Pathological apoptosis by xanthurenic acid, a tryptophan metabolite: activation of cell caspases but not cytoskeleton breakdown

Abstract

Background: A family of aspartate-specific cysteinyl proteases, named caspases, mediates programmed cell death, apoptosis. In this function, caspases are important for physiological processes such as development and maintenance of organ homeostasis. Caspases are, however, also engaged in aging and disease development. The factors inducing age-related caspase activation are not known. Xanthurenic acid, a product of tryptophan degradation, is present in blood and urine, and accumulates in organs with aging.

Results: Here, we report triggering of apoptotic key events by xanthurenic acid in vascular smooth muscle and retinal pigment epithelium cells. Upon exposure of these cells to xanthurenic acid a degradation of ICAD/DFF45, poly(ADP-ribose) polymerase, and gelsolin was observed, giving a pattern of protein cleavage characteristic for caspase-3 activity. Active caspase-3, -8 and caspase-9 were detected by Western blot analysis and immunofluorescence. In the presence of xanthurenic acid the amino-terminal fragment of gelsolin bound to the cytoskeleton, but did not lead to the usually observed cytoskeleton breakdown. Xanthurenic acid also caused mitochondrial migration, cytochrome C release, and destruction of mitochondria and nuclei.

Conclusions: These results indicate that xanthurenic acid is a previously not recognized endogenous cell death factor. Its accumulation in cells may lead to accelerated caspase activation related to aging and disease development.

Figure 1

Caspase-3 activation and apoptosis upon xanthurenic acid (XAN) accumulation in vascular smooth muscle cells (VSMC). a, VSMC after one week of growth in MEM without XAN (left panel), and in the presence of XAN at a concentration of 5 μg/ml (right panel). The nuclear DNA fragmentation at 40 μg/ml, see Fig. 2b. b, Concentration of xanthurenic acid in the cell extract used for Western blot analysis. XAN was determined in the cell extracts as described previously [11]. c, Apoptotic cell death measured as a ratio of the cells with condensed or fragmented nuclei to total nuclei. d, Caspase-3 activity measured by caspase-3 substrate Ac-DEVD-pNa cleavage, e, Western blot of active caspase-3 in VSMC after exposure to XAN. Procaspase-3 (CPP32) was cleaved in the presence of XAN with the formation of pl7 and pl2. f, Immunofluorescence staining for caspase 3 pl7 in control cells (left panel) and in VSMC exposed to 40 μg/ml of XAN for one week (right panel).

Figure 2

Cleavage of caspase-3 substrates PARP, DFF45/ICAD, and gelsolin in VSMC exposed to xanthurenic acid (XAN) for one week. DFF45 and PARP were slightly upregulated in the presence of XAN. a, Western blot of DFF45/ICAD. b, Nuclear fragmentation in control cells (left panel) and VSMC grown in the presence of XAN at 40 μg/ml for one week (right panel). c, Western blot of PARP. d, Western blot of gelsolin.

Figure 3

Detection of apoptotic proteins by immunofluorescence in human retinal pigment epithelium cells after exposure to 20 μM xanthurenic acid for one week (magnification 200-fold). Panels a-c from left to right represent immunodetection of apoptotic protein with respective antibody, staining of nucleus with Hoechst, and merge of both. No staining for these enzymes was observed in control cells. a, Detection of active caspase-3 pl7. b, Detection of PARP p85. c, Detection of active caspase-9. Panel d, Detection of N-half of gelsolin bound to the cytoskeleton. From left to right: without xanthurenic acid, with 10 μM xanthurenic acid, with 20 μM xanthurenic acid. e, Detection of cleaved gelsolin containing polyphosphoinositide binding domain by antibody directed against GPIP1 in apoptotic cells. From left to right: first two panels, control cells; Hoechst staining, no staining with antibody directed against GPIP1. Next two panels: cells incubated for one week with 20 μM xanthurenic acid; Hoechst staining, staining with antibody against GPIP1.

Figure 4

Analysis of mitochondria in human pigment retinal epithelial cells exposed to xanthurenic acid. Panels a-c (magnification 200-fold), Cytochrome C release in the presence of 20 μM xanthurenic acid for one week. a, Staining for cytochrome C. b, Hoechst staining. c, Merge of both. Panels d-f (magnification 800-fold), Mitochondria were stained with Mitotracker CMXRos, and cells were stained with antibody against gelsolin (N-half). d, Control cells, e, Cells exposed to 5 μM xanthurenic acid (see Methods), f, Cells exposed to 20 μM xanthurenic acid. Lack of staining with Mitotracker CMXRos, co-staining for cytochrome C (orange), and N-half of gelsolin (green). Panels g-h (magnification 4000-fold), g, Single control mitochondrion stained for cytochrome C. h, Single mitochondrion stained with Mitotracker CMXRos. i, Same as in j, but staining with Mitotracker CMXRos. j, Staining for cytochrome C in cells exposed to 20 μM xanthurenic acid.

Figure 5

Analysis of caspase-8 and caspase-8 substrate-proteins, plectin and BID, in pig VSMC. a, Western blot analysis of caspase-8 in the cells after exposure to xanthurenic acid (XAN) for one week. Procaspase-8 was cleaved in the presence of XAN with the formation of pl8. Panels b-d, (magnification 800-fold) immunodetection of caspase-8 and plectin and BID in control cells (left panels), and after exposure to 40 μM of xanthurenic acid for 96 hours (right panels), b, detection of caspase-8. c, detection of plectin. d, detection of BID.