This application will explore the critical role of two MAP kinase cascades, namely the: (i) extracellular signal-regulated kinases (ERKs); and (ii) c-Jun kinases (JNKs), as integral components of the biochemical circuitry which governs whether T-lymphocytes proliferate, become anergized or enter a program of cell death (apoptosis). The principal hypothesis is that the dynamic equilibrium between the ERK and JNK cascades regulates key AP-1, NFAT/AP-1 and NF-kappaB response elements (RE) which determine above cellular choices. The long-term goal is to lay the foundation for understanding the basic circuitry which may be involved in the functional decline in senescent T-cells.
Specific Aim 1 will dissect the roles of the MAPK cascades on composite NFAT/AP-1 or Rel/AP-1 response elements in the IL-2 promoter, including the action of the JNK cascade in delivering """"""""signal 2"""""""", which is dependent on TCR/CD28 co-ligation. T-cell lines will be used to determine, through stable, tetracycline-regulated expression of combinations of DA or DN afferent kinases in the ERK and JNK pathways, how these cascades synergize to stimulate NFAT/AP-1 or Rel/AP-1 RE in the IL-2 promoter. They will study cooperativity between Ras, Rac and Vav as the molecular basis for synergy between TCR and CD28 towards JNK activation. The application will extend these studies in reporter transgenic mice, in which the CD28RE/AP-1 RE functions upstream of a luciferase gene. The application also proposes to determine whether interference in MAPK function by deletion of the genes for CD28 and c-rel impact NFAT/AP-1 or Rel/AP-1 RE in the murine IL-2 promoter. In the absence CD28 co-ligation, these response elements may not be properly activated and T-cells become energized. The application proposes to determine the effect of the MAPK cascades on the expression of an inhibitory transcription factor, Nil-2a, which interferes in AP-1 activation in the IL-2 promoter.
Specific Aim 2 will study the integrated action of MAPK cascades in apoptosis pathways in T-cells, including effects on: (i) commitment to apoptosis by regulating FasL and TNF-alpha expression; and (ii) effects on execution of apoptosis by regulating the phosphorylation and expression of modulators of apoptosis. In studies on commitment towards apoptosis, they will use T-cell lines with stable inducible expression of DA or DN regulators of MAPK cascades, or T-cells activated by stress stimuli (UV irradiation , gamma-irradiation and DNA damaging drugs) to determine the effects on the TNF-alpha and FasL promoters. The effects of stress stimuli on induction of apoptosis and expression of these genes will be compared in normal murine T-lymphocytes and lymphocytes from gld and c-rel knock-out (KO) mice.
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