogens / Q000494:pharmacology; D002356:Cartilage / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000494:pharmacology*; D017364:Collagenases / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D003609:Dactinomycin / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D007375:Interleukin-1 / Q000494:pharmacology*; D008297:Male; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000502:physiology; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
D000199:Actins; D002273:Carcinogens; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D002762:Cholecalciferol; D003609:Dactinomycin; D019319:Okadaic Acid; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D017364:Collagenases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate