Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1 beta, and okadaic acid

J Cell Biochem. 1996 Dec 15;63(4):395-409. doi: 10.1002/(SICI)1097-4644(19961215)63:4%3C395::AID-JCB2%3E3.0.CO;2-O.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / metabolism
  • Animals
  • Blotting, Northern
  • Carcinogens / pharmacology
  • Cartilage / cytology
  • Cartilage / enzymology*
  • Cartilage / growth & development
  • Cells, Cultured
  • Cholecalciferol / pharmacology*
  • Collagenases / genetics*
  • Collagenases / metabolism*
  • DNA, Complementary / genetics
  • Dactinomycin / pharmacology
  • Electrophoresis, Agar Gel
  • Enzyme Inhibitors / pharmacology*
  • Gene Expression Regulation, Enzymologic*
  • Genes, Reporter
  • Interleukin-1 / pharmacology*
  • Male
  • Nucleic Acid Synthesis Inhibitors / pharmacology
  • Okadaic Acid / pharmacology*
  • Phosphoprotein Phosphatases / physiology
  • Phosphorylation
  • Plasmids
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Protein Kinase C / physiology
  • Protein-Tyrosine Kinases / physiology
  • RNA, Messenger / analysis
  • RNA, Messenger / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Staurosporine / pharmacology
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription, Genetic
  • Transfection


  • Actins
  • Carcinogens
  • DNA, Complementary
  • Enzyme Inhibitors
  • Interleukin-1
  • Nucleic Acid Synthesis Inhibitors
  • RNA, Messenger
  • Cholecalciferol
  • Dactinomycin
  • Okadaic Acid
  • Protein-Tyrosine Kinases
  • Protein Kinase C
  • Phosphoprotein Phosphatases
  • Collagenases
  • Staurosporine
  • Tetradecanoylphorbol Acetate