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Activation of Caspase-8 and -12 Pathway by 7-Ketocholesterol in Human RPE Cells
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Gerard Lizard
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gerard.lizard{at}u-bourgogne.fr Gerard Lizard
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Activation of Caspase-8 and -12 Pathway by 7-Ketocholesterol in Human Retinal Pigment Epithelial Cells: Involvement or Not of Caspase-3? We read with attention the article published in the December 2006 issue of IOVS by Luthra et al.1 entitled "Activation of caspase-8 and caspase-12 pathways by 7-ketocholesterol in human retinal pigment epithelial cells" which investigated the mode of cell death induced by 7-ketocholesterol (7KC) in human retinal pigment epithelial cells (ARPE-19). Whereas this paper brings new insights in the understanding of the mode of cell death triggered by 7KC used at 40 mg/ml (induction of a reticulum stress through caspase-12), the involvement of caspases-3 and -8 reported in this investigation is not well defined, and would require more appropriate methods to clearly establish the activation and the part taken by these enzymes in 7KC-induced cell death. Indeed, in the present investigation, the activities of caspases-3, -8 and -9 were determined with the FLICA method commonly used to determined the percentages of caspase positive cells by various methods of microscopy or by flow cytometry.2 Up to now, flow cytometry is the only valuable method allowing to evaluate caspase activity on whole cells. In these conditions, caspase activity is determined in arbitrary fluorescent units. However, to our knowledge, the in situ quantification of caspase activity on adherent cells by microcopical methods is not suitable, and the method used is not referenced. Appropriate methods would require to incubate cellular extracts of untreated and 7KC-treated cells with appropriate fluorescent substrats of caspases-3, -8 and -9, to measure the fluorescence at various times by fluorimetry, and to further calculate the resulting activity of these caspases. However, even in these conditions, it would be difficult to affirm that these caspases are effectively activated. Indeed, with FLICA methods, as well as with fluorescent methods used to measure caspase activities, most of the substrats are not specific. Thus, the substrat used to identify caspase-3 probably recognizes caspase-7, and we can not exclude that the substrats reacting with caspase-8 and -9 do not react with caspase-10, and –2, respectively.3,4 For these reasons, subsequent analyses of proteins on polyacrylamide gel associated with Western blotting are generally performed to confirm the involvement of particular caspases in an apoptotic mode of cell death. Without these biochemical analyses, the data presented do not exclude the possible involvement of caspase-7, -10 and -2 in 7KC-treated ARPE-19 cells. However, as the involvement of caspase-9 is excluded since similar fluorescence levels were observed with FLICA in 7KC-treated cells and in DMSO-treated cells (vehicle control), a possible activation of caspase-2 is unlikely in ARPE-19 cells. Whereas the activation of caspase-8 has been reported under treatment with 7KC,5 data obtained with FLICA have not been confirmed by other biochemical methods. Moreover, a possible involvement of caspase-7 (instead of caspase-3) can not be rejected. Indeed, caspase-7 activation has been identified in 7KC-treated U937 cells,6 and in our hands data obtained on 7KC-treated ARPE-19 cells do not support an activation of caspase-3. Thus, when ARPE-19 cells were cultured in the presence of 7KC (20 mg/ml) for 6, 12, 24 and 48 h the characterization of cell death was performed by various methods in part described in a poster presented at the ARVO congress in 2006 by Malvitte L et al. (Malvitte L, et al. IOVS 2006;47:ARVO E-Abstract 2581) by using morphological and biochemical criteria: characterization of the aspect of the nuclei by transmission electron microscopy and by conventional fluorescence microscopy after staining with Hoechst 33342 in order to distinguish between viable, apoptotic and necrotic cells,7 analysis of the DNA fragmentation pattern on agarose gel, identification of caspase-3 positive cells with FLICA, and analysis of caspase-3 activity with fluorometric methods as well as by Western blotting. In these conditions, cells with large nuclei evocating oncotic cells were observed whereas cells with condensed and/or fragmented nuclei were rarely found. No internucleosomal DNA fragmentation were detected. As morphological nuclear changes characteristic of apoptosis are mainly triggered by effector caspases such as caspase-3,8 our morphological observations were in agreement with an absence of caspase-3 activation. It is noteworthy that the lack of caspase-3 activity identified by fluorometry, and the absence of active caspase-3 revealed by FLICA, and by Western blotting (no cleavage of procaspase-3 into subunits p17 and p19) support an absence of caspase-3 activation in ARPE-19 cells treated with 7KC. In order to validate data obtained in human retinal pigment epithelial cells, U937 cells were treated with VP-16 (100 mM, 6 h) and in these conditions, with the different methods used in ARPE-19 cells and with the same reagents, a typical mode of cell death by apoptosis (presence of cells with condensed and/or fragmented nuclei, internucleosomal DNA fragmentation) associated with caspase-3 activation was observed. Thus, contrary to the data reported by Luthra et al.,1 our data rather support an absence of caspase-3 activation in 7KC-treated ARPE-19 cells. So, at the opposite of 7KC-induced cell death observed on numerous cell types (U937, THP-1, bovin and human endothelial cells, human smooth muscle cells, and MCF-7 stably transfected with caspase-3),5,6,9,10 our data suggest that the mode of cell death induced by 7KC in ARPE-19 cells is caspase-3-independent. Thus, in the paper of Luthra et al.,1 the possible activation of caspase-3 via the caspase-12 (since the caspase-9 is not activated) remains questionable, whereas the cleavage of caspase-12 observed by Western blot favors the hypothesis of an activation of the reticulum stress which is in agreement with our previous investigation performed with 7KC in human smooth muscle cells.11 So, whereas an induction of the reticulum stress in 7KC-treated ARPE-19 cells can be considered as convincing, the relationship between caspase-12 and caspase-3 is not clear, mainly because caspase-3 activity, which is only based on FLICA analysis, is not sufficient and therefore not credible to demonstrate an involvement of this caspase. Ultimately, the difficulties to precise the characteristics of cell death induced by 7KC underline that oxysterols induce a particularly complex and original mode of cell death which consequently requires the simultaneous use of numerous criteria to avoid any ambiguity. Laure Malvitte1,2 1Inserm UMR 866 / IFR Santé STIC, Equipe
Biochimie Métabolique et Nutritionnelle, Faculté des Sciences Gabriel, Dijon, France References 1. Luthra S, Fardin B, Dong J, et al. Activation of
caspase-8 and caspase-12 pathway by 7-ketocholesterol in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2006;47:5569-5575. |
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