Thakran Shalini, Zhang Qiuhua, Morales-Tirado Vanessa, Steinle Jena J
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States.
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, United States.
Invest Ophthalmol Vis Sci. 2014 Dec 18;56(1):177-84. doi: 10.1167/iovs.14-15550.
Previously, we reported that pioglitazone prevented insulin resistance and cell death in type 2 diabetic retina by reducing TNFα and suppressor of cytokine signaling 3 (SOCS3) levels. Numerous reports suggest prominent vasoprotective effects of insulin growth factor binding protein-3 (IGFBP-3) in diabetic retinopathy. We hypothesized that pioglitazone protects against retinal cell apoptosis by regulating IGFBP-3 levels, in addition to reducing TNFα. The current study explored potential IGFBP-3 regulatory pathways by pioglitazone in retinal endothelial cells cultured in high glucose.
Primary human retinal endothelial cells (REC) were grown in normal (5 mM) and high glucose (25 mM) and treated with pioglitazone for 24 hours. Cell lysates were processed for Western blotting and ELISA analysis to evaluate IGFBP-3, TNFα, and cleaved caspase 3 protein levels.
Our results show that treatment with pioglitazone restored the high glucose-induced decrease in IGFBP-3 levels. This regulation was independent of TNFα actions, as reducing TNFα levels with siRNA did not prevent pioglitazone from increasing IGFBP-3 levels. Pioglitazone required protein kinase A (PKA) and DNA-dependent protein kinase (DNA PK) activity to regulate IGFBP-3, as specific inhibitors for each protein prevented pioglitazone-mediated normalization of IGFBP-3 in high glucose. Insulin growth factor binding protein-3 activity was increased and apoptosis decreased by pioglitazone, which was eliminated when serine site 156 of IGFBP-3 was mutated suggesting a key role of this phosphorylation site in pioglitazone actions.
Our findings suggest that pioglitazone mediates regulation of IGFBP-3 via activation of PKA/DNA PK pathway in hyperglycemic retinal endothelial cells.
此前，我们报道吡格列酮通过降低肿瘤坏死因子α（TNFα）和细胞因子信号转导抑制因子3（SOCS3）水平，预防2型糖尿病视网膜的胰岛素抵抗和细胞死亡。众多报道表明胰岛素生长因子结合蛋白-3（IGFBP-3）在糖尿病视网膜病变中具有显著的血管保护作用。我们推测，吡格列酮除了降低TNFα水平外，还通过调节IGFBP-3水平来保护视网膜细胞免受凋亡。本研究探讨了吡格列酮在高糖培养的视网膜内皮细胞中潜在的IGFBP-3调节途径。
原代人视网膜内皮细胞（REC）在正常（5 mM）和高糖（25 mM）环境中培养，并用吡格列酮处理24小时。对细胞裂解物进行蛋白质印迹和酶联免疫吸附测定（ELISA）分析，以评估IGFBP-3、TNFα和裂解的半胱天冬酶3蛋白水平。
我们的结果表明，吡格列酮治疗可恢复高糖诱导的IGFBP-3水平降低。这种调节独立于TNFα的作用，因为用小干扰RNA（siRNA）降低TNFα水平并不能阻止吡格列酮增加IGFBP-3水平。吡格列酮需要蛋白激酶A（PKA）和DNA依赖性蛋白激酶（DNA PK）的活性来调节IGFBP-3，因为每种蛋白的特异性抑制剂均可阻止吡格列酮介导的高糖环境中IGFBP-3的正常化。吡格列酮可增加IGFBP-3活性并减少细胞凋亡，当IGFBP-3的丝氨酸位点156发生突变时，这种作用被消除，这表明该磷酸化位点在吡格列酮作用中起关键作用。
我们的研究结果表明，吡格列酮通过激活高血糖视网膜内皮细胞中的PKA/DNA PK途径介导IGFBP-3的调节。
