A Promoter Polymorphism Regulates NFKB1 Gene Transactivity in Human Endothelial Cells under Laminar Shear Stress: 10:15AM-10:30AM: 596

Exercise-induced flow-mediated shear stress modulates endothelial function through upregulation of downstream endothelial nitric oxide synthase (eNOS) gene expression. Laminar shear stress (LSS) induces eNOS gene expression in endothelial cells through the heterodimeric transcription factor, nuclear factor kappa B (NF-□B). Recently, a 4 bp DNA sequence (ATTG) variation (-94ins/delATTG) has been identified in the promoter of the NFKB1 gene, which encodes p105/p50 isoforms of NF-□B. Although the NFKB1 promoter polymorphism influences the NFKB1 gene expression level in cancer cells, the functional significance of the polymorphism has not been elucidated in Human Umbilical Vein Endothelial Cells (HUVEC) under LSS.

PURPOSE: To determine whether the NFKB1 promoter polymorphism regulates transcriptional activity of the gene in human endothelial cells under LSS, and further, whether the polymorphism is associated with changes in forearm blood flow (FBF) after aerobic exercise training (EXTR).

METHODS: HUVEC, genotype NFKB1^I/D, were transiently transfected with a luciferase (luc) reporter gene under the control of NFKB1 promoters containing or lacking the polymorphism (NFKB1(I)- and NFKB1(D)-luc, respectively). Transfected cells were exposed to a physiological level of LSS (15 dyne/cm²) for 24 hrs using a cone-plate shear apparatus followed by dual-luc if erase reporter assays. The data presented are relative to the activity of NFKB1(I)-luc under non-LSS conditions and are the mean of four independent experiments. For the human study, eleven healthy sedentary middle-aged to older individuals were grouped by the NFKB1 genotype (II vs. ID/DD). The subjects underwent six months of EXTR, and reactive hyperemic FBF was measured using plethysmography before and after the EXTR. Allele/genotype comparisons were made using independent t-test.

RESULTS: In non-LSS HUVEC cells, the promoter polymorphism did not affect NFKB1 promoter activity. However, NFKB1 (I) and NFKB1 (D) promoter activity response to LSS was significantly different (p<0.05); NFKB 1(I) promoter activity decreased (0.74±0.2) while NFKB1(D) increased (1.51±0.2) in response to LSS. In the human studies, the ID/DD genotype group (n=8) showed a tendency for greater improvement in peak FBF compared with II homozygotes group (n=3) after EXTR (II, 0.88±0.3vs. ID/DD, 1.98±0.4%, P = 0.12).

CONCLUSION: The NFKB1 -94 ins/del ATTG promoter polymorphism is functional in the response of HUVEC to flow shear stress, and may be associated with adaptability in endothelial function with exercise intervention.