The unfolded protein response in lens epithelial cells from galactosemic rat lenses.
OBJECTIVE
Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats.


METHODS
Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit.


RESULTS
Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation.


CONCLUSIONS
The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.
Mulhern
Michael L
ML
Department of Ophthalmology and Visual Sciences, University of Nebraska, Omaha, NE 68198-5840, USA.
Madson
Christian J
CJ
Danford
Andrew
A
Ikesugi
Kengo
K
Kador
Peter F
PF
Shinohara
Toshimichi
T
D051701:Activating Transcription Factor 4 / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002386:Cataract / Q000378:metabolism / Q000473:pathology*; D002478:Cells, Cultured; D003459:Crystallins / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000473:pathology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005690:Galactose / Q000008:administration & dosage; D005693:Galactosemias / Q000378:metabolism / Q000473:pathology*; D005978:Glutathione / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006943:Hyperglycemia / Q000378:metabolism / Q000473:pathology; D007003:Hypoglycemia / Q000378:metabolism / Q000473:pathology; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D007908:Lens, Crystalline / Q000378:metabolism / Q000473:pathology*; D018832:Molecular Chaperones / Q000378:metabolism; D018384:Oxidative Stress; D011489:Protein Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D051742:Transcription Factor CHOP / Q000378:metabolism
C497110:Atf4 protein, rat; D003459:Crystallins; C497251:Ddit3 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP; C051684:GRP78 protein, rat; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D017382:Reactive Oxygen Species; D051701:Activating Transcription Factor 4; D051742:Transcription Factor CHOP; D020169:Caspases; D005978:Glutathione; D005690:Galactose