Enhanced HtrA2/Omi expression in oxidative injury to retinal pigment epithelial cells and murine models of neurodegeneration.
OBJECTIVE
To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H(2)O(2)-induced oxidative stress in human RPE, in the Ccl2(-)(/)(-)Cx3cr1(-)(/)(-) double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice.


METHODS
Oxidative stress was induced in ARPE-19 cells by 1 mM H(2)O(2) for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice.


RESULTS
H(2)O(2)-induced oxidative damage resulted in HtrA2/Omi translocation from mitochondria to cytosol, leading to RPE cell apoptosis via a caspase-mediated pathway. Treatment of RPE cells with UCF-101 reduced the cytosolic translocation of HtrA2/Omi, attenuated caspase-3 activation, and decreased apoptosis. After specific HtrA2 downregulation, increased cell viability was measured in H(2)O(2)-treated ARPE-19 cells. Retina of DKO mice exhibit increased oxidative stress and upregulation of HtrA2/Omi. Fewer and abnormal mitochondria were found in HtrA2/Omi(-)(/)(-) photoreceptors and RPE.


CONCLUSIONS
These findings suggest that HtrA2/Omi is related to RPE apoptosis due to oxidative stress, which may play an important role in the integrity of mitochondria and the pathogenesis of AMD.
Ding
Xiaoyan
X
Section of Immunopathology, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-1857, USA.
Patel
Mrinali
M
Shen
Defen
D
Herzlich
Alexandra A
AA
Cao
Xiaoguang
X
Villasmil
Rafael
R
Klupsch
Kristina
K
Tuo
Jingsheng
J
Downward
Julian
J
Chan
Chi-Chao
CC
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D000073883:CX3C Chemokine Receptor 1; D053148:Caspase 3; D002460:Cell Line; D002470:Cell Survival; D018932:Chemokine CCL2 / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011744:Pyrimidinones / Q000494:pharmacology; D034622:RNA Interference; D019707:Receptors, Chemokine / Q000235:genetics; D012162:Retinal Degeneration / Q000201:enzymology* / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D013871:Thiones / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism
D000073883:CX3C Chemokine Receptor 1; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; C471885:Cx3cr1 protein, mouse; D004791:Enzyme Inhibitors; D024101:Mitochondrial Proteins; D011744:Pyrimidinones; D019707:Receptors, Chemokine; D013871:Thiones; C501517:UCF 101; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D006861:Hydrogen Peroxide; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse; C505409:Casp3 protein, mouse; D053148:Caspase 3