Activation of the unfolded protein response in aged human lenses.
Cataract formation is a multifactorial disease, induced by a variety of stressors. The endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is known to produce reactive oxygen species (ROS) leading to apoptosis. The present study aimed to investigate whether activation of the UPR occurs in human lenses, using human lens epithelial cell (HLEC) lines and lenses obtained from an eye bank, from individuals aged between 50 and 90 years. In vitro analysis was performed using calcimycin (10 µM) as an ER stressor. The level of ER stress was measured by the production of ROS, staining for cell death, detection of binding immunoglobulin proteins (BIP) and levels of other UPR proteins, including inositol-requiring enzyme-1 (IRE), activating transcription factor (ATF) 6 and PKR-like eukaryotic initiation factor 2a kinase (PERK). These parameters were examined in HLECs exposed to calcimycin for 12, 24, 48 and 72 h. Fluorescent activated cell sorting analysis of the levels of ROS and apoptosis revealed an increase following 24 h calcimycin exposure. The reverse transcription quantitative polymerase chain reaction results demonstrated a gradual increase in the mRNA levels of BIP, IRE1, ATF6 and PERK between 12 and 72 h. A similar effect was observed in the protein levels, which also demonstrated a gradual increase in the levels of endoplasmic oxidoreductin-1-like (Ero1-L)-β and protein disulfide isomerase, but a lower level of Ero1-Lα. Activation of the UPR involved the apoptotic pathway, revealed by increased levels of C/EBP homologous protein, ATF4 and caspase-4. Additionally, the antioxidant protein levels were also suppressed. The investigation of aged human lenses revealed a similar increase in the protein expression of UPR. These results indicated that activation of the UPR‑induced ROS production suppressed the antioxidant status and triggered the apoptotic pathway, ultimately leading to the formation of age-related cataracts.
Department of Ophthalmology, Liaocheng People's Hospital, Liaocheng, Shandong 252000, P.R. China.
He-Zhen
HZ
Tang
Department of Ophthalmology, Liaocheng People's Hospital, Liaocheng, Shandong 252000, P.R. China.
Li-Min
LM
Yang
D051702:Activating Transcription Factor 6 / Q000235:genetics / Q000378:metabolism; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D002460:Cell Line; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008875:Middle Aged; D010088:Oxidoreductases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D051742:Transcription Factor CHOP / Q000378:metabolism; D056811:Unfolded Protein Response / Q000235:genetics / Q000502:physiology*; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism
C497111:ATF6 protein, human; D051702:Activating Transcription Factor 6; D061207:Calcium Ionophores; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D051742:Transcription Factor CHOP; D000001:Calcimycin; C405327:ERO1A protein, human; D010088:Oxidoreductases; C460640:EIF2AK3 protein, human; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D004722:Endoribonucleases