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To facilitate our studies on the mechanisms controlling collagenase production at a molecular level in rabbit synovial fibroblasts, we have constructed a cDNA library using mRNAs isolated from cells induced with crystals of monosodium urate monohydrate. We have screened this library with cDNA probes made from induced and control mRNA populations. From among 30 clones that hybridized preferentially to the induced-cell probe, 4 contained collagenase sequences. The largest, a clone of 650 base pairs, was identified by its ability to hybrid select a mRNA that could be translated in a cell-free system into a product that was precipitable with monospecific antibody to collagenase. Using this clone to probe blots of RNA from induced cells, we detected the appearance of a collagenase mRNA of 2.7 kilobases within 5 hr of addition of urate. The level of collagenase mRNA continued to increase for 35-40 hr, when it was 60 to 90 times more abundant in induced cells than in control cells. The increase in mRNA levels correlated with an increase in immunoreactive collagenase protein that was detectable in culture medium by 10 hr.

We studied mechanisms governing production of the neutral proteinase collagenase by synovial cells. We used a model system of monolayer cultures of rabbit synovial fibroblasts stimulated to produce collagenase by treatment with phorbol myristate acetate or crystals of monosodium urate monohydrate. mRNAs from these and untreated cells were translated in a wheat germ cell-free system. Collagenase was not present in the culture medium or in the in vitro translation products of mRNA from untreated cells but was present in both the medium and translation products of stimulated cells, as analyzed by gel electrophoresis and immunoprecipitation with monospecific antibody. Induction of collagenase was prevented by treatment of the cells with alpha-amanitin (2 mug/ mL), an inhibitor of mRNA synthesis. We have concluded that the induction of collagenase synthesis by either phorbol myristate acetate or urate crystals is due to an increased level of translatable mRNA.

Portions of rabbit genomic clones containing exons cross-hybridize with human synovial cell and human foreskin fibroblast mRNAs. These cross-hybridizing genomic fragments have been subcloned into pBR328 and may be useful probes in isolating cDNA and genomic clones from human tissue, and perhaps from other species as well. In addition, DNA sequence data on a 530 bp cDNA clone for rabbit synovial-cell collagenase indicate that this clone is composed primarily of 3'-untranslated region. As such, it is probably not useful in cross-species hybridizations.

We have determined the nucleotide sequence of a collagenase mRNA from rabbit synovial cells from which the primary structure of the encoded protein was deduced. This proteinase is 51% homologous to the enzyme that activates it from the zymogen form, rabbit synovial cell activator/stromelysin. Rabbit collagenase and activator/stromelysin thus share comembership in a gene family that includes human skin collagenase; the human and rabbit metalloproteinase, activator/stromelysin; and an oncogene-induced proteinase from rat named transin. The mRNA sequence of collagenase enabled us to completely map the structure of its gene, which is 9.1 kilobases and is composed of 10 exons and 9 introns. This is the first report of the structure of a collagenase gene. We show that it has striking similarity to additional members of this metalloproteinase gene family, transin genes I and II of rat. We have further sequenced genomic DNA flanking the collagenase gene and have identified nucleic acid elements of possible importance in gene regulation.