Chondrocytes are capable of participating in breakdown of their own matrix in response to inflammatory mediators and in repair of the damaged collagen network. A cell culture model has been established employing human chondrocytes in monolayer culture as target cells for the effects of potential intercellular mediators produced by inflammatory cells which might modulate chondrocyte functions such as synthesis of a cartilage matrix macromolecules. We found that one inflammatory mediator, IFN-gamma, a product of T-lymphocytes, suppresses synthesis of type II collagen as well as types I and III collagens and fibronectin and levels of cellular procollagen mRNAs in chondrocytes. Interleukin 1 (IL-1), a monocyte product which stimulates the synthesis of PGE2 and neutral proteases, increases synthesis of types I and III collagens and associated procollagen mRNA levels when the synthesis of PGE2 which inhibits collagen synthesis, is blocked by indomethacin. In contrast, IL-1 suppresses levels of type II and type IX procollagen mRNAs thereby inducing loss of cartilage-specific phenotype.
The aim of this proposal is to determine how these positive and negative signals control collagen gene expression. Preliminary evidence indicates that some of these effects are modulated at the transcriptional level. Using recombinant preparations of IL-1 and IFN- gamma and cultured human chondrocytes as well as other human tissue cells and mouse cells as targets, control of collagen gene expression will be studied by: (1) assays of nuclear transcription in vitro and stability in cellulo of procollagen mRNAs; (2) DNAase I hypersensitivity and S1 nuclease protection experiments using DNA probes containing promoter and enhancer sequences of collagen genes; (3) analysis of cis-acting elements by transient transfection experiments using fusion construct of 5'flanking DNA (promoter + enhancer regions of different collagen genes coupled to a reporter gene such as chloramphenicol acetyl transferase (CAT); (4) analysis of potential trans-acting factors by DNA- protein gel mobility shift assay and/or DNA footprinting techniques. Since IL-1 has opposite effects on type II vs type I and type III collagen gene expression, a major goal will be to determine whether similar or different trans-acting factors and/or cis-acting elements are involved. These inflammatory mediators may induce inappropriate repair of cartilage matrix in vivo and these studies should determine whether their effects are mediated primarily at the transcriptional or post-transcriptional level.
