Rm. Grumbles et al., REGULATION OF RAT INTERSTITIAL COLLAGENASE GENE-EXPRESSION IN GROWTH CARTILAGE AND CHONDROCYTES BY VITAMIN-D-3, INTERLEUKIN-1-BETA, AND OKADAIC ACID, Journal of cellular biochemistry, 63(4), 1996, pp. 395-409
The interstitial collagenase produced by the rat growth plate chondroc
ytes is the homologue of the human collagenase-3, or matrix metallopro
teinase-13. This enzyme is responsible for the loss of collagen during
hypertrophy of chondrocytes and for the degradation of transverse sep
ta in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) h
ad an 8-fold higher level of collagenase mRNA in the hypertrophic vers
us proliferative zone of growth plate cartilage. Intramuscular injecti
on of 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3; 1.0 mu g/kg body wei
ght) in rachitic rats increased collagenase mRNA another 1.5-fold in t
he hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)(
2)D-3 and interleukin (IL)-1 beta in cultured proliferative chondrocyt
es was studied by means of steady-state mRNA and half-life determinati
on of mRNA using the transcriptional inhibitor actinomycin D, and nucl
ear run-on transcription analyses. Treatment of cells with 1,25-(OH)(2
)D-3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8-
and 13-fold, respectively. Additionally, the collagenase mRNA half-lif
e was increased by 1,25-(OH)(2)D-3 and IL-1 beta. In the presence of a
protein kinase C inhibitor, staurosporine, 1,25-(OH)(2)D-3 induction
of collagenase mRNA was blocked. Here the addition of phorbol 12-myris
ate 13-acetate (PMA) to activate protein kinase C increased collagenas
e 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 mRN
A abundance 10-fold. The rate of the rat collagenase gene transcriptio
n in nuclei was increased with 1,25-(OH)(2)D-3, 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)(2)D-3, IL-1 beta, and PMA increased
reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, ther
e are multiple nuclear and cytoplasmic mechanisms by which cartilage m
odulators regulate rat interstitial collagenase gene expression. (C) 1
996 Wiley-Liss, Inc.