ROLE OF NOVEL TRANSCRIPTION FACTORS IN THE PATHOGENESIS OF RHEUMATOID ARTHRITIS

在类风湿关节炎（RA）患者中，常住的滑膜细胞

In patients with rheumatoid arthritis (RA), resident synovial cells of the pannus, as well as infiltrating mononuclear cells, produce a wide variety of bioactive molecules that contribute to the development and severity of the disease. Interleukin 1alpha(IL-1alpha) and interleukin 1beta (IL- 1beta) are proinflammatory cytokines that play a central role in the immune imbalance and tissue destruction associated with RA. Expression of the IL- 1alpha and beta genes is ultimately controlled by transcriptional regulatory proteins that bind to specific sequences within the genes and transduce intracellular second messenger signals. We recently identified a novel family of nuclear proteins, collectively termed NFIL-1betaA (betaA), that recognize the same highly conserved sequence within the IL- 1beta promotor and appear to be required for IL-1beta gene expression. The beta family may represent several distinct proteins that contain at least one DNA-binding polypeptide. One specific isoform of betaA that is exclusively expressed in fibroblasts and endothelial cells contains both a 36 kDa (p36) and a 90 kDa (p90) DNA-binding polypeptide. The broad goals of this proposal are to study the specific betaA isoform(s) expressed by synovial fibroblasts, assess their functional role in directing IL-1beta gene expression, and to examine the regulation of p36 and p90 expression in rheumatoid tissues. Specifically, we will obtain molecular probes for p36 and p90 by cloning the cDNAs for these proteins, using two distinct approaches; (a) to screen a lambdagt-11 expression library using radiolabelled double stranded DNA oligonucleotides containing the betaA binding sequence, and (b) to purify p36 and p90 crude nuclear extracts by DNA affinity chromatography in order to obtain partial amino acid sequence data. The partial amino acid sequence information will then be used to generate oligonucleotide probes for cDNA library screening or polymerase chain reaction cloning. Subsequently, we will examine the regulation of p36 and p90 expression in normal and rheumatoid synovial fibroblasts. The ability of several selected antirheumatic drugs (that are known to suppress IL-1 production) to modulate betaA expression will also be evaluated. Lastly, synovial tissue samples will be examined for betaA expression by in situ hybridization using antisense mRNA probes, and by immunohistochemical staining using the betaA antisera, in order to determine the relative levels of betaA produced by the distinct cell types within the diseased joint that can concurrently express IL-1. These studies should greatly contribute to our understanding of how IL-1 production is regulated at the molecular level in synovial fibroblasts.