Hosler Matthew R, Wang-Su Shuh-Tuan, Wagner B J
Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07101-1709, USA.
Invest Ophthalmol Vis Sci. 2006 May;47(5):2045-52. doi: 10.1167/iovs.05-0650.
The durability of the ubiquitin proteasome pathway in the mammalian lens makes this enzyme system a potential contributor to certain cataracts and posterior capsular opacification (PCO). The present study addresses proteasome involvement in TGF-beta induced, cataract-associated gene activation in human lens cells.
HLE B-3 cells were treated with TGF-beta, in combination with the proteasome inhibitors MG-132 or lactacystin. TGF-beta target gene expression was measured by semiquantitative RT-PCR. Annexin-FITC staining and flow cytometry were used to assess apoptosis levels. Western blot analyses were performed with anti-SnoN and anti-Smad2 antibodies.
TGF-beta induced the expression of alpha-smooth muscle actin, fibronectin, and TGF-beta-inducible gene mRNA in HLE B-3 cells and primary cultured human lens cells from donor tissues. TGF-beta also induced a time-dependent decrease in the level of the Smad repressor SnoN. Gamma-glutamyl-cysteine synthetase (gamma-GCS) mRNA levels decreased in the presence of TGF-beta. Proteasome inhibitor cotreatment blocked the induction of alpha-SMA mRNA, the loss of SnoN protein, the decrease in gamma-GCS mRNA, and TGF-beta-induced apoptosis.
The HLE B-3 cell line and primary cultured human lens cells respond similarly to TGF-beta treatments by activating cataract-related gene expression. This response in both of these model systems is blocked by inhibiting the proteasome. This suggests that the proteasome can mediate cataract and PCO-associated changes and therefore is a novel target of medical therapy.
泛素蛋白酶体途径在哺乳动物晶状体中的持久性使该酶系统成为某些白内障和后囊膜混浊（PCO）的潜在促成因素。本研究探讨蛋白酶体在转化生长因子-β（TGF-β）诱导的人晶状体细胞白内障相关基因激活中的作用。
用TGF-β联合蛋白酶体抑制剂MG-132或乳胞素处理HLE B-3细胞。通过半定量逆转录聚合酶链反应（RT-PCR）检测TGF-β靶基因表达。采用膜联蛋白-FITC染色和流式细胞术评估细胞凋亡水平。用抗SnoN和抗Smad2抗体进行蛋白质印迹分析。
TGF-β诱导HLE B-3细胞以及来自供体组织的原代培养人晶状体细胞中α-平滑肌肌动蛋白、纤连蛋白和TGF-β诱导基因mRNA的表达。TGF-β还诱导Smad阻遏蛋白SnoN水平随时间下降。在TGF-β存在的情况下，γ-谷氨酰半胱氨酸合成酶（γ-GCS）mRNA水平降低。蛋白酶体抑制剂共同处理可阻断α-SMA mRNA的诱导、SnoN蛋白的丢失、γ-GCS mRNA的降低以及TGF-β诱导的细胞凋亡。
HLE B-3细胞系和原代培养的人晶状体细胞对TGF-β处理的反应相似，均通过激活白内障相关基因表达。在这两种模型系统中的这种反应均可通过抑制蛋白酶体来阻断。这表明蛋白酶体可介导白内障和PCO相关的变化，因此是药物治疗的新靶点。
