Caspase-8, -12, and -3 activation by 7-ketocholesterol in retinal neurosensory cells.
OBJECTIVE
To determine the caspase pathways involved with 7-ketocholesterol (7kCh)-induced apoptosis in rat R28 cells.


METHODS
R28 cells were exposed to 7kCh with or without low-density lipoprotein (LDL) and z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by a trypan blue dye exclusion assay. Caspase-3, -8, -9, and -12 activities were measured by fluorochrome caspase assays. ARPE-19 cells were used as control for caspase-3 inhibition experiments.


RESULTS
R28 cultures showed decreased cell viability on 7kCh exposure compared with controls (P < 0.001), and this was reversed with LDL and LDL + z-VAD-fmk (P < 0.001). The 7kCh-treated R28 cultures had increased caspase-8 activity compared with controls (P < 0.001). This activity was blocked partially with LDL (P < 0.01) or LDL + z-VAD-fmk (P < 0.001) but not with z-VAD-fmk alone. Caspase-12 activity was increased after 7kCh treatment compared with controls (P < 0.01), and this activity was increased further with the addition of LDL. Caspase-3 activity in R28 cultures increased with 7kCh treatment compared with controls (P < 0.001). In R28 cultures, the z-VAD-fmk treatment did not blocked 7kCh-induced caspase-3 activity but did block activity in ARPE-19 cultures (P < 0.001). Caspase-9 was not activated by 7kCh treatment.


CONCLUSIONS
In R28 cells, 7kCh-induced apoptosis involves the caspase-3 along with the caspase-8 and caspase-12 pathways. LDL partially blocked 7kCh-induced caspase-8 activity but increased caspase-12 activities, suggesting that caspase-8 and caspase-12 pathways are independent of each other. The z-VAD-fmk inhibitor blocked caspase-3 activities in the homogeneous ARPE-19 cultures but not in the heterogeneous R28 cultures.
Neekhra
Aneesh
A
Department of Ophthalmology, School of Medicine, University of California, Irvine, California, USA. mkenney@uci.edu
Luthra
Saurabh
S
Chwa
Marilyn
M
Seigel
Gail
G
Gramajo
Ana L
AL
Kuppermann
Baruch D
BD
Kenney
M Cristina
MC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053454:Caspase 12 / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D053938:DNA Fragmentation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007653:Ketocholesterols / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D051381:Rats; D012160:Retina / Q000166:cytology*; D014343:Trypan Blue
D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; D007653:Ketocholesterols; D008077:Lipoproteins, LDL; C460579:benzyloxycarbonyltyrosyl-valyl-alanyl-aspartic acid fluoromethyl ketone; C506443:Casp12 protein, rat; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; D053454:Caspase 12; D053148:Caspase 3; D053181:Caspase 8; D014343:Trypan Blue; C003001:7-ketocholesterol