Scleroderma is a disorder manifested by an overproduction of dermal collagen, by a high frequency of chromosome breaks, and by autoantibody production. These seemingly unrelated phenomena may be linked to the mechanism of action of Scl-70, a topoisomerase I (topo I) which reacts with autobodies in scleroderma. Like other topo I enzymes, which nick DNA and stimulate transcription, Scl-70/topo I is activated by phosphorylation. Two major goals of this research are: (a) to investigate the relation between Scl-70/topo I activation, chromosome breaks, and collagen gene expression in scleroderma, and (b) to learn more about the mechanisms responsible for increased collagen gene transcription. A previously characterized Scl-kinase and DNA footprinting and sequencing will be used to investigate the effects of phosphorylation on the DNA binding specificity of Scl-70/topo I. Cultured fibroblasts will be employed to determine if agents which increase or decrease procollagen mRNA levels (serum factors and retinoids, respectively) also affect levels of endogenous Scl-70/topo I phosphorylation. Conversely, the effects of the topo I inhibitor camptothecin on procollagen mRNA levels will be investigated by dot blot hybridization. DNase I-fractionated chromatin from scleroderma fibroblasts and controls will be compared for differences in the distribution, absolute levels and extent of phosphorylation of Scl- 70/topo I. We will also look for increases in the DNase I sensitivity of collagen genes in scleroderma chromatin. Pulse field gradient gel electrophoresis combined with Southern blotting, hybridization with gene probes, and end-labeling techniques will be employed to determine the extent of DNA breakage and gene disruption in scleroderma. Mobility shift studies will be employed to determine if there are more topo I- induced breaks in the collagen regulatory DNA of scleroderma patients than normals. Finally, restriction endonuclease mapping will be performed to rule out collagen gene rearrangement. These in vitro studies will be corroborated by cytogenetic analysis. The proposed investigations will allow us to distinguish among several possible mechanisms of collagen overproduction in scleroderma and will provide evidence for or against a direct role for Scl- 70/topo I. Based on the outcome of these studies it may be possible to design more specific and effective therapies for this disorder.
| Douvas, A; Sobelman, S (1991) Multiple overlapping homologies between two rheumatoid antigens and immunosuppressive viruses. Proc Natl Acad Sci U S A 88:6328-32 |
