- Research article
- Open Access
Pathological apoptosis by xanthurenic acid, a tryptophan metabolite: activation of cell caspases but not cytoskeleton breakdown
© Malina et al, licensee BioMed Central Ltd. 2001
- Received: 4 April 2001
- Accepted: 4 July 2001
- Published: 4 July 2001
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.
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.
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.
- Xanthurenic Acid
- Senile Cataract
- Tryptophan Metabolite
- Tryptophan Degradation
Xanthurenic acid is formed upon tryptophan degradation by indoleamine-2,3 dioxygenase (IDO). The end products of this degradation pathway are, alternatively, nicotinate and xanthurenic acid. IDO activity is stimulated by superoxide radicals, liposaccharides and interferon-γ . Kynurenine aminotransferase (KAT), the enzyme directly responsible for xanthurenic acid formation from 3-hydroxykynurenine, is found in the cytoplasm and mitochondria, and is highly expressed in the retina [2,3]. Xanthurenic acid is present in blood and urine at concentrations of 0.7 and 5-10 μM, respectively [4,5]. A several - fold increase is observed in vitamin B6 deficiency and some diseases such as tuberculosis [6,7]. Xanthurenic acid's presence in the blood is linked to malaria development, and in the lenses to senile cataract formation [8,9,10]. Xanthurenic acid binds covalently to proteins, leads to their unfolding, and to cell death . Here, we report that xanthurenic acid induces cell death associated with caspase-3, -8, and -9 activation, nuclear DNA cleavage, and cytochrome C release. However, cell death is not associated with cytoskeleton breakdown, usually observed because of actin depolymerization by caspase-3-cleaved gelsolin .
Xanthurenic acid leads to caspase-3 activation in smooth muscle cells
Xanthurenic acid provokes degradation of caspase-3 substrates DFF-45, PARP, and gelsolin
Caspase 3 is required for the degradation of DFF45/ICAD with formation of the carboxy-terminal fragment p11, which is necessary for DNA cleavage [17,18]. In cells exposed to xanthurenic acid, DFF45 was cleaved with generation of the p11 fragment, recognized with an antibody directed against full-length DFF45 (Fig. 2a). Processed DFF45 leads to internucleosomal cleavage, and indeed the DNA of cells exposed to xanthurenic acid was fragmented as shown by Hoechst 33342 staining and fluorescence microscopy (Fig. 2b). The amount of PARP protein was increased in xanthurenic acid-exposed cells, and PARP was degraded to the apoptotic p85 fragment (Fig. 2c, 3b), which was reported to be formed upon caspase-3 cleavage . Also gelsolin was cleaved in cells in a xanthurenic acid concentration-dependent manner with formation of p41, the amino terminal part of gelsolin, called "N-half" (Fig. 2d. Fig. 3d,e). The latter is a product of caspase-3 activity [12, 20]. The p41 fragment was further degraded, indicating that xanthurenic acid activated additional enzyme(s) involved in gelsolin processing. It was reported that N-half leads to depolymerization of actin filaments .
Apoptosis induced by xanthurenic acid did not cause cytoskeleton depolymerization
In the presence of xanthurenic acid the caspase-3 cleaved gelsolin did not cause cytoskeleton breakdown (Fig. 3d). To the contrary, the elongated cytoskeleton was strongly stained for N-half of gelsolin, and the staining increased with xanthurenic acid concentration (Fig. 3d), the condition which led to activation of caspase-3 (Fig. 3a) and caspase-9 (Fig. 3c). N-half contains two polyphosphoinositide (PIP) binding domains. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate form a stable complex with gelsolin, which prevents caspase-3 and -9 activation . We prepared a part of N-half containing amino acid residues 162-187 (human sequence of gelsolin), which contains a PIP binding domain in position 162-169, and raised a polyclonal antibody against the peptide, called GPIP1. This antibody stained the cytoskeleton of xanthurenic acid-exposed cells but not of the control cells (Fig. 3e). This indicates that after xanthurenic acid-dependent gelsolin cleavage the sequence containing GPIP1 binds to the cytoskeleton. The cleavage of gelsolin in the presence of xanthurenic acid did not lead to breakdown of the cytoskeleton (Fig. 3e), in contrast to experiments where gelsolin was overexpressed .
Xanthurenic acid induces mitochondrial damage
Effector caspases in demolition phase of xanthurenic acid-induced apoptosis
In the presence of xanthurenic acid the cell death was associated with DFF p11 formation, which is characteristic for caspase-3 activity. Recently, it was reported that executioner caspases-3, 6, and 7 play non-redundant roles during the demolition phase of apoptosis. Caspase-6 and caspase-7 are not involved in DNA degradation but in lamin A degradation, and caspase-7 activation provokes PARP cleavage . Our Western blot analysis showed that in the presence of xanthurenic acid lamin A cleavage does not occur in VSMC suggesting that these caspases are not activated in the presence of this compound. This suggests that the cleavage of PARP occurred due to caspase-3 activation. Plectin, a cytolinker responsible for the mechanical stability of the cytoskeleton, is cleaved by caspase-8 at ASP 2395. The cytoskeleton lost intermediary fibers due to plectin cleavage, and cells lost their integrity. Full-length caspase-8 co-localises with mitochondria and active caspase-8 is translocated to plectin .
Our results indicate that an accumulation of the tryptophan metabolite, xanthurenic acid, leads to cleavage of caspases substrates and apoptosis. Unexpectedly, xanthurenic acid-induced apoptosis is associated with an abnormal function of cleaved gelsolin. The results indicate that xanthurenic acid is an important factor involved in aging and disease development.
We used the following polyclonal antibodies from Santa Cruz Biotechnology Inc. CA, USA: antibodies against full length caspase-3, caspase-8, DFF 45/ICAD, and PARP, amino terminus of gelsolin, plectin, cytochrome C, and carboxy terminus of BID. Immunocytochemistry was performed using primary antibodies against active caspase-3 pl7 (BD PharMingen, San Diego, CA, USA, and Promega, Madison, USA), active caspase-9 (BioLabs Inc., New England, UK), and anti-PARP p85 (Promega). Secondary IgG-Texas Red, fluoresceine (FITC)-conjugated antibodies and Mitotracker CMXRos were from Molecular Probes, Leiden, The Netherlands. Other reagents were from Sigma if not specified.
Preparation of polyclonal antibody directed against GPIP1
GPIP1, a peptide comprising 25 aminoacids corresponding to residue 162-187 (NH2-KSGLKYKKGGVA-SGFKHVVPNEVVV-COOH) in human gelsolin sequence (Swiss-Prot P06396) was synthetized (95% purity) by MWG AG Biotechnology, Ebersberg, Germany. 200 μg of the peptide were injected 3 times into rabbits in 3 weeks intervals. Sera were used for Western blots and immunoflourescence in a 1:100 dilution.
Primary vascular smooth muscle cells (VSMCs) were prepared from porcine aorta. Retinal pigment epithelium (RPE) cells obtained from a 59 years old eye donor were provided by Dr. M. Boenke, Department of Ophthalmology, University Hospital, Bern. The cells were cultivated in Minimal Essential Medium (MEM) with Earle's salts (Life Sciences, Basel, Switzerland). Cells were grown under a humidified atmosphere of 5% CO2 in air at 37°C in MEM supplemented with 10% fetal bovine serum, penicillin (10 U/ml), streptomycin (10 μg/ml) and fungizone (250 ng/ml). When confluent, they were incubated for one week in MEM or MEM supplemented with xanthurenic acid. A 20 mM stock solution of xanthurenic acid was prepared in 0.5 M NaHCO3, and diluted in 0.05 M NaHCO3.
Cytotoxicity and apoptosis assay
Cells were observed with differential interference contrast optics on a Zeiss Avionert 405 M inverted microscope. Cell viability was determined by staining the cells with Hoechst 33342 and propidium iodide (PI) (Juro, Switzerland) using 50 μg/ml of each dye. Fragmented, apoptotic, nuclei were observed with excitation at 350 nm, and necrotic nuclei at 530 nm. The extent of apoptosis was estimated by examination of 300 nuclei from each sample.
Cell lysis and immunobloting
Cell were washed twice with cold 0.01 M phosphate buffer, pH 7.4. For Western blotting, cells were lysed in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1 %Triton X-100, and the following protease inhibitors: 1 mM phenyl-methylsulfonyl fluoride, and leupeptin, aprotinin, and pepstatin, each at 1 μg/ml. The concentration of proteins was calculated from the absorption maximum at 280 nm, as described previously , and concentration of xanthurenic acid from its absorptiom maximum at 342 nm (εM 6500). The lysate was centrifuged for 10 min at 14 000 g, and the supernatant was boiled in loading-buffer for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE containing 10 or 12.5% acrylamide. After transfer to Hybond ECL membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden) the proteins were probed with the appropriate antibodies. Chemilunimescence ECL system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for the detection of peroxidase-conjugated secondary antibody.
Caspase 3 cleavage activity is based on the spectrophotometric detection of the chromophore p-nitroaniline at 405 nm after cleavage from the substrate Ac-Asp-Glu-Val-Asp-p-nitroaniline (Ac-DEVD-pNa) (Bachem, Basel, Switzerland). Caspase activity was measured after 1 hour of incubation of 200 μM Ac-DEVD-pNa at 37°C with the cell extract containing 25 mM HEPES (pH 7.5), 300 mM NaCI, 10 mM KCl, 1.5 mM MgCl2, 10% glycerol, 0.1 mM DTT, 1 mM phenylmethylsulfonylfluoride, and aprotinin, leupeptin, and pepstatin, each at 1 μg/ml.
Cells grown on glass coverslips were fixed for 10 min at room temperature in 4% paraformaldehyde in 0.1 M PIPES, pH 6.8, washed in PBS and permeabilized for 5 min in PIPES containing 0.05% saponin (65 μl per coverslip), washed in PBS, incubated with cold aceton for additional fixing and permeabilisation, and again washed in PBS. Cells were incubated for 1.5 hour with the first antibody diluted in PBS containing 1% bovine serum albumine, and after washing incubated for 1.5 hour with the secondary antibody. The coverslips were then washed in PBS and incubated for 10 min with 65 μl of 4% paraformaldehyde solution containing 1 μl of Hoechst 33342 dye (1 mg/ml), washed in PBS, and incubated with Antifade Kits (Molecular Probes, Leiden, The Netherlands) according to the supplier's instruction. Staining of mitochondria was performed using Mitotracker CMXRos, as follows: confluent cells culture were pre-incubated without or with xanthurenic acid in MEM medium for 3 hours. The medium was removed and replace with medium containing 100 nM Mitotracker CMXRos. After an incubation for 45 min Mitotracker CMXRos was removed and replaced by MEM medium. The cell were cultivated for the next 20 hours, stained additionally with antibody against gelsolin (as above) and then observed by fluorescence microscopy.
This work was supported by grant awarded to H. Z. M. by the Swiss National Foundation (32-59183.99). We thank Drs. E. Berrou and M. Boenke for helpful discussions and Mrs D. Zuercher for retinal cell culture preparation.
- Carlin JM, Ozaki Y, Byrne GI, Brown RR, Borden EC: Interferons and indoleamine 2,3-dioxygenase: role in antimicrobial and antitumor effects. Experientia. 1989, 45: 535-541.View ArticlePubMedGoogle Scholar
- Tobes MC: Kynurenine-oxoglutarate aminotransferase from rat kidney. Methods Enzymol. 1987, 142: 217-224.View ArticlePubMedGoogle Scholar
- Mosca M, Cozzi L, Breton J, Avanzi N, Toma S, Okuno E, et al: Cloning of rat and human kynurenine aminotransferase. Adv Exp Med Biol. 1996, 398: 449-454.View ArticlePubMedGoogle Scholar
- Williams SA, Monti JA, Boots LR, Cornwell PE: Quantitation of xanthurenic acid in rabbit serum using high performance liquid chromatography. Am J Clin Nutr. 1984, 40: 159-167.PubMedGoogle Scholar
- Cavill IA: Estimation of the tryptophan metabolites xanthurenic acid, 3-hydroxykynurenine and kynurenine. Clin Chim Acta. 1967, 18: 285-289. 10.1016/0009-8981(67)90170-2.View ArticlePubMedGoogle Scholar
- Yeh JK, Brown RR: Effects of vitamin B-6 deficiency and tryptophan loading on urinary excretion of tryptophan metabolites in mammals. J Nutr. 1977, 107: 261-271.PubMedGoogle Scholar
- Nair S, Maguire W, Baron H, Imbruce R: The effect of cycloserine on pyridoxine-dependent metabolism in tuberculosis. J Clin Pharmacol. 1976, 16: 439-443.View ArticlePubMedGoogle Scholar
- Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, et al: Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito [see comments]. Nature. 1998, 392: 289-292. 10.1038/32667.View ArticlePubMedGoogle Scholar
- Malina HZ, Martin XD: 3-hydroxykynurenine transamination leads to the formation of the fluorescent substances in human lenses. Eur J Ophthalmol. 1996, 6: 250-256.PubMedGoogle Scholar
- Malina HZ, Martin XD: Xanthurenic acid derivative formation in the lens is responsible for senile cataract in humans. Graefes Arch Clin Exp Ophthalmol. 1996, 234: 723-730.View ArticlePubMedGoogle Scholar
- Malina HZ: Xanthurenic acid provokes formation of unfolded proteins in endoplasmic reticulum of the lens epithelial cells. Biochem Biophys Res Commun. 1999, 265: 600-605. 10.1006/bbrc.1999.1716.View ArticlePubMedGoogle Scholar
- Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, et al: Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science. 1997, 278: 294-298. 10.1126/science.278.5336.294.View ArticlePubMedGoogle Scholar
- Schlegel J, Peters I, Orrenius S, Miller DK, Thornberry NA, Yamin TT, et al: CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem. 1996, 271: 1841-1844. 10.1074/jbc.271.4.1841.View ArticlePubMedGoogle Scholar
- Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G, Alnemri ES: Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc Natl Acad Sci USA. 1996, 93: 14486-14491. 10.1073/pnas.93.25.14486.PubMed CentralView ArticlePubMedGoogle Scholar
- Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997, 91: 479-489.View ArticlePubMedGoogle Scholar
- Stennicke HR, Jurgensmeier JM, Shin H, Deveraux Q, Wolf BB, Yang X, et al: Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem. 1998, 273: 27084-27090. 10.1074/jbc.273.42.27084.View ArticlePubMedGoogle Scholar
- Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S: caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998, 391: A43-50. 10.1038/34112.View ArticleGoogle Scholar
- Tang D, Kidd VJ: Cleavage of DFF-45/ICAD by multiple caspases is essential for its function during apoptosis. J Biol Chem. 1998, 273: 28549-28552. 10.1074/jbc.273.44.28549.View ArticlePubMedGoogle Scholar
- Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier G, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994, 371: 346-347. 10.1038/371346a0.View ArticlePubMedGoogle Scholar
- Geng YJ, Azuma T, Tang JX, Hartwig JH, Muszynski M, Wu Q, et al: Caspase-3-induced gelsolin fragmentation contributes to actin cytoskeletal collapse, nucleolysis, and apoptosis of vascular smooth muscle cells exposed to proinflammatory cytokines. Eur J Cell Biol. 1998, 77: 294-302.View ArticlePubMedGoogle Scholar
- Azuma T, Koths K, Flanagan L, Kwiatkowski D: Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression. ] Biol Chem. 2000, 275: 3761-3766. 10.1074/jbc.275.6.3761.View ArticleGoogle Scholar
- Koya RC, Fujita H, Shimizu S, Ohtsu M, Takimoto M, Tsujimoto Y, et al: Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release. J Biol Chem. 2000, 275: 15343-15349. 10.1074/jbc.275.20.15343.View ArticlePubMedGoogle Scholar
- Slee EA, Adrain C, Martin SJ: Executioner caspases-3, -6 and -7 perform distinct, non-redundant, roles during the demolition phase of apoptosis. J Biol Chem. 2000, 276: 7320-7326. 10.1074/jbc.M008363200.View ArticlePubMedGoogle Scholar
- Stegh AH, Herrmann H, Lampel S, Weisenberger D, Andra K, Seper M, et al: Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis. Mol Cell Biol. 2000, 20: 5665-5679. 10.1128/MCB.20.15.5665-5679.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Li H, Zhu H, Xu CJ, Yuan J: Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998, 94: 491-501.View ArticlePubMedGoogle Scholar
- Tang D, Lahti JM, Kidd VJ: Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. J Biol Chem. 2000, 275: 9303-9307. 10.1074/jbc.275.13.9303.View ArticlePubMedGoogle Scholar
- Zou H, Li Y, Liu X, Wang X: An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem. 1999, 274: 11549-11556. 10.1074/jbc.274.17.11549.View ArticlePubMedGoogle Scholar
- Peter ME, Krammer PH: Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis. Curr Opin Immunol. 1998, 10: 545-551. 10.1016/S0952-7915(98)80222-7.View ArticlePubMedGoogle Scholar
- Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al: Two CD95 (APO-1/Fas) signaling pathways. Embo J. 1998, 17: 1675-1687. 10.1093/emboj/17.6.1675.PubMed CentralView ArticlePubMedGoogle Scholar
- Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, et al: Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999, 144: 281-292. 10.1083/jcb.144.2.281.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.