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Decreased SIRT-1 Impairs HIF-1α Nuclear Translocation in Response to Hypoxia: A Potential Mechanism of Emphysema
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A7118 - Decreased SIRT-1 Impairs HIF-1α Nuclear Translocation in Response to Hypoxia: A Potential Mechanism of Emphysema
Author Block: M. To1, N. Honda1, A. Hitani1, I. Kano1, K. Ito2, K. Haruki1; 1Department of Laboratory Medicine, Dokkyo Medical University Koshigaya Hoapital, Saitama, Japan, 2National Heart and Lung Institute, Imperial College, London, United Kingdom.
Background: SIRT-1 is a protein to promote cytoprotection upon exposure to wide range of cellular stress, and also known to be decreased in COPD lung. We previously demonstrated that hypoxia induced-HIF-1α nuclear translocation was reduced in COPD patients and this poor cellular adaptation to hypoxia might play a role in the pathogenesis of COPD, especially emphysema. In this study, we aimed at elucidating the role of SIRT1 on HIF-1α protein stabilization, HIF-1α nuclear translocation and VEGF expressions upon hypoxic condition.
Materials and Methods: SIRT-1 is knocked-down by transfection with siRNA of SIRT-1 to A549 cells, and the cells were incubated under hypoxia condition (1% O2, 5% CO2, and 94% N2) for indicated periods. HIF-1α proteins in nuclei and whole cell extracts were measured by SDS/PAGE Western blot. In the condition, mRNA of HIF-1α, VEGF and MMP-9 were quantified by quantitative real time PCR.
Results: Transfection of SIRT-1 siRNA induced a 72% reduction in SIRT-1 protein at 48h after transfection. SIRT-1 inhibition by RNAi resulted in significant decreases in hypoxia induced-HIF-1α nuclear translocations (%of SiNeg: 2hrs; 68.7±5.5%, 4hrs; 49.5±18.4%, 8hrs; 55.0±7.0%, 16hrs; 70.0±9.5%, n=4-6), but that did not affect either HIF-1α mRNA expressions or HIF-1α protein stabilisation in all indicated hypoxia exposure periods. VEGF expressions under hypoxia were significantly suppressed by SIRT-1 knock down (SiNeg: 1.81±0.41, SiSIRT-1: 1.29±0.31, n=6, p=0.026). In contrast, MMP-9 expressions under hypoxia increased by SIRT-1 knock down (SiNeg: 1.30±0.21, SiSIRT-1: 2.01±0.27, n=4, p=0.029).
Conclusion: SIRT-1 reduction in COPD seems to be involved in pathogenesis of emphysema via impaired nuclear translocations of HIF-1α under hypoxia and subsequent decrease of VEGF expressions.
Author Block: M. To1, N. Honda1, A. Hitani1, I. Kano1, K. Ito2, K. Haruki1; 1Department of Laboratory Medicine, Dokkyo Medical University Koshigaya Hoapital, Saitama, Japan, 2National Heart and Lung Institute, Imperial College, London, United Kingdom.
Background: SIRT-1 is a protein to promote cytoprotection upon exposure to wide range of cellular stress, and also known to be decreased in COPD lung. We previously demonstrated that hypoxia induced-HIF-1α nuclear translocation was reduced in COPD patients and this poor cellular adaptation to hypoxia might play a role in the pathogenesis of COPD, especially emphysema. In this study, we aimed at elucidating the role of SIRT1 on HIF-1α protein stabilization, HIF-1α nuclear translocation and VEGF expressions upon hypoxic condition.
Materials and Methods: SIRT-1 is knocked-down by transfection with siRNA of SIRT-1 to A549 cells, and the cells were incubated under hypoxia condition (1% O2, 5% CO2, and 94% N2) for indicated periods. HIF-1α proteins in nuclei and whole cell extracts were measured by SDS/PAGE Western blot. In the condition, mRNA of HIF-1α, VEGF and MMP-9 were quantified by quantitative real time PCR.
Results: Transfection of SIRT-1 siRNA induced a 72% reduction in SIRT-1 protein at 48h after transfection. SIRT-1 inhibition by RNAi resulted in significant decreases in hypoxia induced-HIF-1α nuclear translocations (%of SiNeg: 2hrs; 68.7±5.5%, 4hrs; 49.5±18.4%, 8hrs; 55.0±7.0%, 16hrs; 70.0±9.5%, n=4-6), but that did not affect either HIF-1α mRNA expressions or HIF-1α protein stabilisation in all indicated hypoxia exposure periods. VEGF expressions under hypoxia were significantly suppressed by SIRT-1 knock down (SiNeg: 1.81±0.41, SiSIRT-1: 1.29±0.31, n=6, p=0.026). In contrast, MMP-9 expressions under hypoxia increased by SIRT-1 knock down (SiNeg: 1.30±0.21, SiSIRT-1: 2.01±0.27, n=4, p=0.029).
Conclusion: SIRT-1 reduction in COPD seems to be involved in pathogenesis of emphysema via impaired nuclear translocations of HIF-1α under hypoxia and subsequent decrease of VEGF expressions.
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