UPR Activation and the Down-Regulation of α-Crystallin in Human High Myopia-Related Cataract Lens Epithelium.

To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts.The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6.In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control.These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract

Differences in Unfolded Protein Response Pathway Activation in the Lenses of Three Types of Cataracts.

To investigate the activation of three unfolded protein response (UPR) pathways in the lenses of age-related, high myopia-related and congenital cataracts.Lens specimens were collected from patients during small incision cataract surgery. Lenses from young cadaver eyes were collected as normal controls. Real-time PCR and Western blotting were performed to detect the expression of GRP78, p-eIF2α, spliced XBP1, ATF6, ATF4 and p-IRE1α in the lenses of normal human subjects and patients with age-related, myopia-related or congenital cataracts.In the lenses of the age-related and high myopia-related cataract groups, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased. Additionally, in the congenital cataract group, the protein levels of p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78 and ATF4 were greatly increased. However, the protein and gene expression levels of ATF6 were not up-regulated in the congenital cataract group compared with the normal control group.The UPR is activated via different pathways in the lenses of age-related, high myopia-related and congenital cataracts. UPR activation via distinct pathways might play important roles in cataractogenesis mechanisms in different types of cataracts

Up-regulation of p-eIF2α protein level and ATF4 gene expression level in the lens epithelium of HMC patients.

<p>RNA and protein were extracted from HMC, HMNC, ARC or normal control (Con) human epithelium specimens. Western blotting was performed to detect the protein expression of p-eIF2α (Fig 4A), and β-actin was used as the internal control. Real-time PCR was performed to detect the relative gene expression level of ATF4 in each group (Fig 4B), and 18S was used as the internal control gene (mean ± SD, <i>n</i> = 3). *<i>P</i> < 0.05; **<i>P</i> < 0.001.</p

Up-regulation of ER chaperone GRP78 mRNA and protein levels in the lens epithelium of HMC patients.

<p>RNA and protein were extracted from human epithelium specimens of HMC, HMNC, ARC or normal control (Con) samples. Western blotting was performed to detect the protein expression of GRP78 (Fig 2A), and β-actin was used as the internal control. Real-time PCR was performed to detect the gene expression level of GRP78 in each group (Fig 2B), and 18S was used as the internal control gene (mean ± SD, n = 3). *P < 0.05; **P < 0.001.</p

Up-regulated p-IRE1α protein expression level and spliced XBP1 gene expression level in the different cataract lens groups RNA and protein were extracted from human lens specimens.

<p>Western blotting was performed to detect the protein expression of p-IRE1α (Fig 2A, Fig 2B), and β-actin was used as the internal control; real-time PCR was performed to detect the relative gene expression levels of spliced XBP1 in each group (Fig 2C), and 18S rRNA was used as the internal control gene (mean ± SD, <i>n</i> = 3). *<i>P</i> < 0.05; **<i>P</i> < 0.001.</p

Relative αA- and αB-crystallin mRNA and soluble protein expression in the lens epithelium of HMC.

<p>RNA and soluble protein were extracted from human epithelium specimens of HMC, HMNC, ARC or normal control (Con) samples. Real-time PCR was performed to detect the mRNA levels of αA- and αB-crystallin (Fig 1A) in each group, and 18S was used as the internal control gene. Western blot assays were performed to detect the soluble αA-and αB-crystallin levels (Fig 1B), and β-actin was used as the internal control (mean ± SD, <i>n</i> = 3). *<i>P</i> < 0.05 and **<i>P</i> < 0.001.</p

Up-regulated cleaved ATF6 protein and gene expression levels in the different cataract lens groups.

<p>RNA and protein were extracted from human lens specimens. Western blotting was performed to detect the protein expression of cleaved ATF6 (Fig 4A and 4B), and β-actin was used as the internal control; real-time PCR was performed to detect the relative gene expression level of ATF6 in each group (Fig 4C), and 18S rRNA was used as the internal control gene (mean ± SD, <i>n</i> = 3). *<i>P</i> < 0.05; **<i>P</i> < 0.001.</p

Up-regulation of p-IRE1α protein level and spliced XBP1 gene expression level in the lens epithelium of HMC patients.

<p>RNA and protein were extracted from HMC, HMNC, ARC or normal control (Con) human epithelium specimens. Western blotting was performed to detect the protein expression of p-IRE1α (Fig 3A), and β-actin was used as the internal control. Real-time PCR was performed to detect the relative gene expression levels of spliced XBP1 in each group (Fig 3B), and 18S was used as the internal control gene (mean ± SD, <i>n</i> = 3). *<i>P</i> < 0.05; **<i>P</i> < 0.001.</p

Up-regulated expression of the ER chaperone GRP78 at the mRNA and protein levels in the different cataract lens groups RNA and protein were extracted from human lens specimens.

<p>Western blotting was performed to detect the protein expression of GRP78 (Fig 1A and 1B), and β-actin was used as the internal control; real-time PCR was performed to detect the gene expression level of GRP78 in each group (Fig 1C), and 18S rRNA was used as the internal control gene (mean ± SD, <i>n</i> = 3). *<i>P</i> < 0.05; **<i>P</i> < 0.001.</p