The long term objective of the proposed research is to characterize mechanisms of intracellular signalling and gene regulation that are operative in kidney tubular cells in inflammatory diseases, a prominent examples of which is acute renal allograft rejection. Previous studies have demonstrate the induction of vascular cell adhesion molecule-1 (VCAM-1) in renal proximal tubular epithelial cells (RPTEC) during episodes of acute allograft rejection. VCAM-1 may play a critical role in acute rejection because its counter-receptor, VLA-4, is expressed on lymphocytes and monocytes, the presence of which is a hallmark of rejection. Essentially nothing is known about the intracellular mechanisms that regulate the VCAM-1 gene in RPTEC. The present studies seek to identify regulatory sequences in the VCAM-1 gene promoter that are involved in the transcriptional up-regulation of this gene in RPTEC in response to pro-inflammatory cytokines such as TNF-alpha. The potential role NF-kappaB and protein kinases that regulate NF-kappaB will be assessed. The approach to be employed will be a combination of Northern blot analyses, transient transfections of VCAM-1 reporter gene constructs, gel electrophoretic mobility shift assays, and immunocomplex kinase assays. Previous studies have implicated the mitogen activated protein kinases (MAP3Ks) MEKK1 and NIK in the activation of NF-kappaB. These studies raise the possibility that additional MAP3Ks might also have the capacity to activate NF-kappaB, particularly since NF-kappaB can be activated by many different stimuli. Proposed studies will examine whether other MAP3Ks might activate NF-kappaB and identify the mechanisms that might be involved. Finally, MEKK1 can also activate the MAP2K MKK4 which, in turn, activates the stress induced kinase JNK. Mutagenesis will be employed to determine specific protein sequences that are involved in the interaction between MEKK1 and MKK4. It is anticipated that these studies will identify mechanisms that are involved VCAM-1 gene regulation in RPTEC and contribute to the understanding of the NF-kappaB and JNK pathways, two pathways believed to be of critical importance in the response of cells to stress.
