Lung inflammation involving activated T-cells is a characteristic feature of asthma, sarcoidosis, hypersensitivity pneumonitis and chronic allograft rejection after lung transplantation. T-cell activation triggers intracellular phosphorylations that lead to nuclear translocation of transcription factors in the NF-kB/rel-homology family. In the nucleus of activated T-cells, global changes in chromatin structure allow access of transcription factors to their cognate binding sites. The IL-2 enhancer contains binding sites for NF-kB, AP-1, Oct-I and the purine-box regulator that binds to the antigen receptor response element/NF-AT target site. Purine-box regulator proteins of 45 kDa and 90 kDa were affinity-purified from the nucleus of activated Jurkat T-cells and partial amino acid sequence data were used to clone NP45 and NF9O cDNAs. NF45 and NF90 interact with the DNA-dependent protein kinase, Ku70 and Ku80. NF90 also interacts with dsRNA-activated protein kinase, PKR, and suppresses translation of specific mRNAs. Interleukin enhancer binding factor 3 (ILF3) is a longer form of NF90 that interacts with Protein Arginine Methyltransferase 1. NF45 and NF90 are autoantigens in murine lupus and human autoimmune diseases. Posttranslational modifications of NF45 and NF90/ILF3 during T-cell activation will be characterized and correlated with IL-2 expression. Triptolide is a diterpenoid triepoxide that inhibits NF-kB and IL-2 transcription and T-cell activation through mechanisms that do not involve calcineurin. Triptolide reacts covalently with several nuclear proteins. Immunosuppressive and antiproliferative mechanisms of triptolide will be investigated by characterizing targets of triptolide and triptolide inhibition of transcription in vitro. The developmental phenotypes and immune system functions of mice with targeted disruptions of NF45 and NF90/ILF3 will be determined. The murine NF45 and NF90/ILF3 genes have been sequenced in entirety, targeting vectors generated, and embryonic stem cells screened for homologous recombination. Appropriate ES clones will be injected into blastocysts at the Stanford Transgenic Core facility. Increased understanding of T-cell activation and its modulation by immunosuppressant drugs will guide future therapies for lung inflammation, autoimmune diseases, cancer and AIDS.
Showing the most recent 10 out of 21 publications
