The Role of Calcineurin and NFAT in the Regulation of Insulin Gene Transcription

Lawrence, Michael C., The Role of Calcineurin and NFAT in the Regulation of Insulin Gene Transcription. Doctor of Philosophy (Biomedical Sciences), December 2001, 185 pp., 41 illustrators, references, 222 titles. In an effort to understand glucose and hormone regulated insulin gene transcription elicited by increased intracellular calcium, a novel pathway has been identified. This pathway involves the calcium/calmodulin-dependent phosphatase 2B (calcinuerin) and nuclear factor activated T-cells (NFAT), which in the studies herein, have been determined to up-regulate insulin gene transcription in response to glucose and glucagon-like peptide-1 (GLP-1) in pancreatic β-cells. Three NFAT elements within the first 410 base pairs of the rat I insulin gene promoter were first identified, two of which are conserved (by presence and location) among mammals including dogs, mice, and humans. The presence of NFAT in rat insulinoma β-cells (INS-1) and rat pancreatic islets was detected by immumobotting, immunofluorescence, and RT-PCR. Electrophoretic mobility shift assays displayed NFAT-specific DNA-binding activity that could be competed with unlabeled NFAT probe when incubated with INS-1 cells or rat islet nuclear extracts and shifted with extracts pre-incubated in the presence of either anti-calcineurin or anti-NFAT antibodies. Transfection experiments with either the -410 rat I (rIsnI-Luc) or the NFAT-Luc promoter-reported showed increased promoter activity when stimulated by glucose or cell depolarization (increases intracellular calcium) and displayed a synergistically enhanced response when co-stimulated with glucose and GLP-1. The GLP-1 induced responses were mimicked by forskolin and concentration-dependently inhibited by each of two selective but distinct protein kinase A (PKA) inhibitors, H-89 and myristoylated PKI (14-22) amide. The selective calcineurin-inhibitor FK506, as well as the chelatin of intracellular Ca2+ by BAPTA, also abolished the effects of high glucose and GLP-1. Moreover, co-transfection experiments with a constitutively active form of calcineurin and the promoter-reporters (rISnI-Luc and NFAT-Luc) showed increased reporter activity over controls. Furthermore, two-point base pair mutations in any of the three identified NFAT sites within the rat insulin I promoter resulted in a significant (p [less than] 0.05) reduction in the combined effect of glucose and GLP-1. These studies establish the presence of NFAT in insulin-secreting cells, its ability to bind elements within the insulin gene promoter, and show that glucose and GLP-1 synergistically enhance NFAT-mediated insulin gene transcription by PKA- and calcineurin-dependent pathways in pancreatic β-cells.