The P.I. has made the novel observation that macrophage (mac) expression of Ia is inhibited by nitric oxide (NO), and that this inhibition occurs at the level of gene transcription. CD4+ T-lymphocytes may only be activated by Ia-positive antigen-presenting cells such as macrophages. Thus, any inhibitor of Ia-expression will also inhibit T-lymphocyte immune responses. Ia-expressing macrophages participate in several diseases including certain glomerulonephritides, autoimmune nephritis, and kidney transplant rejection. NO is present in the kidney during these diseases and may play a regulatory role. In addition, NO is responsible for the eradication of certain tumors and infections, such as those caused by Leishmania major, Listeria monocytogenes and Mycobacterium tuberculosis. Excessive production of NO, and unnecessary tissue damage by this toxic molecule, may be prevented by NO inhibiting mac Ia-expression, and thus the subsequent event leading to NO production by macrophages. Determining how mac Ia expression is regulated by NO may offer better understanding of these illnesses and more thoughtful and effective treatment. The overall goal of this proposal is to understand how NO regulates the transcription of IA in macrophages.
Specific aim 1 will determine a) which elements of the IA promotor are involved in this inhibition by NO, and b) whether NO alters the binding,of transcription factors to these element. Altered binding could result from the absence of a necessary transcription factor or the presence of a negative regulating protein. Gel shift, DNA footprinting, and the construction of truncation mutant of the IA promotor attached to a reporter gene (CAT) will be used to address specific aim 1. Binding of IFNgamma to it's receptor leads to the phosphorylation of STAT91, which then forms a homo-dimer (GAF), enters the nucleus and binds to a specific transcription element called GAS. GAS, which is not present in the 5' flanking region of IA, may regulate gene product, such as fos, which then activate the IA gene.
In specific aim #2, gel shift assays, DNA footprinting and CAT assays will be used to evaluate the effect of NO on activation of the fos gene as a prototype of a GAF regulated gene. The effect of NO on the phosphorylation of STAT91 will be examined. These studies would be the first to suggest a link between the GAF/GAS pathway and IFNgamma signalling to the IA gene.
| Penfield, J G; Wang, Y; Li, S et al. (1999) Transplant surgery injury recruits recipient MHC class II-positive leukocytes into the kidney. Kidney Int 56:1759-69 |
