Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells.
OBJECTIVE
To determine whether caspase or cathepsin pathways are activated in human retinal pigment epithelial cells (ARPE-19) after exposure to 7-ketocholesterol (7kCh).


METHODS
ARPE-19 cells were exposed to 7kCh with or without z-VAD-fmk, a pan-caspase inhibitor. Caspase-3, -8, and -9 activities were measured by a fluorochrome inhibitor of caspase (FLICA) assay. Caspase-12 activity was detected by Western blotting. RT-PCR was performed for 18s, mortalin-2, cathepsins B, D, and L/V2.


RESULTS
At 24 hours, 7kCh-treated cultures had increased caspase-8 (P < 0.001) and caspase-3 (P < 0.001) activities compared with vehicle-treated cultures. 7kCh-induced caspase-3 activation was blocked by z-VAD-fmk (P < 0.001). Caspase-9 was not activated by 7kCh treatment (P > 0.05). Procaspase-12 was cleaved into its active form after treatment with 7kCh for 24 hours. At 6 hours, the RNA level for mortalin-2, a pro-survival gene, was upregulated. ARPE-19 cells did not express RNA for cathepsins B, D, or L/V2 under any conditions.


CONCLUSIONS
In ARPE-19 cells, 7kCh-induced apoptosis uses the receptor-mediated caspase-8 pathway and the endoplasmic reticulum stress-induced caspase-12 pathway but not the mitochondrial caspase-9 pathway. The cathepsin pathways are not involved in 7kCh-induced cell death. These data demonstrate that 7kCh causes a loss of cell viability through caspase-dependent apoptosis and can act as an oxidative stressor leading to retinal pigment epithelial cell atrophy. Elucidating the specific apoptotic pathways involved may have therapeutic potential for AMD and other retinal diseases.
Luthra
Saurabh
S
Department of Ophthalmology, School of Medicine, University of California, Irvine, California 92868, USA.
Fardin
Babak
B
Dong
Joyce
J
Hertzog
Dieter
D
Kamjoo
Sami
S
Gebremariam
Simon
S
Butani
Virit
V
Narayanan
Raja
R
Mungcal
Jeanie K
JK
Kuppermann
Baruch D
BD
Kenney
M Cristina
MC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D015153:Blotting, Western; D053454:Caspase 12 / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism*; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002790:Cholesterol 7-alpha-Hydroxylase / Q000037:antagonists & inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007653:Ketocholesterols / Q000494:pharmacology*; D024101:Mitochondrial Proteins; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; C492001:HSPA9 protein, human; D007653:Ketocholesterols; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002790:Cholesterol 7-alpha-Hydroxylase; D002403:Cathepsins; C505530:CASP8 protein, human; D053454:Caspase 12; D053148:Caspase 3; D053181:Caspase 8; C003001:7-ketocholesterol