Silencing of insulin receptor substrate–1 increases cell death in retinal Müller cells

[摘要] Purpose: To determine whether β-adrenergic receptors require insulin receptor substrate (IRS)-1 activity to regulate apoptosis in retinal Müller cells.Methods: Müller cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium grown in normal (5 mm) or high glucose (25 mM) conditions. The medium was supplemented with 10% fetal bovine serum and antibiotics. Cells were allowed to reach 80%–90% confluence. After becoming appropriately confluent, cells were placed in medium with reduced serum (2%) for 18–24 h to eliminate any effects of fetal bovine serum. Cells were then transfected with 10 ug of IRS-1 small hairpin RNA (shRNA). Forty-eight hours following transfection, cells were lysed and harvested for protein analysis using western blotting. In additional experiments, some cells were treated with 10 uM salmeterol for 24 h following transfection with IRS-1 shRNA. To determine whether IRS-1 directly regulates apoptotic events in the insulin-signaling pathway in retinal Müller cells, a cell death assay kit was used. In tumor necrosis factor (TNF)α inhibitory studies, cells were treated with 5 ng/ml of TNFα alone for 30 min or 30 min pretreatment with TNFα followed by salmeterol for 4 h.Results: Müller cells treated with 5 ng/ml TNFα in 25 mM glucose significantly increased phosphorylation of IRS-1Ser307. Treatment with the selective beta-2-adrenergic receptor agonist, salmeterol, significantly decreased phosphorylation of IRS-1Ser307. Following IRS-1 shRNA transfection+salmeterol treatment, Bcl-2–associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-1 shRNA also decreased cell death and increased protein levels of B-cell lymphoma-extra large (Bcl-xL), an anti-apoptotic factor.Conclusions: In these studies, we show for the first time that salmeterol, a beta-2-adrenergic receptor agonist, can reduce retinal Müller cell death through IRS-1 actions. These findings also suggest the importance of IRS-1 in beta-adrenergic receptor signaling in the prevention of cell death in retinal Müller cells.

[学科分类] 生物化学/生物物理