Biochemistry Division, National Institute of Nutrition, Hyderabad, India.
Invest Ophthalmol Vis Sci. 2013 Nov 19;54(12):7674-82. doi: 10.1167/iovs.13-12715.
Small heat shock proteins (sHsps) have a critical role under stress conditions to maintain cellular homeostasis by their involvement in protein-folding and cytoprotection. The hyperglycemia in diabetes may impose cellular stress on the retina. Therefore, we investigated the expression of sHsps, phosphoregulation of αB-crystallin (αBC), and their localization in the diabetic rat retina.
Diabetes was induced in rats and maintained on hyperglycemia for a period of 12 weeks. The expression of sHsps, HSFs, and phosphorylated sHsps was analyzed by quantitative (q) RT-PCR and immunoblotting. The solubility of sHsps was analyzed by detergent solubility assay. Cellular localization of sHsps and phosphorylated αBCs was examined by immunohistochemistry.
Of 10 sHsps, five sHsps were detected in the rat retina. Among those, increased expression for αA-crystallin (αAC), αBC, and Hsp22, and decreased expression for Hsp20 were seen in the diabetic retina, whereas Hsp27 mRNA levels were increased, while protein levels were decreased. While the expression of HSFs was either unaltered or decreased, expression of hypoxia inducible factor-1α (HIF-1α) was increased in the diabetic retina. The phosphorylation of αBC at Ser45 and Ser19 was increased in the retina of diabetic rats. However, phosphorylation of αBC at Ser59 was decreased in the soluble fraction with a concomitant increase in the insoluble fraction. Moreover, diabetes activated the p38MAPK signaling cascade by increasing the p-p38 MAPK in the retina. Further, diabetes induced the aggregation of Hsp27, αAC, αBC, and pS59-αBC in the retina. A strong immunoreactivity of Hsp27, αAC, αBC, and phosphorylated αBC was localized in different retinal layers of diabetic rats.
The results indicate an upregulation of αAC, αBC, and Hsp22, but their solubility was compromised in the diabetic retina. There was increased phosphorylation at Ser59, Ser45, and Ser19 of αBC under diabetic conditions. Localization of sHsps and their phosphorylated forms was dispersed to many layers of the retina in diabetes. These results suggest that sHsps may be protecting the retinal neurons in chronic diabetes.
小分子热休克蛋白（sHsps）在应激条件下通过参与蛋白质折叠和细胞保护作用对于维持细胞内稳态起着关键作用。糖尿病中的高血糖可能会对视网膜造成细胞应激。因此，我们研究了 sHsps 的表达、αB-晶体蛋白（αBC）的磷酸化调节及其在糖尿病大鼠视网膜中的定位。
在大鼠中诱导糖尿病，并在高血糖状态下维持 12 周。通过定量（q）RT-PCR 和免疫印迹分析 sHsps、HSFs 和磷酸化 sHsps 的表达。通过去污剂溶解性测定分析 sHsps 的可溶性。通过免疫组织化学检测 sHsps 和磷酸化的αBC 的细胞定位。
在大鼠视网膜中检测到 10 种 sHsps 中的 5 种。其中，糖尿病视网膜中αA-晶体蛋白（αAC）、αBC 和 Hsp22 的表达增加，Hsp20 的表达减少，而 Hsp27 的 mRNA 水平增加，而蛋白水平降低。HSFs 的表达要么不变，要么减少，而缺氧诱导因子-1α（HIF-1α）的表达在糖尿病视网膜中增加。αBC 在 Ser45 和 Ser19 的磷酸化在糖尿病大鼠的视网膜中增加。然而，在可溶性部分中，αBC 在 Ser59 的磷酸化减少，同时不溶性部分增加。此外，糖尿病通过增加视网膜中的 p-p38MAPK 使 p38MAPK 信号级联激活。进一步，糖尿病诱导 Hsp27、αAC、αBC 和 pS59-αBC 在视网膜中的聚集。Hsp27、αAC、αBC 和磷酸化的αBC 的强烈免疫反应性在糖尿病大鼠的不同视网膜层中定位。
结果表明，αAC、αBC 和 Hsp22 的表达上调，但在糖尿病视网膜中的可溶性受到损害。在糖尿病条件下，αBC 在 Ser59、Ser45 和 Ser19 处的磷酸化增加。sHsps 及其磷酸化形式的定位在糖尿病中分散到视网膜的许多层中。这些结果表明，sHsps 可能在慢性糖尿病中保护视网膜神经元。
