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【摘要】 Cadmium is a potent nephrotoxin that has been shown to induce apoptosis in some cells but also to prevent it under certain circumstances. In several clinical situations and experimental models of injury to the renal glomerulus, pathological proliferation of mesangial cells is followed by resolution involving mesangial cell apoptosis. We investigated the effects of Cd 2+ on rat mesangial cells induced to undergo apoptosis through either the extrinsic receptor-mediated pathway or the intrinsic mitochondrial-dependent pathway. Camptothecin initiated the intrinsic pathway with activation of caspase-9 and caspase-dependent cleavage of procaspase-3. Tumor necrosis factor- (TNF- ) initiated caspase-8 activity and cleavage of pro-caspase-3 at the convergence point of the two pathways. However, pro-caspase-8 levels were low, and caspase-9 was also activated in response to TNF-, characteristic of what have been termed type II cells. With both TNF- and camptothecin, concurrent exposure to 10 µM CdCl 2 suppressed DNA laddering, nuclear condensation, and pro-caspase-3 cleavage. It also decreased activity of both caspase-8 and caspase-9, prevented caspase-8-dependent cleavage of the proapoptotic factor Bid, and suppressed release of cytochrome c from mitochondria. At this 10-µM concentration, Cd 2+ was unique among a number of metal ions in preventing DNA fragmentation. We conclude that Cd 2+ is anti-apoptotic in rat mesangial cells, acting by a mechanism that may involve general caspase inhibition. This may have consequences for the resolution of nephritis in situations of mesangial cell hyperproliferation.
【关键词】 tumor necrosis factor caspases metal ions
CADMIUM IS A HIGHLY TOXIC metal with a long biological half-life that targets lung, liver, kidney, and bone ( 6, 13, 35, 55 ). It is considered a human carcinogen by the International Agency for Research on Cancer ( 22 ), and it produces malignant tumors in testes, prostate, and lungs of experimental animals ( 35, 51, 52 ). It is not a strong mutagen, but it acts as a promoter through mitogenic effects on gene expression (reviewed in Ref. 5 ). Cadmium can induce apoptosis in isolated T lymphocytes ( 11 ) and cultured LLC-PK1 cells ( 31 ) and lead to apoptotic cell damage in canine proximal tubules ( 17 ) and rat testicular tissue ( 56 ). Most observations of Cd 2+ -mediated cell death are consistent with the caspase-dependent intrinsic pathway of apoptosis. Several investigators have reported the release of cytochrome c and the activation of caspase-9 in cell lines treated with Cd 2+ ( 26, 54 ). However, there have also been reports of Cd 2+ -induced caspase-independent cell death. For example, in Cd 2+ -treated MRC-5 cells, apoptosis-like nuclear changes were mediated by AIF ( 41 ).
Complicating the issue, investigators have also shown an anti-apoptotic effect of Cd 2+. Cadmium was shown to block apoptosis induced by a variety of agents in Chinese hamster ovary (CHO) cells and this inhibitory effect of Cd 2+ was linked to the inhibition of caspase-3 ( 57 ). The IC 50 for caspase-3 inhibition was 8.7 µM in intact CHO cells and 31 µM in cell-free systems ( 57 ). Another study reported the inhibition of pro-caspase-3 cleavage by Zn 2+ in etoposide-mediated apoptosis in Molt-4 cells ( 36 ). Apoptosis is initiated during the resolution phase of glomerulonephritis to counteract mesangial hypercellularity, allowing normal structure and function to return to the glomerulus ( 15, 21, 48 ). Impaired apoptosis is associated with increased renal pathology in systemic lupus nephritis ( 43 ). Therefore, the possibility that Cd 2+ can inhibit mesangial cell apoptosis becomes significant.
Preliminary experiments in our laboratory showed that Cd 2+ inhibited DNA fragmentation occasionally induced by serum withdrawal in mesangial cells. Although serum withdrawal is an established model of apoptosis, presumably due to growth factor deprivation ( 2, 20, 27, 34 ), and has been reported to initiate apoptosis in mesangial cells ( 1, 29, 50 ), it may not be an effective stimulus for studies with rat mesangial cells (RMC). Several studies report that a high proportion of RMC remain viable even after 24 h in serum-free conditions ( 3, 50 ), and we could not consistently induce DNA fragmentation with this approach. Therefore, we selected two potent inducers of apoptosis, tumor necrosis factor- (TNF- ) and camptothecin (CPT), to explore more well-defined pathways. The extrinsic pathway is triggered by TNF- ( 4, 33 ) and the intrinsic pathway by CPT ( 7, 45 ). An advantage of using agents such as TNF- and CPT is that one can distinguish effects of Cd 2+ on the two key pathways in apoptosis.
EXPERIMENTAL PROCEDURES
Materials and reagents. FBS and culture media were obtained from GIBCO BRL Life Technologies (Burlington, ON). Accessories for cell culture were obtained from VWR International. CPT, aprotinin, pepstatin A, leupeptin, cytochrome c, neutral red, and (4,5-dimethyl azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were products of Sigma (St. Louis, MO). DNase-free RNase, DNA quick-spin columns, proteinase K, and Klenow polymerase were products of Roche Applied Science (Laval, PQ). SB-203580 was purchased from Calbiochem (La Jolla, CA). Polyclonal rabbit anti-caspase-3 antibody (no. 9662), the monoclonal antibody to the p17 fragment of caspase-3 (no. 9664), and anti-cytochrome c antibody (no. 4272) were products of Cell Signaling (Beverly, MA). Caspase-8 (sc-7890) antibody was from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG2b was purchased from Southern Biotechnology Associates (Birmingham, AL). A rabbit antibody to mouse recombinant cleaved Bid designated AR-53 was a gift from Dr. S. Krajewski, Burnham Institute. HRP-conjugated secondary antibodies, anti-mouse IgG and anti-rabbit IgG, and enhanced chemiluminescence (ECL Plus) Western blotting system were purchased from Amersham Biosciences (Baie d'Urfé, PQ). Caspase-8 and caspase-9 enzymatic activity kits, and the caspase inhibitors z-LEHD-fmk and z-IETD-fmk, were products of R&D Systems (Minneapolis, MN). Vectashields mounting medium with 4',6-diamino-2-phenylindol (DAPI) was purchased from Vector Laboratories (Burlingame, CA). [ - 32 P]-dCTP (3,000 Ci/mil) was from New England Nuclear Life Science Products (Boston, MA).
Cell culture. RMC were isolated as described previously ( 53 ). Cells were cultured in RPMI 1640 medium with penicillin G, streptomycin, and 10% FBS in a humidified atmosphere of 5% CO 2 at 37°C and passaged by trypsinization at 5 x 10 5 cells per 10-cm plate. All experiments were conducted on cells between passage 9 and 18.
Induction of apoptosis. Before TNF- treatment, RMC were rendered quiescent at 70-80% confluence with medium containing 0.2% FBS for 48 h. Quiescent cells were then washed with serum-free medium and given 2 µM SB-203580 (an inhibitor of the anti-apoptotic kinase p38) in serum-free medium 30 min before the addition of TNF- (50 ng/ml) with or without 10 µM CdCl 2. In experiments using z-IETD-fmk (caspase-8 inhibitor), the inhibitor was added along with SB-203580 30 min before addition of TNF- and CdCl 2. Nonquiescent RMC were used for all CPT treatments. Cells at 80% confluence were washed twice with serum-free medium and treated with 25 µM CPT in serum-free medium, with or without 10 µM CdCl 2. When used, z-LEHD-fmk (caspase-9 inhibitor) was added 30 min before CPT and CdCl 2.
DNA isolation and detection. Cells were scraped into a 15-ml polystyrene tube and centrifuged at 200 g for 5 min. The resulting cell pellet was washed twice with PBS, and 500 µl of lysis buffer (20 mM Tris·HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.5% SDS, and 100 µg/ml proteinase K) were added to the pellet at 50°C overnight. The cell lysate was transferred into a microcentrifuge tube and incubated with 10 mg/ml RNase at 37°C. DNA was collected by two extractions with phenol/chloroform/isoamyl alcohol (25:24:1) followed by an overnight precipitation at -80°C with anhydrous ethanol and sodium acetate. One microgram of mesangial cell DNA was transferred to a microcentrifuge tube, and the volume was adjusted to 5 µl. Fifteen microliters of reaction buffer [2 µl of 10 x Klenow buffer (0.5 M Tris·HCl, pH 7.6, 0.1 M MgCl 2 ), 0.2 µl of [ - 32 P]-dCTP (3,000 Ci/mmol), 0.2 µl Klenow polymerase (10 U/ml), and 12.6 µl water] were added and incubated at room temperature for 10 min. Radiolabeled DNA was purified using a Sephadex G-25 spin column. Equal amounts of DNA were separated by agarose gel electrophoresis on a 1.8% gel.
Detection of apoptotic nuclei. Cells growing in 12-well plates were incubated with 1 ml of fixative (80% vol/vol methanol in PBS) for 30 min. Fixative was aspirated and the plate was oven-dried at 50°C for 20 min. A drop of mounting medium containing DAPI was added to each well and nuclear morphology was examined using a fluorescence microscope.
Caspase enzyme activity assays. Cells for caspase-8 and caspase-9 activity measurements were grown in 10-cm dishes, scraped into 15-ml polystyrene tubes, and centrifuged at 200 g for 5 min. The resulting cell pellet was washed with PBS, protein concentrations were determined by the method of Peterson ( 37 ), and caspase-8 and caspase-9 activities were measured using fluorometric kits from R&D Systems according to the manufacturer's instructions.
Preparation of postmitochondrial extracts. RMC (1 x 10 7 ) were scraped from plates, washed with PBS, and resuspended in 500 µl of an extraction buffer consisting of 220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 mM MgCl 2, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin, on ice for 10 min. The cells were then homogenized with 10 strokes using a prechilled glass Dounce homogenizer and type B pestle. The homogenized cell mixture was transferred to a microcentrifuge tube and centrifuged at 900 g for 10 min at 4°C. The supernatant was then centrifuged at 16,100 g for 20 min at 4°C. The resulting supernatant from this centrifugation, which contains cytosolic proteins and microsomal components, was considered the postmitochondrial fraction.
Western blotting. Proteins were extracted as described for caspase enzymatic assays. For caspase blotting, 50 µg of protein were separated by SDS-PAGE on a 12.5% gel. Proteins were blotted overnight onto a nitrocellulose membrane via electrophoretic transfer. The membrane was blocked with 5% wt/vol nonfat milk in TBS-Tween (0.1% vol/vol Tween 20) for 1 h, washed with TBS-Tween, and incubated overnight with the anti-caspase-3 antibody (1:1,000 dilution in TBS-Tween containing 5% wt/vol nonfat milk). The secondary antibody was an HRP-conjugated anti-rabbit IgG (1:10,000 in PBS-Tween containing 5% wt/vol nonfat milk). Detection was with ECL Plus. Similar procedures were used for Bid and cytochrome c except that separation was on 15% gels, 10 µg mitochondrial or postmitochondrial protein were used for cytochrome c detection, 30 µg protein for Bid/tBid blotting, and the anti-tBid antibody was used at 1:4,000 dilution.
Viability assays. For MTT assay, cells were grown in 24-well plates and after a particular treatment were washed in serum-free, phenol red-free RPMI 1640. MTT (1 mg/ml) was added in RPMI 1640 at 300 µl/well and incubated at 37°C for 1 h. MTT solution was replaced with 300 µl of DMSO. After 30 min at room temperature, absorbance was read at 570 nm with background subtraction at 650 nm. For neutral red assay, cells in 24-well plates were washed with serum-free RPMI 1640 and incubated in 300 µl/well neutral red solution (50 µg/ml in serum-free medium) for 2 h. Plates were then shaken gently with 50% ethanol/1% glacial acetic acid for 10 min. The absorbance was read at 540 nm with background subtraction at 650 nm.
Statistics. Statistical significance was determined either by ANOVA using the Bonferroni test when multiple treatments were compared or by the Student's t -test when only two treatment means were compared.
RESULTS
Previous time and concentration dependence studies showed that mesangial cells responded to an 8-h exposure to 10 µM Cd 2+ with peak changes in c- fos expression and activation of Erk and Jnk MAPKs before loss of viability ( 9, 10, 53 ). Therefore, we chose these conditions as producing a robust response for studying effects on apoptosis. Because the conditions of Cd 2+ exposure themselves did not produce detectable apoptosis in RMC (data not shown), two models of induced apoptosis were examined. Tetrazolium blue and neutral red viability assays were performed under the experimental conditions of these models. Compared with cells growing in 10% FBS, quiescent cells or those maintained in serum-free conditions show decreases in both assays, probably indicating lower metabolic activity ( Fig. 1 ). TNF- treatment in serum-free medium shows a similar decrease to serum-free conditions alone, in both assays. Inclusion of Cd 2+ causes a decrease to 50% of serum-free conditions, whether TNF- is present or not. Qualitatively similar results are obtained when Cd 2+ is added in serum-free conditions, with or without CPT, to cells previously growing in complete medium. These methods do not distinguish apoptosis and necrosis and do not allow us to determine whether cells are undergoing different fates in the presence of CPT, TNF-, or Cd 2+.
Fig. 1. Viability assays of cells treated with Cd 2+ or apoptotic agents. Top : TNF- experiments. Quiescent cells were left untreated, transferred to serum-free (SF) medium, or stimulated with 10% FBS for 8 h. Alternatively, cells were incubated for 6 h (neutral red assay) or 7 h (MTT assay) in SF medium containing 50 ng/ml TNF-, either with or without 10 µM CdCl 2. Some cells were also treated with CdCl 2 alone. MTT or neutral red solutions were added in fresh medium without other additives for either 1 h (MTT) or 2 h (neutral red). Values are compared with the absorbance in the quiescent cultures taken as 100%. Bottom : camptothecin (CPT) experiments. Control cells in complete medium were either left untreated or transferred to SF medium or fresh 10% FBS for 8 h. Alternatively, cells were treated with 25 µM CPT with or without 10 µM CdCl 2, or with CdCl 2 alone, and then with MTT or neutral red for the same times as with TNF- in top. Absorbances are expressed relative to the control cells taken as 100%. Top and bottom : values are means ± SD from 4 independent experiments. MTT, filled bars; neutral red, open bars.
Intrinsic and extrinsic pathways to apoptosis are generally distinguished. To activate the extrinsic, death receptor-mediated apoptotic pathway, mesangial cells were treated with TNF- (50 ng/ml) in serum-free medium. Inhibition of p38 was previously shown to augment TNF- -induced apoptosis in RMC compared with TNF- treatment alone ( 16 ), an observation we confirmed here (data not shown). Therefore, the p38 MAP kinase inhibitor SB-203580 (2 µM) was included in the TNF- experiments. CPT (25 µM in serum-free medium) was used to activate the intrinsic pathway. Both treatments produced DNA laddering characteristic of apoptosis at 8 h ( Fig. 2 ). The serum-free condition itself was without noticeable effect. Inclusion of 10 µM CdCl 2 in both treatments suppressed laddering ( Figs. 1 and 2 ). A number of other metal cations (Cu 2+, Zn 2+, Hg 2+, Ni 2+, Co 2+, Mn 2+, and Fe 3+ ) were tested for their ability to affect laddering in both models, all at 10 µM for 8 h. Hg 2+ and Zn 2+, in particular, caused a noticeable decrease with TNF- treatment, whereas Cu 2+ and Hg 2+ were partially effective with CPT ( Fig. 3 ). However, only Cd 2+ caused complete suppression and did so in both models.
Fig. 2. Induction and inhibition of DNA fragmentation in rat mesangial cells. Autoradiograms of agarose gels of end-labeled DNA are shown. Equal amounts of total DNA were loaded in each lane. Left : quiescent cells were transferred to SF medium for 8 h without any other addition ( lane i ) or containing 50 ng/ml TNF- plus 2 µM SB-203580 ( lane ii ), or with 10 µM caspase-8 inhibitor z-IETD-fmk ( lane iii ) or 10 µM CdCl 2 ( lane iv ). Right : cells in complete medium were maintained as such ( lane v ) or transferred to SF medium alone ( lane vii ) or SF medium with 25 µM CPT without ( lane vi ) or with ( lane viii ) 10 µM CdCl 2, all for 8 h. The migration of DNA size markers on the left-most gel is shown. The gels are typical of 3 experiments each.
Fig. 3. Suppression of DNA fragmentation by Cd 2+. A : all cultures were treated with TNF- and SB-203580 in SF medium for 8 h, without further addition (left-most lane, -) or in the presence of the indicated metal. All metals were in the divalent form except Fe, which was trivalent, and all were included as their chloride salts to a final concentration of 10 µM. B : control cells in complete medium (C) or cells kept in SF medium for 8 h (SF) are compared with cells treated for 8 h in SF medium with CPT alone (-) or in the presence of the indicated metal as in A. Equal amounts of total DNA were loaded in each lane. Autoradiograms of agarose gels of end-labeled DNA are shown. Left : migration of DNA size markers.
Because the absence of DNA laddering does not preclude apoptosis, apoptotic nuclei with chromatin condensation were visualized by DAPI fluorescent staining ( Fig. 4 ). Serum-free conditions caused slight but not significant increases in basal levels of cells scored as positive for apoptosis. Cadmium caused a decrease from 21 ± 6 to 7 ± 2% ( P < 0.01) positive nuclei in TNF- -treated cultures and from 29 ± 7 to 1.7 ± 0.6% ( P < 0.001) in CPT-treated cultures. In both cases, the Cd 2+ -treated cells showed values comparable to the serum-free controls ( Fig. 4 B ).
Fig. 4. Effect of Cd 2+ on chromatin condensation. Nuclei were stained with DAPI and those with chromatin condensation characteristic of apoptosis were seen as brighter fragmented spots on low-power fields. A : examples of fields comparing CPT treatment (25 µM) alone ( a ) or with 10 µM CdCl 2 ( c ) for 8 h, used for scoring apoptosis. b : Higher magnification of the boxed region in a. White arrowheads indicate examples of nuclei scored as apoptotic and indicate the same pair of cells in both panels. B : percentage of apoptotic cells from fields such as those shown in A. Bars are means ± SD from 3 independent experiments. In the set of TNF- experiments, a total of between 2,000 and 5,700 cells were scored under each set of conditions. In the CPT experiments, between 5,500 and 6,500 cells were scored. Filled bars: cells were grown in 0.2% FBS for 48 h (except untreated, maintained in complete medium) and then transferred to SF medium for 8 h or SF medium containing 50 ng/ml TNF- plus SB-203580 (treated) with or without 10 µM CdCl 2. Open bars: cells were grown in complete medium (untreated), then transferred to SF medium for 8 h, or SF medium containing 25 µM CPT (treated) with or without 10 µM CdCl 2. Between bars of the same color, a differs from c at P < 0.001, and b differs from c at P < 0.01.
Because activation of caspase-3 is a point of convergence of intrinsic and extrinsic pathways, inhibition of both TNF- -induced and CPT-induced apoptosis could result from suppression of caspase-3 cleavage. Polyclonal caspase-3 antibody detected pro-caspase-3, as well as cleavage products occurring after 8-h treatment with TNF- or CPT ( Fig. 5 A ). A monoclonal antibody to the p17 cleavage product further identified this fragment. Inclusion of Cd 2+ caused a marked decrease in cleavage products. Time courses showed occurrence of the cleaved fragments by 4 h following each treatment, increasing thereafter and again largely suppressed by Cd 2+ ( Fig. 5, B and C ).
Fig. 5. Suppression of procaspase-3 cleavage by Cd 2+. A : Western blots of caspase-3 in extracts of cells held in SF medium for 8 h ( lane i ) or treated with TNF- plus SB-203580 without ( lane ii ) or with 10 µM CdCl 2 ( lane iii ), using a polyclonal anti-caspase-3 antibody. Lanes iv to vi are as lanes i to iii, respectively, but blotted with an antibody specific to the p17 cleavage product. Cells were also held in SF medium ( lane vii ) or treated with CPT without ( lane viii ) or with 10 µM CdCl 2 ( lane ix ). Lanes x to xii are as lanes vii to ix, respectively, but blotted with the p17-specific antibody. B : cells were treated with TNF- plus SB-203580 in SF medium with or without 10 µM Cd 2+ for the times indicated below in C. The Western blot was probed with the polyclonal anti-caspase-3 antibody. C : as in B, except that treatment was with CPT with or without Cd 2+. A : SF controls. B : positions of caspase-3 and its cleavage products are indicated. Left : molecular weight markers.
Because Cd 2+ thus prevents activation of caspase-3, we examined the activity of the upstream caspases-8 and -9. As caspase-8 is the apical caspase in the extrinsic pathway of apoptosis ( 19, 46 ), we expected to see increased cleavage of procaspase-8 following TNF- treatment. Indeed, the 18-kDa fragment was prominent after TNF- treatment and was decreased following TNF- given in the presence of the caspase-8 inhibitor z-IETD-fmk ( Fig. 6 A ). The production of caspase-8 cleavage products was inhibited as effectively by including 10 µM CdCl 2 as by the caspase-8 inhibitor. Caspase-8 activity was also detected using a fluorometric assay that employs a caspase-8-specific substrate ( Fig. 6 B ). Inhibition to below basal levels occurred in the presence of the caspase-8 inhibitor z-IETD-fmk, whereas TNF- /SB-203580 treatment caused only a twofold increase above untreated control cells. The low level of caspase-8 activation suggests that mesangial cells may be so-called type II cells with low levels of caspase-8 protein (see DISCUSSION ). Nevertheless, caspase-8 activation was abolished by Cd 2+ ( Fig. 6 B ), in keeping with suppression of caspase-8 cleavage.
Fig. 6. Caspase-8 in TNF- -treated cells. A : representative Western blot of cell extracts with anti-caspase-8 antibody. Quiescent cells were treated with TNF- for 8 h, either alone or in the presence of 10 µM caspase-8 inhibitor z-IETD-fmk or 10 µM CdCl 2. Arrows indicate the positions of the 55-kDa pro-caspase-8 and the major cleavage product at 18 kDa. Equal protein loading was confirmed with GelCode blue staining of the gels and Ponceau S staining of the membrane (not shown). B : caspase-8 activity was measured in extracts from quiescent, untreated cells, or cells treated for 8 h with TNF- /SB-203580 in the presence of increasing amounts of the caspase-8 inhibitor z-IETD-fmk. In a separate set of experiments, cells were treated with TNF- in the presence or absence of 10 µM CdCl 2. The inhibitor studies are from duplicate wells of a single experiment. The TNF- Cd data represent means ± SD from 3 independent experiments. *Difference at P < 0.01.
We next examined the effect of Cd 2+ treatment on caspase-9 activity. Activity measured against the caspase-9 substrate was increased more than 20-fold on exposing cells to CPT. This activity was inhibited in a dose-dependent manner by the caspase-9 inhibitor z-LEHD-fmk ( Fig. 7 A ), again supporting specificity of the reaction. It was also completely eliminated by incubating the cells with 10 µM Cd 2+. If caspase-9 acting through activation of caspase-3 is an important pathway for apoptosis in mesangial cells, then we would expect that inhibition of caspase-9 would suppress caspase-3 cleavage. On the contrary, under conditions where z-LEHD-fmk completely inhibited caspase-9, it had no effect on caspase-3 cleavage induced by CPT ( Fig. 7 B ). Thus, although Cd 2+ can inhibit caspase-9 activity, CPT must lead to caspase-3 cleavage through an alternative mechanism that is also Cd 2+ sensitive ( Fig. 5 ).
Fig. 7. Caspase-9 activity in CPT-treated cells. A : caspase-9 activity was measured in extracts from untreated cells, or cells treated for 8 h with CPT in the presence of different amounts of the caspase-9 inhibitor z-LEHD-fmk. In a separate set of experiments, cells were treated with CPT in the presence or absence of 10 µM CdCl 2. The inhibitor studies are from duplicate wells of a single experiment. The CPT data represent means ± SD from 3 independent experiments. *Difference at P < 0.01. B : Western blot of whole cell extracts with polyclonal caspase-3 antibody. Cells were either left untreated or treated with CPT for 8 h in the presence of different amounts of the caspase-9 inhibitor z-LEHD-fmk. At inhibitor concentrations shown in A to effectively block caspase-9 activity, there is no apparent effect on caspase-3 cleavage products.
The possibility of cross talk between caspase-8 and caspase-9 pathways could provide additional routes to caspase-3 cleavage. The caspase-8 inhibitor z-IETD-fmk had no effect on CPT-mediated DNA cleavage (data not shown). However, caspase-9 was increased about sixfold by TNF- ( Fig. 8 ). This activation, too, was nearly completely ihibited by Cd 2+. Inhibition of caspase-8 only partially prevents caspase-3 cleavage after TNF- treatment, whereas the caspase-9 inhibitor or both inhibitors used in combination do so nearly completely ( Fig. 9 ). On the other hand, following CPT treatment, the combination of inhibitors failed to prevent caspase-3 cleavage (data not shown), consistent with Fig. 7 B.
Fig. 8. Caspase-9 activity in TNF- -treated cells. Caspase-9 activity was measured in extracts from untreated cells (taken as 100%), or cells treated for 8 h with TNF- in the presence or absence of 10 µM CdCl 2, or with TNF- plus the caspase-8 inhibitor z-IETD-fmk (8i; 10 µM). Values are averaged from 2 experiments. Basal activity in untreated quiescent cells is taken as 100%.
Fig. 9. Effect of caspase inhibitors on TNF- -stimulated caspase-3 cleavage. Quiescent cells were either held in SF conditions or treated with TNF- for 8 h. The 17-kDa cleavage product is suppressed or eliminated by inclusion of the caspase-8 inhibitor, z-IETD-fmk (8i; 10 µM) and the caspase-9 inhibitor, z-LEHD-fmk (9i; 100 µM), alone or in combination. The blot is typical of 3 experiments.
These results are consistent with destabilization of mitochondria by caspase-8 mediated by truncated Bid (tBid) ( 30 ). Therefore, we examined the effect of Cd 2+ on tBid formation following serum withdrawal and TNF- treatment. Compared with quiescent cells or those held in serum-free conditions, TNF- caused an increase in cleavage to tBid ( Fig. 10 A ). Cadmium suppressed this increase, maintaining tBid at the level seen in quiescent cells. TNF- also caused a shift of cytochrome c from the mitochondrial compartment to the postmitochondrial fraction ( Fig. 10 B ). This was partially suppressed by both Cd 2+ and the caspase-8 inhibitor z-IETD-fmk. Cadmium treatment in the absence of TNF- was without effect on mitochondrial cytochrome c.
Fig. 10. Suppression of Bid cleavage and cytochrome c release by Cd 2+. A : quiescent cultures (Q) were either left untreated or transferred to SF medium alone or containing TNF- /SB-203580, with or without 10 µM CdCl 2, for 8 h. Bid (22 kDa) and tBid (15 kDa) were identified by Western blotting, and -actin is shown on the same blot as a gel-loading control. The experiment was performed 3 times with similar results. B : cells in the first 4 lanes were treated as in A. In addition, some cells were treated with 10 µM CdCl 2 alone or with TNF- /SB-203580 plus 10 µM caspase-8 inhibitor, z-IETD-fmk (TNF + 8i). Mitochondrial and postmitochondrial cytosolic proteins were separated as described in EXPERIMENTAL PROCEDURES. Western blots of cytochrome c are shown from the same cell preparations. The experiment was repeated several times with consistent results.
DISCUSSION
While many studies have shown proapoptotic actions of Cd 2+ ( 11, 18, 23, 24, 26, 47, 54 ), the present work, in common with several others ( 42, 57 ), shows an antiapoptotic effect of this metal. Before focusing on specific pathways, we had observed that treatment with 10 µM CdCl 2 inhibited both DNA laddering and caspase-9 activity induced by serum withdrawal in RMC. Because serum withdrawal is a rather nonspecific manuever, our present objective was to study Cd 2+ 's effect on the two major pathways leading to apoptosis: one involving death receptors (called the extrinsic pathway), and the other involving the mitochondria (the intrinsic pathway).
RMC treated with 50 ng/ml TNF- plus 2 µM SB-203580 underwent apoptosis in a manner consistent with activation of the extrinsic pathway. The caspase-8 activity and DNA laddering induced by this treatment are both inhibited by 1 µM z-IETD-fmk indicating that TNF- -induced apoptosis in RMC is mediated via caspase-8, similar to other models of death receptor-mediated apoptosis. Furthermore, TNF- treatment results in the proteolytic activation of pro-caspase-3, indicating that this effector caspase plays a role in TNF- -mediated apoptosis. In the presence of Cd 2+, the biochemical and morphological changes induced by TNF- were inhibited significantly.
RMC treated with CPT also underwent apoptosis. However, the mechanism was not dependent on caspase-8 because CPT-induced DNA fragmentation could not be prevented with a caspase-8 inhibitor. There is significant caspase-9 activity after CPT treatment compared with the untreated cells, and procaspase-3 is proteolytically cleaved by CPT treatment, thus holding to the intrinsic apoptotic model. However, inhibiting CPT-induced caspase-9 activity by the caspase-9 inhibitor z-LEHD-fmk did not prevent the proteolytic processing of pro-caspase-3 ( Fig. 7 ). Based on the evidence showing that pro-caspase-9 activation leads invariably to pro-caspase-3 activation ( 8, 28, 44 ), and our observation that CPT activates pro-caspase-9, it is unlikely that the activation of pro-caspase-3 in CPT-treated RMC is solely by a caspase-9-independent pathway. It is possible that there are two pathways carrying out the activation of pro-caspase-3 in CPT-treated RMC. One pathway may involve the activation of pro-caspase-3 by caspase-9 while another pathway may involve the activation of pro-caspase-3 by an unidentified factor which is independent of caspase-9. We cannot say whether these pathways operate in conjunction or whether the canonical caspase-9 to caspase-3 pathway is a default pathway, the inhibition of which augments the activity of the other. That Cu 2+ causes partial suppression of DNA laddering following CPT treatment but not after TNF- treatment may afford an interesting opportunity to distinguish these pathways, although we have not pursued this avenue in the present studies. Nevertheless, the important finding is that similar to its effect on the extrinsic pathway, Cd 2+ inhibited the biochemical and morphological features induced by CPT.
RMC appear to be more sensitive to CPT-induced apoptosis, because TNF- exposure induces 21% apoptotic cells and some of this (7%; Fig. 4 ) can be accounted for by serum withdrawal, whereas CPT causes 29% apoptosis. Furthermore, we found caspase-8-dependent caspase-9 activity in TNF- -treated RMC ( Fig. 8 B ), indicating caspase-9 is an important participant in RMC apoptosis. Cells have been classified as type I and type II, the latter having lower amounts of procaspase-8 and less activation of capsase-8 at the death receptor ( 39, 40 ). Activation of the intrinsic pathway downstream of caspase-8 activation is a crucial event in propagating the apoptotic signal from the death receptor in type II cells. This would seem to be the case in RMC. That the caspase-9 inhibitor z-LEHD-fmk inhibits caspase-3 cleavage following TNF- treatment ( Fig. 9 ) reinforces the involvement of caspase-9 even after activation of the extrinsic pathway. A caveat here, though, is that the substrate-based caspase inhibitors used here nevertheless show some cross-inhibition between caspases-8 and -9 ( 14, 38, 49 ).
Although caspase-9 has cleavage sites for direct activation of caspase-8, recruitment of caspase-9 to the apoptosome is also mediated through caspase-8-dependent formation of tBid ( 32 ). In the present study, tBid appears to be involved following TNF- stimulation. It is interesting that serum withdrawal alone leads to caspase-8 cleavage (data not shown), although not progression to downstream events such as chromatin condensation and DNA laddering. Presumably in the absence of TNF-, other serum factors are necessary to sustain progression. Morphology and viability assays indicate that the cells abort the apoptotic program in the absence of serum, without immediate conversion to necrosis. However, the point we wish to make here is that Cd 2+ inhibits Bid cleavage and cytochrome c release following TNF- stimulation, suggesting inhibition of a caspase-8-dependent mechanism.
A possible mechanism by which Cd 2+ inhibits extrinsic and intrinsic pathway-mediated apoptosis is by inhibiting the caspases. In the TNF- /extrinsic pathway model, the inhibition of the proteolytic activation of procaspase-3 is expected as Cd 2+ inhibits its upstream activator, caspase-8. In the CPT/intrinsic pathway model, the inhibition of pro-caspase-3 activation can be explained by the observation that Cd 2+ inhibits caspase-9. In addition, the inhibition of caspase-9 activation may be due to the inability to form the apoptosome, because Cd 2+ prevents cleavage of Bid to the mitochondrion destabilizing species tBid. However, serum withdrawal can also induce cytochrome c release ( 12, 25 ). Therefore, whether the inhibition of CPT-induced caspase-9 activity is due to prevention of mitochondrial cytochrome c release or due to another mechanism remains to be answered. Cd 2+ has a high affinity for -SH groups and therefore can bind cellular polypeptides that contain these groups. Given that caspases are cysteine-dependent enzymes, the binding of Cd 2+ to the R-S - of the active-site cysteine could inhibit enzymatic activity. This hypothesis is supported by the report from Yuan et al. ( 57 ) that the IC 50 for caspase-3 inhibition by Cd 2+ is 8.7 µM in intact CHO cells. However, the same study reports an IC 50 of 31 µM Cd 2+ in cell-free systems. This difference between intact cells and the cell-free system indicates that if caspase inhibition is important in vivo, there must be additional antiapoptotic mechanisms that are triggered by Cd 2+ in intact cells.
In summary, this study has provided several details regarding the relationship between Cd 2+ and apoptosis in RMC. 1 ) Cd 2+ inhibits apoptosis induced by different stimuli in RMC. 2 ) Cd 2+ inhibits apoptosis via both the extrinsic and intrinsic pathways, although the intrinsic pathway appears to be more important in RMC. 3 ) Cd 2+ inhibits the initiator caspases, caspase-8 and caspase-9, and the activation of the effector caspase, caspase-3. 4 ) Cd 2+ inhibits cleavage of Bid and cytochrome c release from mitochondria following activation of caspase-8. 5 ) The net effect on DNA laddering is specific to Cd 2+ among a number of metal ions tested at similar concentration. Dysregulation of mesangial cell apoptosis by Cd 2+ may exacerbate chronic renal disease by promoting proliferative forms of glomerulonephritis.
GRANTS
This work was supported by a grant from the Canadian Institutes of Health Research.
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作者单位：Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada