Salvador-Silva M, Ricard C S, Agapova O A, Yang P, Hernandez M R
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Neurosci Res. 2001 Oct 1;66(1):59-73. doi: 10.1002/jnr.1197.
The small heat shock proteins (sHSP), alpha B-crystallin and Hsp27 are chaperone molecules that maintain the integrity of intermediate filament (IF) network and prevent unfolding of cellular proteins induced by stress. In the optic nerve head (ONH) of eyes with glaucoma, reactive astrocytes expressed Hsp27, perhaps in response to stress related to elevated intraocular pressure. In this study, we determined the effect of elevated hydrostatic pressure (HP) in the synthesis, distribution and co-localization of alpha B-crystallin and Hsp27 with IF in cultured ONH astrocytes. Astrocyte monolayers were pressurized to 60 mm Hg (92% air 8% CO(2)) and incubated at 37 degrees C for 6, 24 or 48 hr. Controls were exposed to ambient pressure. Cells were analyzed by immunocytochemistry, Western blot and immunoprecipitation using antibodies to Hsp27, alpha B-crystallin, vimentin or GFAP. Control astrocytes seemed flat, polygonal with short processes. alpha B-crystallin appeared granular in the perinuclear area and filamentous in the cell periphery. Fine granular Hsp27 was distributed throughout the cytoplasm. GFAP and vimentin co-localized with Hsp27 in the cytoplasm. Astrocytes exposed to HP were star-shaped with long processes. Hsp27 was condensed in large granules around the nucleus. GFAP and vimentin co-localized with Hsp27 and alpha B-crystallin in the perinuclear area. Western blot and metabolic labeling detected increased synthesis of Hsp27, GFAP and vimentin but no change in alpha B-crystallin. These results indicated that GFAP and vimentin associate with Hsp27 and alpha B-crystallin in ONH astrocytes. HP affected the integrity of the cytoskeleton consistent with morphological changes. Small HSP may reinforce and maintain IF integrity in response to HP.
小热休克蛋白（sHSP）、αB晶状体蛋白和Hsp27是伴侣分子，可维持中间丝（IF）网络的完整性，并防止应激诱导的细胞蛋白展开。在青光眼患者的视神经乳头（ONH）中，反应性星形胶质细胞表达Hsp27，这可能是对与眼压升高相关的应激的反应。在本研究中，我们确定了升高的静水压力（HP）对培养的ONH星形胶质细胞中αB晶状体蛋白和Hsp27的合成、分布以及与IF共定位的影响。将星形胶质细胞单层加压至60 mmHg（92%空气+8% CO₂），并在37℃下孵育6、24或48小时。对照组暴露于常压。使用针对Hsp27、αB晶状体蛋白、波形蛋白或胶质纤维酸性蛋白（GFAP）的抗体，通过免疫细胞化学、蛋白质印迹和免疫沉淀分析细胞。对照星形胶质细胞看起来扁平，呈多边形，有短突起。αB晶状体蛋白在核周区域呈颗粒状，在细胞周边呈丝状。细颗粒状的Hsp27分布于整个细胞质中。GFAP和波形蛋白在细胞质中与Hsp27共定位。暴露于HP的星形胶质细胞呈星形，有长突起。Hsp27在细胞核周围浓缩成大颗粒。GFAP和波形蛋白在核周区域与Hsp27和αB晶状体蛋白共定位。蛋白质印迹和代谢标记检测到Hsp27、GFAP和波形蛋白的合成增加，但αB晶状体蛋白无变化。这些结果表明，在ONH星形胶质细胞中，GFAP和波形蛋白与Hsp27和αB晶状体蛋白相关联。HP影响细胞骨架的完整性，与形态学变化一致。小HSP可能在响应HP时加强并维持IF的完整性。
