干细胞之家 - 中国干细胞行业门户第一站

 

 

搜索

点这里关注干细胞之家微信公众号

朗日生物希瑞干细胞
查看: 9522|回复: 98
go

Regulation of caspase-3 and -9 activation in oxidant stress to RTE by forkhead t

Rank: 1

积分
威望
0  
包包
0  
发表于 2009-4-22 08:08 |显示全部帖子
作者:Gur P. Kaushal,, Ling Liu, Varsha Kaushal, Xiaoman Hong, Oksana Melnyk, Rohit Seth, Robert Safirstein, and Sudhir V. Shah作者单位:1 Department of Medicine, Central Arkansas Veterans Healthcare System, and 2 Department of Biochemistry, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
; V( R4 {& G+ j) ~' Q                  
, q! b% n( L. ^3 e/ K# Y                  - h4 @; U8 M  G3 e
         
/ z$ f; ], D$ E. x                        
0 @  x. a' P) E+ ]            ; ~7 `& C7 _) ]7 L
            
4 P8 C1 A, V9 a            
2 s2 |+ p! F' E0 q9 r. D' u            5 C. d& K+ t2 N
                     
+ R4 H' Q  J2 }' r" K0 T8 K+ O. `1 {        8 c- d- Q) \3 u( ?0 r5 R9 w
        & O1 |5 C- f$ Q' c( G
        8 E* M3 n* c5 S. L/ w
          【摘要】
! J" ^, E% ?: g2 q4 i      Cytotoxicity to renal tubular epithelial cells (RTE) is dependent on the relative response of cell survival and cell death signals triggered by the injury. Forkhead transcription factors, Bcl-2 family member Bad, and mitogen-activated protein kinases are regulated by phosphorylation that plays crucial roles in determining cell fate. We examined the role of phosphorylation of these proteins in regulation of H 2 O 2 -induced caspase activation in RTE. The phosphorylation of FKHR, FKHRL, and Bcl-2 family member Bad was markedly increased in response to oxidant injury, and this increase was associated with elevated levels of basal phosphorylation of Akt/protein kinase B. Phosphoinositol (PI) 3-kinase inhibitors abolished this phosphorylation and also decreased expression of antiapoptotic proteins Bcl-2 and BclxL. Inhibition of phosphorylation of forkhead proteins resulted in a marked increase in the proapoptotic protein Bim. These downstream effects of PI 3-kinase inhibition promoted the oxidant-induced activation of caspase-3 and -9, but not caspase-8 and -1. The impact of enhanced activation of caspases by PI 3-kinase inhibition was reflected on accelerated oxidant-induced cell death. Oxidant stress also induced marked phosphorylation of ERK1/2, P38, and JNK kinases. Inhibition of ERK1/2 phosphorylation but not P38 and JNK kinase increased caspase-3 and -9 activation; however, this activation was far less than induced by inhibition of Akt phosphorylation. Thus the Akt-mediated phosphorylation pathway, ERK signaling, and the antiapoptotic Bcl-2 proteins distinctly regulate caspase activation during oxidant injury to RTE. These studies suggest that enhancing renal-specific survival signals may lead to preservation of renal function during oxidant injury.
! K- d: W2 l' D% a          【关键词】 LLCPK cells Akt phosphorylation Bim PI kinase inhibitors) e. g& R" [0 A+ S+ W& m7 N
                  CASPASES ARE A FAMILY OF STRUCTURALLY related cysteine proteases that play a central role in the execution of apoptosis ( 20, 49, 62 ). On receiving a proapoptotic stimulus, the caspases are proteolytically processed to the active forms from their normally synthesized inactive proenzymes. Activated caspases mediate apoptosis by cleaving and inactivating intracellular proteins that are essential for cell survival and proliferation ( 20, 22, 49, 62 ). This cleavage of target proteins contributes to the morphological changes observed in apoptosis. Multiple pathways can result in activation of executioner caspases depending on the nature of the death-inducing stimulus. At present, there are two relatively well-characterized cell death pathways that result in the activation of the downstream caspase-3. One is receptor mediated ( 53 ) and the other is mitochondrial dependent ( 29 ). The receptor-dependent pathway is initiated by activation of cell death receptors (Fas and tumor necrosis factor- ), leading to activation of procaspase-8, which in turn cleaves and activates procaspase-3. The mitochondrial-dependent pathway is triggered by cytochrome c release and other proapoptotic proteins from the mitochondria. Cytosolic cytochrome c recruits procaspase-9, Apaf-1, and dATP and promotes caspase-9 activation. Activated caspase-9 then cleaves and activates downstream procaspase-3 to its active form ( 29 ). However, it is not known whether oxidant-induced activation of caspases involves both of these signaling pathways in renal injury.
. k5 b* N3 m1 q" H# f2 h
- \6 f; _& i% P  R& \  {1 }Reactive oxygen species (ROS) have been implicated in the pathogenesis of a variety of renal diseases, including ischemic and toxic acute renal failure ( 4, 5, 43 ). ROS-induced cell death has been reported in a wide variety of cells, including renal tubular epithelial cells (RTE) ( 1, 14, 24, 31, 38, 57, 58 ) and nonrenal cells ( 25, 40 ). Studies from other laboratories and that from ours have shown an important role of caspases in RTE injury ( 15, 21, 23, 33 - 36, 38, 41, 44, 46 ). Because oxidants have been implicated in a wide spectrum of RTE injury, understanding the relationship between oxidants and caspases and cellular pathways that control their activation would provide important information related to RTE injury.* o3 M! k+ I' [; a8 S  g- k6 J' J1 R' v
  X' `4 b$ r2 X, b
Cells mount responses to death stimuli that can overcome the insult and maintain cell viability. Thus cell fate in response to severe stress will depend on the balance between antiapoptotic and proapoptotic signaling pathways. Oxidative stress can provoke cellular responses in which proapoptotic pathways are activated as well as those that participate to defend against oxidative injury ( 25, 40 ). Thus the extent of oxidant-induced cellular injury will depend on the balance between the activation of caspase cascade and induction of survival factors capable of blocking the activation of caspases.
0 t& k* U; E( M, \* t3 [7 D: a& e! d$ ?
At present there is limited information on the role of cytoprotective as well as proapoptotic signals that regulate cell fate in oxidant injury to renal cells. We have previously shown that Akt phosphorylation plays an important role in cisplatin-induced caspase activation in RTE ( 34 ). The present study provides evidence that downstream targets of Akt phosphorylation, Bcl-2 family members, and MAP kinases play distinct roles in the regulation of caspase activation in H 2 O 2 -induced RTE injury. We provide evidence that the regulation of caspase-3 and caspase-9 activation in oxidant injury is controlled by Akt-mediated phosphorylation of proapoptotic Bcl-2 family member Bad and forkhead transcription factors FKHR and FKHRL1 that control expression of the proapoptotic protein Bim. In addition, our studies demonstrate that, while activation of mitogen-activated protein kinases is a common response to oxidant injury, only induction of ERK1/2 activation in oxidant injury contributes to the regulation of caspase activation.
/ h2 A& n& J. v6 }+ k8 j! _
& V5 o) O* [$ M% ~4 P: B1 ZMETHODS
$ S' h9 b7 k& U' `) m' q
8 S1 Z1 g- Y: L0 h! w. K$ F2 RCell culture and reagents. LLC-PK 1 cells obtained from American Type Culture Collection were cultured as in our previous studies ( 34 ). The cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM L -glutamine, 20 mM HEPES, and 2 mM nonessential amino acids. Cultures were maintained in a humidified incubator gassed with 5% CO 2 -95% air at 37°C and fed with fresh medium at intervals of 48-72 h. Experiments were performed with cells grown to 80% confluence. Caspase substrates were purchased from Peptide International and antibodies to caspases and proteins of Bcl-2 family were from Santa Cruz Biotechnology and Cell Signaling Technology (Beverly, MA). Antibodies to Akt, Ser473-phosphorylated Akt, Bad, Ser136-phosphorylated Bad, Ser112-phosphorylated Bad, phospho-ERK1/2 (phospho p44/42 MAP kinase), ERK (p44/42 MAP kinase), Ser256-phosphorylated forkhead transcription factor P-FKHR, Thr-32- and Thr-24-phosphorylated forkhead transcription factor P-FKHRL1, and an Akt kinase assay kit were obtained from Cell Signaling Technology. Antibodies to Bim, cytochrome c, and apoptosis-inducing factor (AIF) were from Santa Cruz Biotechnology. Antibody to cytochrome c oxidase subunit IV (Cox IV) was from Molecular Probes. MEK inhibitor U-0126, JNK inhibitor SP-600125, p38 inhibitor SB-202190, LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a quercetin derivative and a specific inhibitor of phosphoinositol (PI) 3-kinase, and wortmannin, a potent inhibitor of PI 3-kinase, were obtained from Calbiochem.
3 p; e$ m  [5 ~$ {! |+ ?# d* M. a  F4 E3 E
Induction of oxidant injury. Cell medium was replaced with fresh DMEM containing serum, glutamine, pyruvate, nonessential amino acids, glucose, 3.7 g/l NaHCO 3, and 20 mM HEPES at pH 7.4, and cells were incubated either with or without H 2 O 2 of various concentrations (50-400 µM) for the period of time indicated (1-8 h). In initial studies, we determined the optimum exposure time and the suitable concentration of H 2 O 2. To determine a role of inhibition of Akt phosphorylation and signaling by MAP kinases in H 2 O 2 -induced cell death, cells were preincubated with LY-294002 or wortmannin, inhibitors of Akt phosphorylation, and MAP kinases, respectively, for 10 min and then exposed to H 2 O 2 (100 µM).
0 K( I6 B- D( \: G4 z9 b( _$ t3 p/ `' ?
Determination of caspase activity. Cells were harvested by centrifugation, and the pellets were washed twice in cold PBS. The washed cell pellets were lysed with 20 mM HEPES, pH 7.5, containing 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS), 2 mM DTT, 0.1% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A at 4°C. The supernatants obtained were used to determine the activities of caspase-1, -3, -8, and -9 by fluorometric assay using the following amino-4-methyl coumarin (AMC)-tagged substrates: YVAD-AMC for caspase-1, DEVD-AMC for caspase-3, IETD-AMC for caspase-8, and LEHD-AMC for caspase-9. These substrates are specifically cleaved by the respective caspases at the Asp residue, releasing the fluorescent AMC tag ( 34, 52 ). The enzyme extracts containing 50 µg protein were incubated with 100 mM HEPES, pH 7.4, containing 10% sucrose, 0.1% CHAPS, 10 mM DTT, and 50 µM caspase substrate in a total reaction volume of 0.25 ml. The reaction mixture was incubated for 60 min at 30°C. At the end of incubation, the amount of liberated fluorescent group AMC was determined using a fluorescent Spectrofluorometer (PerkinElmer) with an excitation wavelength of 380 nm and an emission wavelength of 460 nm ( 34, 52 ). AMC was used as a standard. Based on the standard curve made with a fluorescence reading with free AMC, the data for caspase activity are expressed as nanomoles of AMC liberated when 50 µg of protein extract were incubated with 50 µM substrate for 60 min at 30°C.9 C$ G& W1 {7 k2 O
, p0 I; y) T4 \/ i
Phosphorylation of Akt, Bad, and forkhead proteins. Akt phosphorylation was determined by using an Akt Kinase Assay kit (Cell Signaling Technology) following the manufacturer's instructions. Briefly, cells were lysed with cell lysis buffer supplied with the assay kit. Total Akt in cell extracts was immunoprecipitated with anti-Akt antibody conjugated to agarose beads. The immunoprecipitated Akt was then incubated with glycogen synthase kinase-3 (GSK-3) protein in a kinase assay containing ATP. After incubation and centrifugation, the supernatant was used to assay phospho-GSK-3 by Western blot analysis using anti-phospho-GSK-3 / antibodies. Akt bound to the agarose beads was released by boiling in SDS-sample buffer and quantitated by Western blotting using anti-Akt antibody. Phosphorylation of Bad, ERK1/2, and forkhead transcription factors (FKHR and FKHRL1) was determined using the respective phospho-specific antibodies.& ?/ q6 k$ q& L6 m' a0 j
9 E% K& a- N0 O+ N' ?! ~
Western blot analysis. The cell lysates were prepared as described above for caspase assay, and 100-µg protein samples were subjected to reducing SDS-gel electrophoresis. SDS-PAGE was performed as previously described ( 56 ) using 8% polyacrylamide gels. The resolved proteins were electrophoretically transferred to Immobilon polyvinylidine difluoride (Millipore) membrane and processed further for antibody staining as described by Towbin et al. ( 56 ). After this transfer, membranes were washed in a buffer containing 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 for 5 min and then in the same buffer containing 5% nonfat dry milk for 1 h at room temperature. The membranes were then incubated for 2 h with primary antibodies (1:1,000 dilution) in the same buffer with 5% dry milk. At the end of this time, the membranes were thoroughly washed (at least 4 times) with 50 mM Tris·HCl, pH 7.5, containing 0.05% Tween 20. The membrane filters were then incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit antibody diluted 1:3,000 in Tris buffer containing 5% dry milk. After incubation, membrane filters were washed in the same buffer containing 0.05% Tween 20 and developed by exposure to chemiluminescent substrates (Pierce, Rockford, IL).
. s; {: G& u+ T
! A6 P! W( n' f4 XTransient transfection. LLC-PK 1 cells were transfected with expression constructs pUSE-DN-Akt encoding DN-Akt or the empty vector pUSE in the presence of an expression vector pCMV-Lac Z expressing -galactosidase. pUSE-DN-Akt and pUSE were from Upstate Biotechnology. Transfection was performed using Lipofectamine (Life Technologies) as per the manufacturer's recommendations as described in our previous study ( 34 ). Briefly, cells were plated at a density of 2 x 10 5 in six-well plates. After 24 h, cells were washed three times with serum-free media and incubated for 6 h with a mixture of Lipofectamine and plasmids containing the inserts (1.5 µl of Lipofectamine, 50 µl of serum-free medium, and 1 µg of DNA in 50 µl of serum-free medium) in serum-free media at 37°C with 10% CO 2. Culture medium then was replaced with fresh DMEM containing 10% serum and 1% penicillin-streptomycin mixture, and cells were then treated with H 2 O 2 for the indicated times. After incubation, cells were examined for expression of phospho-Akt by Western blotting and for caspase activity using fluorogenic substrates.5 t; `& Z9 R1 W5 q' B; y

0 u6 M0 B* ]! ^) v! ]' EImmunofluorescence localization of active caspase-3. Cells grown on sterile glass coverslips were treated with 200 µM H 2 O 2 for various time points in the presence and absence of the PI 3-kinase inhibitor LY-294002. After the treatments, the cells were washed in PBS and fixed in 2% parformaldehyde in PBS for 15 min. After the cells were washed twice in PBS, they were permeabilized for 1 h in blocking buffer containing 1% BSA, 1% goat serum, 0.1% saponin, 1 mM CaCl 2, 1 mM MgCl 2, and 2 mM NaV 2 O 5 in PBS. Cells were then incubated in with 1:200 rabbit anti-caspase-3 (active) antibody for 1 h in a 37°C humidified incubator. After three washes in buffer containing 1% BSA and 0.1% saponin in PBS, cells were incubated at 37°C in a humidified incubator for 1 h with 1:500 of Alexa fluor-conjugated secondary antibody (goat anti-rabbit) in blocking solution and again washed with washing buffer. The nuclei were stained with 0.5 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) for 5 min, and cells were washed twice in washing buffer. Coverslips were then mounted on slides using antifade mounting media (Molecular Probes) and visualized for active caspase-3 and morphological changes using a Zeiss Deconvoluted Microscope.; o5 P- x+ L" h9 `4 l6 J
3 o0 E% g- j; G7 g. J
Subcellular fractionation. LLC-PK 1 cells were harvested and cell pellets were washed in PBS. Mitochondrial and cytosolic fractions were isolated essentially as previously described ( 3, 17 ). Briefly, cells were suspended in isotonic mitochondrial buffer containing 210 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM HEPES, pH 7.5, supplemented with protease inhibitor cocktail, and homogenized for 30-40 strokes with a Dounce homogenizer at 4°C. To establish the number of strokes necessary for cell permeabilzation, the trypan blue exclusion method (0.4% trypan blue solution in PBS diluted 1:10 with cell suspension) was used to discriminate between permeabilized (stained) cells and intact cells (unstained). The suspension was then transferred to Eppendorf centrifuge tubes and centrifuged at 500 g for 5 min to pellet nuclei and unbroken cells. The supernatant was then centrifuged at 10,000 g for 30 min to pellet the mitochondria-enriched fraction. The resulting supernatant was used as the cytosolic fraction. The mitochondrial pellet was further purified by resuspending in isotonic mitochondrial buffer and centrifuging at 10,000 g. Contamination of mitochondria in the cytosolic fraction was determined by immunoblotting for cytochrome oxidase subunit IV, an integral membrane protein of mitochondria.
$ ]* m* G2 }- |* ^, a& o! b/ _
2 f6 E& ^, `4 @Fluorescent-activated cell sorter analysis. Following treatments with 200 µM H 2 O 2 in the presence and absence of LY-294002, cells were harvested by trypsinization, pooled with culture medium containing floating cells, and collected by centrifugation at 500 g for 5 min. The cell pellets were suspended in 70% ethanol in PBS containing 5 mM EDTA and kept at 4°C overnight, collected by centrifugation at 1,500 g for 10 min, and resuspended in 0.5 ml of PBS containing 5 mM EDTA. RNase A was added (50 µl, 10 mg/ml), and the suspension was incubated for 30 min at 25°C. Propidium iodide was added (450 µl, 10 µg/ml), and the samples were analyzed using a FACSCalibur machine (Becton Dickinson). For each culture condition, 5 x 10 5 cells were analyzed. The percentage of cells in sub-G 1 /G 0 (fraction containing cell debris, apoptotic and necrotic cells), S, and G 2 /M phases was determined using a cell-cycle analysis program (Win MDI 2.8). The results shown are representative of three independent experiments." p" d( n7 i# }9 i; x

' e' o! t4 Z0 |# M& c8 I0 }Statistical analyses. Results are means ± SE. Comparison between values was determined by Student's t -test. A P value
1 Q$ U. a: ~) [7 Y) t2 Q- W# {$ B; {5 o* \
RESULTS5 W) ^6 s7 H% z; a! V5 }; O$ s

! h9 t: G0 t' n; A% q" p) ]Phosphorylation of Akt, Bad, FKHR, and FKHRL1 in oxidant injury. To elucidate the role of Akt phosphorylation in caspase activation in oxidant injury, we first examined the activity of Akt in response to H 2 O 2 to LLC-PK 1 cells. Akt kinase assay revealed that H 2 O 2 markedly increased basal Akt kinase activity in a time- and dose-dependent manner ( Fig. 1, A and B ). However, the protein expression of Akt alone was not changed during the course of oxidant injury. Oxidant-induced phosphorylation of Akt was abolished in a dose-dependent manner by LY-294002 (0-10 µM) ( Fig. 2 A ) or by wortmannin (0-0.25 µM) ( Fig. 2 B ), inhibitors of PI 3-kinase. Similar results were obtained when mouse proximal tubular epithelial cells were treated with H 2 O 2 and LY-294002 ( Fig. 2 C ). MAP kinase MEK inhibitor U-0126 and JNK inhibitor SP-600125 did not prevent H 2 O 2 -induced Akt phosphorylation ( Fig. 3 ), indicating that MAP kinases are not involved in phosphorylation of Akt.
6 n: j: X% U% a4 a
8 b8 [. o/ }4 a$ QFig. 1. Activation of Akt in response to oxidant injury. A : time course of activation of Akt in response to H 2 O 2. Cells were treated with 200 µM H 2 O 2 for various time periods as indicated. Cell lysates (100 µg protein) were analyzed for Akt activation by Western blot analysis using an Akt Kinase Assay Kit (Cell Signaling Technology). Total Akt in the cell extracts was immunoprecipitated with Akt antibody, and an Akt kinase assay was performed using GSK-3 fusion protein. Phosphorylated glycogen synthase kinase-3 (GSK-3; P-GSK) was determined by Western blot analysis using phospho-GSK-3 / antibody as described in METHODS. Total Akt in the immunoprecipitate was determined by Western blot analysis using Akt antibody. B : dose-dependent activation of Akt in response to H 2 O 2. Cells were treated for 4 h with various concentrations of H 2 O 2 as indicated. Cell lysates (100 µg protein) were analyzed for Akt activation as described in A. Results shown are representative of 3 independent experiments.# F1 J! l5 Y0 [3 J

$ O; Y4 B) k# e: }" f5 b1 LFig. 2. Effect of PI 3-kinase inhibitors on oxidant-induced Akt phosphorylation. A : dose-dependent inhibition of Akt phosphorylation by phosphoinositol (PI) 3-kinase inhibitor LY-294002 (LY). LLC-PK 1 cells were treated with 200 µM H 2 O 2 in the presence or absence of various concentrations (0-10 µM) of LY for 4 h as indicated. Cell lysates (50 µg protein) were analyzed for Akt activation by Western blot analysis using antibodies to phosphorylated Akt (Ser473) and Akt. B : dose-dependent inhibition of Akt phosphorylation by PI 3-kinase inhibitor wortmannin (WM). Phosphorylated Akt in the cell extracts was determined as described in A. C : mouse proximal tubular epithelial cells (TKPTs) were treated with 200 µM H 2 O 2 in the presence or absence of various concentrations (0-10 µM) of LY for 4 h as indicated. Phosphorylated Akt in the cell extracts was determined as described A. The results shown are representative of 3 independent experiments.
8 q5 s' l5 {  k3 ]5 s6 l4 l, p
Fig. 3. Effect of MAP kinase MEK inhibitor U-0126 and JNK inhibitor SP-600125 on Akt phosphorylation. Cells were treated for 4 h with 100 and 200 µM H 2 O 2 in the presence or absence of various concentrations of U-0126 or SP-600125 as indicated. Cell lysates (50 µg protein) were analyzed for Akt activation by Western blot analysis using antibodies to phosphorylated Akt (Ser473). Control cells were untreated. The results shown are representative of 3 independent experiments.
) X& J5 M7 X4 H0 }/ N
- g' H0 Y) ~0 M5 i4 yBad phoshorylation was markedly increased with H 2 O 2 treatment; however, the level of Bad protein was not changed during the course of oxidant injury ( Fig. 4 A ). These studies indicate that Bad phosphorylation is associated with Akt phosphorylation during H 2 O 2 -induced injury and may be involved in the suppression of caspase activation. Indeed, phosphorylation of Bad was abolished in a dose-dependent manner by LY-294002 (0-10 µM) ( Fig. 4 B ) or by wortmannin (0-0.25 µM) (data not shown). Similarly, H 2 O 2 induced marked phosphorylation of the FKHR and FKHRL1, and this phosphorylation was abrogated by LY-294002 in a time-dependent manner ( Fig. 5 A ), indicating that forkhead factors are downstream targets of Akt phosphorylation in oxidant injury. The phosphorylated forms of forkhead proteins are antiapoptotic, whereas unphosphorylated forms are proapoptotic. Because, in some cell types, forkhead transcription factors induce expression of proapoptotoic protein Bim ( 18, 28, 51 ), we examined the expression of protein levels of Bim in oxidant injury in the presence and absence of LY-294002. Bim protein was greatly reduced during the course of oxidant injury; however, after treatment with LY-294002, Bim accumulated markedly ( Fig. 5 B ).  J" ^* C+ \0 u+ L) V1 v6 n8 W
$ R0 f+ E- g5 k% Q. T" F
Fig. 4. Effects of oxidant injury on phosphorylation of Bad. A : time course of Bad phosphorylation in response to oxidant injury. Cells were treated with 200 µM H 2 O 2 for various time periods as indicated. Cell lysates (100 µg protein) were analyzed for Bad phosphorylation by Western blot analyses using specific antibodies to phosphorylated Bad (Ser136) and Bad, respectively. B : dose-dependent inhibition of Bad phosphorylation by PI 3-kinase inhibitor LY. Cell lysates were analyzed for Bad phosphorylation by Western blot analysis as described in A. Control (C) cells were untreated. The results shown are representative of 3 independent experiments.) z- H& h8 F' ^5 n3 Q
  t3 b& k& O6 j& j5 G& R0 Y7 l/ h
Fig. 5. A : time course activation of forkhead transcription factors FKHR and FKHRL1 and inhibition of their phosphorylation by LY in response to H 2 O 2. Cells were treated with 200 µM H 2 O 2 in the presence or absence of LY (10 µM) for various time periods as shown. Cell lysates (100 µg protein) were analyzed for phospho (P)-FKHR and P-FKHRL1 by Western blot analysis using phospho-specific antibodies to P-FKHR and P-FKHRL1. -Actin control was detected using with -actin antibody. Control cells were untreated. B : time course effect on Bim protein expression in response to 200 µM H 2 O 2 in the presence and absence of LY. Cells were treated as described in A. Cell lysates (50 µg protein) were analyzed by Western blot analysis using antibody specific to Bim as indicated. Control cells were untreated. The results shown are representative of 3 independent experiments.
, l  v3 E6 D* ]4 O* ]* R1 n! G
# p6 m6 Z5 S9 {# Z6 I: eEffect of phosphorylation of Akt, forkhead transcription factors, and Bad on caspase-9 and caspase-3 activation, and cell death in oxidant injury. Because Akt phosphorylation is known to block apoptosis ( 16, 26 ), the inhibition of Akt phosphorylation should potentiate enhanced caspase activation during the course of oxidant injury. Cytochrome c was released from the mitochondria to the cytosol in response to H 2 O 2 injury ( Fig. 6 A ), and treatment with PI 3-kinase inhibitor further increased oxidant-induced release of cytochrome c ( Fig. 6 A ). Immunoblotting for the mitochondrial markers Cox IV and AIF showed significant enrichment of markers in the mitochondrial fraction compared with the cell extract, and these markers were almost absent in the cytosolic and the nuclear fractions ( Fig. 6 B ). LY-294002 (10 µM) resulted in an enhanced and early increase in oxidant-induced caspase-3 and caspase-9 activity (cleavage of DEVD-AMC and LEHD-AMC, respectively) compared with that of H 2 O 2 alone in a time (0-8 h)- ( Fig. 7 A ) and dose (0-400 µM of H 2 O 2 for 4 h)-dependent manner ( Fig. 7 B ). LY-294002 (10 µM) alone (as shown in Fig. 7 B at 0 H 2 O 2 concentration) slightly increased caspase-3 and caspase-9 activation, but this activation is far less than that seen when cells were treated with H 2 O 2 alone or with LY-294002 and H 2 O 2 together.
" D5 B& x4 D7 i2 W% m  o. Q8 h0 n6 a* N. y! g
Fig. 6. A : cytochrome c (Cyt c ) release from the mitochondria to the cytosol in oxidant injury in the presence and absence of LY. Cells were treated for 4 h with 50 and 200 µM H 2 O 2 in the presence or absence of LY. Cells were processed for the isolation of mitochondrial and the cytosolic fractions as described in METHODS, and samples (50 µg protein) of mitochondrial and the cytosolic fractions were subjected to Western blot analysis with an antibody to cytochrome c. B : mitochondrial, cytosolic, and nuclear fractions were isolated as described in the METHODS, and 50 µg of total cell extract ( E ), 10 µg of mitochondrial proteins (M), 50 µg of cytosolic proteins (C), and 10 µg of nuclear proteins (N) were subjected to Western blot analysis with antibodies to the mitochondrial markers Cox IV and AIF. The results shown are representative of 3 independent experiments.
* N5 {3 v1 {) s9 B! U) ]/ Q7 v3 b* m$ {5 D& d
Fig. 7. A : time course effect of LY on oxidant-induced caspase-3 and caspase-9 activation. Cells were treated with 200 µM H 2 O 2 in the presence ( ) or absence ( ) of LY (10 µM) for various time periods as indicated. The caspase activity in cell lysates (50 µg protein) was determined using the caspase-3 substrate DEVD-AMC and caspase-9 substrate LEHD-AMC. Results are means ± SE, n = 4. * P
: n+ t7 C9 R( ~, L- @4 l9 o; x
3 ?% {7 g7 S4 _0 y. @; u+ n( x  |To confirm that inhibition of Akt phosphorylation sensitizes LLC-PK 1 cells to oxidant injury and affects caspase activation, we used a genetic approach to downregulate oxidant-induced Akt activation. We transiently transfected cells with an empty vector and DN-Akt vector (pUSE-DN-Akt) and treated cells with and without H 2 O 2. Western blot analysis confirmed the transfection and showed reduced levels of phospho-Akt in cells transfected with DN-Akt ( Fig. 8 A ). Transfection with DN-Akt makedly increased oxidant-induced caspase-3 and -9 activation ( Fig. 8 B ). Transfection with the empty vector had no effect on caspase activation (data not shown).
& y0 R$ i) |& s% H
1 f- Q$ i* D( Q; |Fig. 8. Effect of DN-Akt on H 2 O 2 -induced Akt phosphorylation and caspase activation. A : Westerm blot analysis of P-Akt levels in LLC-PK 1 cells transiently transfected with empty vector (pUSE) and DN-Akt vector (pUSE-DN-Akt) in the presence and absence of 100 µM H 2 O 2. Cells were cotransfected with control plasmid (pCMV-Lac Z) encoding the -galactosidase. B : cells were transiently transfected with pUSE and pUSE-DN-Akt in the presence of 100 µM H 2 O 2. The caspase activities in cell lysates (50 µg protein) were determined as described in METHODS.
! j) V. \) T; P' V8 }# A0 M) ^1 y/ v' W& c3 ~7 i
Substrate specificity of the caspases may be promiscuous but was overcome by identifying the specific caspases and their activation by using specific antibodies. As shown in Fig. 9, proteolytic processing of procaspase-3 in oxidant injury results in the formation of a 17-kDa subunit of the active caspase-3. Similarly, caspase-9 is proteolytically processed, resulting in 37 (prodomain  large fragment)- and 17-kDa subunits of the active form. LY-294002 (10 µM) resulted in the appearance of active caspase-3 and caspase-9 as early as 1 h of H 2 O 2 treatment and enhanced activation of these caspases at later time points ( Fig. 9 ). These data suggest that inhibition of the PI 3-kinase/Akt pathway in oxidant injury to RTE cells results in not only earlier activation of caspase-3 and -9 but also higher levels of activation. In marked contrast, inhibition of Akt phosphorylation did not affect the oxidant-induced activities of caspase-8 and proinflammatory caspase-1 (data not shown). Thus the receptor-mediated pathway that activates procaspase-8 and -1 is not affected by inhibition of Akt phosphorylation in oxidant-induced injury. These studies indicate that Akt phosphorylation regulates the mitochondrial-dependent caspase-9 and caspase-3 activation in oxidant-induced injury. The response of the disruption of oxidant-induced Akt phosphorylation was also reflected in early and enhanced cell death, as determined by cell viability ( Fig. 10 A ) and fluorescent-activated cell sorter analysis ( Fig. 10 B ). As shown, H 2 O 2 -induced cell death (sub-G 1 fraction containing cell debris, apoptotic and necrotic cells) was markedly increased with LY-294002 treatment. Untreated cells or cells treated with LY-294002 alone had a very low level of cell death. Cells treated with 200 µM H 2 O 2 for 2 and 4 h showed 7.5 and 12.5% cell death, respectively ( Fig. 10 B, c and e ). Addition of 10 µM LY-294002 in 2- and 4-h H 2 O 2 -treated cells increased cell death to 13.1 and 30.9%, respectively ( Fig. 10 B ). H 2 O 2 also increased the cell population in the S phase, suggesting significant damage to DNA at the G 1 phase leading to increased proliferation of cells in the S phase.2 u5 g) F9 y! q' I* r% ]! N- D4 F

3 r' {( B% E* o4 N: d6 ZFig. 9. A : effect of LY and wortmannin on oxidant-induced time course expression and activation of caspase-3. Cells were treated with 200 µM H 2 O 2 in the presence of LY (10 µM) or wortmannin (0.5 µM) for various time periods as indicated. Cell lysates (100 µg protein) were subjected to Western blot analyses using antibodies specific for caspase-3. Control cells (C) were not treated. B : effect of LY on oxidant-induced expression and activation of caspase-9. Cells were treated with 200 µM H 2 O 2 in the presence of LY (10 µM) for various time periods as indicated. Cell lysates (100 µg protein) were subjected to Western blot analyses using antibodies specific for caspase-9. Control cells (C) were not treated. The results shown are representative of 3 independent experiments.
+ s  ]! Q8 f) ~9 [: J$ p! E5 X3 s0 r2 c& }1 f; c. e
Fig. 10. A : effect of LY on H 2 O 2 -induced cell death. Cells were treated with 200 µM H 2 O 2 in the presence ( ) or absence ( ) of LY (10 µM) for the time points indicated. After incubations, cell death was determined by trypan blue exclusion. Results are means ± SE; n = 5. * P 7 ~/ W9 w8 G4 }. b
5 u) P9 j9 o0 x0 J  h
Immunofluorescence localization of active caspase-3 in oxidant injury. To confirm enhanced oxidant-induced caspase activation and cell death, we double stained cells with nucleus-specific DAPI and an antibody that specifically recognizes the active form of caspase-3. Double staining revealed that LY-294002 treatment enhanced oxidant-induced caspase-3 activation as well as cell apoptosis ( Fig. 11 ). It is interesting to note that staining of active caspase-3 is predominantly observed in the apoptotic and fragmented nuclei. Taken together, these studies indicate that enhanced activation of caspase-3 and -9 by inhibition of Akt phosphorylation has an impact on oxidant-induced cell death in renal cells.5 b4 e' Y+ A' X+ B3 P) s5 u  b5 i9 C

9 T" {; l' Y* y& mFig. 11. Enhanced activation of caspase-3 Casp 3) and cell apoptosis by the PI-3 inhibitor LY as revealed by double-staining with 4',6'-diamidino-2 phenylindole (DAPI) and antibody (Ab) to the cleaved form of caspase-3. LLC-PK 1 cells were grown on glass coverslips and treated with 200 µM H 2 O 2 for 4 h in the presence and absence of LY. The cells were fixed in a 4% paraformaldehyde solution in PBS, washed, and permeabilized with 1% saponin as described in METHODS. Cells on cover glasses were then processed for anticaspase-3 antibody (against the cleaved subunit that reacts only with the active form of the enzyme) followed by Alexa Fluor (BD Biosciences)-conjugated secondary antibody (shown in red for active caspase-3). Finally, cover glasses were washed in PBS and treated with 1 µg/ml DAPI solution in PBS (nuclei stain blue), mounted, and visualized in a deconvoluted fluorescence microscope. Arrows indicate some apoptotic nuclei that stain with antibody to the active form of caspase-3. The results shown are representative of 3 independent experiments.
$ F# k  p- Y( S6 e8 F
) M) o  w( z( d* m2 v  ZBcl-2 family members in oxidant injury. The members of the Bcl-2 family play important roles in caspase activation, and therefore we examined the effect of inhibition of Akt phosphorylation on the expression of the Bcl-2 proteins. There was a marked H 2 O 2 -induced increase in the expression of the anti-apoptotic Bcl-2 and BclxL proteins, and levels of these proteins decreased after treatment with LY-294002. Protein levels of the proapoptotic members Bad, Bax, and Bak did not change with LY-294002 treatment, suggesting that these proteins were not affected by inhibition of Akt phosphorylation ( Fig. 12 ).- a5 H4 J4 A  _. A1 a* I' E3 l4 |

7 q: m/ B- G! F; q* @Fig. 12. Time course expression of the members of Bcl-2 family in response to 200 µM H 2 O 2 in the presence and absence of LY. Cell lysates (50 µg protein) were analyzed by Western blot analysis using antibodies specific to members of the Bcl-2 family as indicated. The results shown are representative of 3 independent experiments.
# u8 ~; z: U. `8 g
3 A4 i1 [1 z' J9 D# E( n& L3 @MAP kinases (ERK1/2, JNK, and p38) in oxidant injury and caspase activation. Phosphorylation of ERK1/2, JNK, and p38 was markedly increased with increased exposure to oxidant injury ( Fig. 13 ). Protein levels of ERK1/2, JNK, and p38 were not changed during the period of oxidant injury. MEK inhibitor U-0126, JNK inhibitor SP-6000125, and p38 inhibitor SB-202190 were very effective in inhibiting the phosphorylation of the respective proteins, both in untreated and H 2 O 2 -treated cells ( Fig. 13 ). We next examined the effect of inhibition of ERK1/2, JNK, and p38 phosphorylation on caspase-3 and caspase-9 activation. Inhibition of ERK1/2 signaling by the MEK inhibitor U-0126 increased the activation of caspase-3 [caspase-3 activity (in nmol): 2-h H 2 O 2, 1.1 ± 0.2; 2-h H 2 O 2  U-0126, 1.45 ± 0.3; 4-h H 2 O 2, 1.8 ± 0.4; 4-h H 2 O 2  U-0126, 2.5 ± 0.5] and caspase-9 [caspase-9 activity (in nmol): 2-h H 2 O 2, 1.1 ± 0.2; 2-h H 2 O 2  U-0126, 1.45 ± 0.3; 4-h H 2 O 2, 1.8 ± 0.4; 4-h H 2 O 2  U-0126, 2.5 ± 0.5]. This activation, however, was far less than obtained by inhibition of Akt phosphorylation. There was no effect on caspase-8 and caspase-1 activation (data not shown). The inhibition of JNK and p38 signaling did not affect the oxidant-induced caspase activation (data not shown). The comparison of caspase-3 activation on inhibition of Akt phosphorylation and ERK1/2 was better demonstrated by the identity of the active form of caspase-3 as revealed by Western blot analysis using a specific antibody to caspase-3 ( Fig. 14 ). As shown, inhibition of ERK1/2 signaling by the MEK inhibitor U-0126 minimally produced the active form of caspase-3 compared with the inhibition of Akt phosphorylation by LY-294002. Thus in marked contrast to the inhibition of Akt phosphorylation, inhibition of MAP kinase ERK1/2 had a minimal effect on caspase activation.5 X  G. V% [4 C
  c( j* e' X% J
Fig. 13. Time course of H 2 O 2 -induced activation of ERK1/2, JNK, and p38 and the effect of MEK inhibitor U-0126, JNK inhibitor SP-600125, and p38 inhibitor SB-202190 on their phosphorylation. Cells were treated with 200 µM H 2 O 2, and in the presence or absence of 10 µM U-0126, 20 µM SP-600125, and 20 µM SB-202190 for 4 h, respectively, as indicated. Cell lysates (50 µg protein) from each sample were analyzed for ERK1/2 phosphorylation, JNK phosphorylation, and p38 phosphorylation by Western blot analyses using antibodies to P-ERK1/2, P-JNK, and P-p38 and antibodies to their respective unphosphorylated forms. Control cells were not treated. The results shown are representative of 3 independent experiments.
8 e4 H6 `$ N* b
; N$ ?. a, ]# ?7 K% dFig. 14. Time course of proteolytic processing of procaspase-3 to active caspase-3 in response to H 2 O 2 in the presence and absence of LY or U-0126. Cells were treated with 200 µM H 2 O 2 in the presence of LY (10 µM) or U-0126 (10 µM) or caspase-3 inhibitor DEVD-CHO (10 µM) for various time periods as indicated. Cell lysates (100 µg protein) were subjected to Western blot analyses using antibodies specific for caspase-3. Control cells were untreated. The results shown are representative of 3 independent experiments.
- ]; @) B" s* Z2 b/ x- L- O
! \# N" C2 y# l+ y4 {; cDISCUSSION
; R! o( M8 z* y7 [* _! X& k9 a
. l$ V, z4 V! e; g' @0 C# z: q) D& PMany studies have documented that oxidants induce RTE cytotoxicity ( 1, 14, 24, 31, 38, 58 ). We have previously demonstrated that RTE cells transcribe and express multiple caspases, including the initiator and executioner caspases ( 35 ). Further studies from several laboratories including that from ours have shown that caspases play an important role in hypoxic ( 21, 23, 33, 36, 46 ), ischemic ( 15, 41 ), and cisplatin-induced ( 34 ) RTE injury. In the present study, we provide evidence that both the proapoptotic and survival pathways are triggered after exposure to H 2 O 2. In the proapoptotic pathway, H 2 O 2 activates the mitochondrial-mediated intrinsic cell death pathway that results in the mitochondrial release of cytochrome c and subsequent activation of caspase-9 and caspase-3. The proapoptotic pathway is regulated by the survival pathways mediated by forkhead transcription factors, Bcl-2 proteins, and mitogen-activated kinases.. O' G; Q0 y6 H; V& l% `5 f2 p/ T( e4 \1 l

% v! H( ^0 L% p5 u, V( Z4 WOne of the signaling pathways that blocks apoptosis is mediated by the PI 3-kinase/Akt phosphorylation pathway. Akt, a serine/threonine kinase, is a downstream target of PI 3-kinase ( 16, 26, 61 ). However, the role of Akt phosphorylation in determining cell fate in H 2 O 2 -induced injury to renal tubular cells is not known. Our studies in LLC-PK 1 cells show that H 2 O 2 increases the basal activity of Akt phosphorylation, which is inhibited by PI 3-kinase inhibitors LY-294002 or wortmannin, indicating that Akt phosphorylation is dependent on the activation of PI 3-kinase. Several pathways have been proposed for PI-3/Akt phosphorylation-mediated cell survival ( 16 ). One of the best studied molecules that mediate cell survival by Akt phosphorylation is the proapoptotic Bcl-2 family member Bad. Bad has the ability to directly interact with and bind to antiapoptotic Bcl-2 and Bcl-xL and block their survival function ( 30, 64 ). Phosphorylated Akt can phosphorylate Bad ( 30, 63 ) and renders Bad incapable of binding to Bcl-xL and restores the antiapoptotic function of Bcl-2 ( 16, 30 ). We have demonstrated oxidant-induced increased phosphorylation of Bad that was dependent on Akt phosphorylation.6 U. R1 u% {5 R3 m, w

; a3 R( }5 S* T* h5 a" `We have also provided evidence that phosphorylation of FKHR and FKHRL1 is induced by H 2 O 2 and that this phosphorylation is blocked by inhibition of Akt activation. The mechanism by which forkhead proteins promote cell survival or cell death depends on the state of their phosphorylation, such that phosphorylated forms are antiapoptotic whereas unphosphorylated forms are proapoptotic. Forkhead proteins, when phosphorylated, are retained in the cytosol sequestered by 14-3-3 proteins ( 6, 8 ). In the absence of induction of a survival signal, dephosphorylated forkhead proteins translocate to the nucleus and trigger apoptosis most likely by inducing the expression of genes that are critical for cell death ( 6, 26 ). The structural domains of forkhead proteins are known to contain the "Forkhead" DNA-binding domain ( 27 ) and the conserved Akt phosphorylation sites ( 6, 9 ). In hematopoietic cells, overexpression of constitutively active FKHRL1 can induce Bim expression, which results in apoptosis ( 19, 51 ). In RTE, there is no information on oxidant-induced expression of forkhead proteins or forkhead protein-dependent Bim expression. Our studies demonstrate for first time in RTE that Bim protein levels are elevated on inhibition of Akt-mediated phosphorylation of FKHR and FKHRL1. Recently, Andreucci et al. ( 2 ) have shown Akt-mediated phosphorylation of FKHR and FKHRL1 in an ATP depletion model in LLC-PK 1 cells and in ischemia-reperfusion injury in rats. Now, our studies have shown that inhibitors of PI 3-kinase block oxidant-induced phosphorylation of Akt, Bad, FKHR, and FKHRL1 and elevation of Bim. All of these events contribute to enhanced activation of mitochondrial-dependent activation of caspase-9 and caspase-3, as well as cell death. A recent study has shown that Akt can also phosphorylate human caspase-9, resulting in the reduction of caspase-9 activity ( 10 ). In addition, our studies demonstrated that inhibition of PI 3-kinase/Akt phosphorylation triggers early oxidant-induced activation of caspase-3 and caspase-9 as well as enhanced cell death, indicating the possibility that activation of Akt modulates the response to oxidant injury.
2 R; U+ E: \. ^* u  f+ Z' n3 L
( ~! G" M4 m2 H* gBecause MAP kinases ERK1/2, JNK, and p38 are known to play roles in cell death and survival signaling, we examined and compared their role in caspase activation in oxidant injury to RTE. Phosphorylation of ERK1/2, JNK, and p38 was markedly increased with increasing periods of oxidant injury to RTE. The use of specific inhibitors of ERK1/2, JNK, and p38 facilitated the comparison in caspase activation. Inhibition of ERK1/2 but not of JNK and p38 enhanced activation of caspase-9 and caspase-3 in response to oxidant injury. These findings support previous observations that ERK1/2 plays a role in cell survival ( 7, 19 ). Because ERK1/2 is known to phosphorylate Bad at Ser112 ( 16, 26 ), it may also regulate caspase activation through a mitochondrial-dependent pathway." K4 m' K- z" ?; k
8 Z  N% n: X5 m# W2 E
Our data show that oxidative stress not only triggers the intrinsic cell death pathway but also induces the survival pathway mediated by Akt phosphorylation and, to a lesser degree, Erk1/2 signaling. Blocking these survival pathways sensitized RTE to the proapoptotic effects of H 2 O 2, which significantly enhanced cytochrome c release, caspase activation, and cell death. This suggests that the activation of survival pathways in RTE is an attempt to minimize the cell damage induced by the oxidative stress.
3 `. S- q9 [, d9 F; l' U7 g5 L7 E/ T" C& @
The cellular defenses triggered in response to oxidative stress in RTE are not sufficient to completely block the intrinsic cell death pathway, however. In oxidative stress to RTE, the ultimate outcome of the interplay between pro- and antiapoptotic signaling remains tilted toward cell death. This effect may be due to activation of several proapoptotic responses that are not antagonized by the active survival pathways. For example, the survival response mediated by Akt phosphorylation inactivates selective proapoptotic molecules such as Bad and Bim but not other proapoptotic members of the Bcl-2 family, including Bax, Bak puma, and noxa ( 48, 59 ). Furthermore, oxidative stress elevates p53, which transcriptionally controls the expression of Bax, puma, and noxa ( 11, 13, 39 ). Bax induces mitochondrial dysfunction and activates the intrinsic pathway either by forming a pore by oligomerization in the outer mitochondrial membrane or by opening other membrane channels ( 37, 47, 50 ). A recent study shows that p53 directly activates Bax in the absence of other proteins to permeabilize mitochondria and engage in the intrinsic cell death program ( 12 ). p53 Has been reported to directly bind Bcl-xL and Bcl-2, inactivating their antiapoptotic function and promoting cytochrome c release ( 42 ). In addition, H 2 O 2 may directly trigger mitochondrial membrane permeabilization, contributing to cytochrome c release ( 40, 54, 55 ). Thus in the final outcome, proapototic signals induced by oxidative damage exceed the capability of cellular survival signals.
) g9 Y/ _" Q7 ^7 a+ x* J
# v$ E; O9 j, r; m' b" V4 G1 O3 c0 J/ @: K, sIn summary, our data clearly demonstrate that H 2 O 2 not only triggers caspase activation but also causes induction of the PI 3-kinase/Akt and ERK signaling pathways, providing a beneficial response for cell survival. These observations have not been previously recognized in RTE injury. Another adaptive and protective response against oxidative stress has been recently provided for heme oxygenase ( 32, 45 ). Thus our studies provide additional targets to modulate oxidant-induced injury to RTE. It is possible that enhancing the renal-specific cell survival signals and inhibiting death signals will lead to preservation of renal function in renal injury.0 K# u; j! b0 B
; {- _4 x0 w9 J" s
GRANTS( Y2 O& @9 \! o. s- f# S7 m+ Y
8 p3 R% K3 A0 A# T
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58324 and American Heart Assocation (Affiliate) grants to G. P. Kaushal.
+ x. |/ j: @" N# [, R" T1 A* @; n* |% o8 I% b  @
ACKNOWLEDGMENTS/ b9 D- d! v& p6 J/ f% _

' ~. w7 Z' K( G6 nThe authors thank Dr. Randy Haun for critically reviewing the manuscript, Dr. Didier Portilla for valuable discussions, and Judy Nagle for secretarial assistance. We also thank the Office of Grants and Scientific Publications at the University of Arkansas for Medical Sciences for editorial assistance during the preparation of this manuscript.8 T' C! N5 V4 S
          【参考文献】1 Q' ]  k: z7 `. b% e
Andreoli SP and Mallett CP. Disassociation of oxidant-induced ATP depletion and DNA damage from early cytotoxicity in LLC-PK 1 cells. Am J Physiol Renal Physiol 272: F729-F735, 1997.
" t" N# i) c# a+ y
! W) A3 X" g- J0 C' o
' x- M' a' c% b/ |; m: f. N$ ]5 G3 k' o2 G- ^
Andreucci M, Michael A, Kramers C, Park KM, Chen A, Matthaeus T, Alessandrini A, Haq S, Force T, and Bonventre JV. Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK 1 cells result in phosphorylation of FKHR and FKHRL1. Kidney Int 64: 1189-1198, 2003.8 k1 y  G. F6 v; A- r+ s
& o8 s; W+ A, [
1 [9 s, V; ^5 n  D) M

  ^+ A3 k' W* b8 e: L1 {/ s, ^Arnoult D, Parone P, Martinou JC, Antonsson B, Estaquier J, and Ameisen JC. Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli. J Cell Biol 159: 923-929, 2002., S2 Z; v8 B5 v/ C9 J+ d: l0 b+ _

* v/ ~6 b( v, m. U; m5 P; k4 l& M
7 s' i% n+ T! o! r( \# e
: M  `0 m6 t. Z" ]' |Baliga R, Ueda N, Walker PD, and Shah SV. Oxidant mechanisms in toxic acute renal failure. Am J Kidney Dis 29: 465-477, 1997., s6 H/ ~; Y0 C* f5 F: O

0 Y! ]9 j% C& u0 N) [$ a0 L; r" j3 o$ S' h: {# r0 y+ Q
* g- _- l1 U% s8 a+ g" g- C* B
Basnakian AG, Kaushal GP, and Shah SV. Apoptotic pathways of oxidative damage to renal tubular epithelial cells. Antioxid Redox Signal 4: 915-924, 2002.
# e- L5 |0 g5 {9 e) D* z6 ~( D$ f' N+ A; y/ z
2 `! \3 z& L1 I9 [& T. _

$ l- I2 k3 W& A5 |$ ]" R1 jBirkenkamp KU and Coffer PJ. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem Soc Trans 31: 292-297, 2003.
* c2 U* a4 N5 z0 P! A: }( J% v% N" U. _; S8 z
4 t! ]. J# T8 P  z5 t  s% }
4 _$ a- Z% I* X' \
Bonni A, Brunet A, West AE, Datta SR, Takasu MA, and Greenberg ME. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286: 1358-1362, 1999.
) C2 D0 y) f$ u6 \; A6 ]$ a: ^1 ^+ E
% X! `0 k9 k: v) R. A3 l5 Q2 r
. g- p, F: X0 `; s$ [; o( E
Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, and Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857-868, 1999.3 a( G3 e1 y* e, R! n, j
1 f; f) W& }& M6 Z+ \, e* M
' }' f6 c7 w: s7 o* R( N) a

7 D  V! m7 ^( O% {$ x0 b2 ]$ kBurgering BM and Kops GJ. Cell cycle and death control: long live Forkheads. Trends Biochem Sci 27: 352-360, 2002.
! h9 ~! U, i8 a9 X
* d. A  |. M+ r+ F8 `& o% }& x' d4 x4 h$ W/ z3 O! s

" y& D- w/ [/ ?" q% E& ACardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, and Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science 282: 1318-1321, 1998.$ l3 H; J3 B; u' n2 P* |: P

  I5 i, g2 d. {1 l0 X; T6 T& _8 _+ `
! Q8 t, s7 q0 x1 b' F/ J
Chen K, Albano A, Ho A, and Keaney JF Jr. Activation of p53 by oxidative stress involves platelet-derived growth factor- receptor-mediated ataxia telangiectasia mutated (ATM) kinase activation. J Biol Chem 278: 39527-39533, 2003.
- t) d/ i# _8 \2 q: |( a. a7 h" O# @% C  m/ C5 x! i4 k

' S! o4 Z9 s. Z* w% @5 L! \  P, d8 m6 i$ Y2 g# n
Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, and Green DR. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303: 1010-1014, 2004.
- c- T, r6 w+ e: r7 U2 O3 y# Y# x$ d- \
, x# J6 Q$ n- A6 D. X8 C

% l9 }4 h2 i7 F6 c0 V' `Cregan SP, MacLaurin JG, Craig CG, Robertson GS, Nicholson DW, Park DS, and Slack RS. Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons. J Neurosci 19: 7860-7869, 1999.+ Z/ W3 A9 N2 j
& i: a% L) t9 K
0 Y5 A$ l# X# i1 S6 q6 W
* p/ {" C, |( z( Q
Cuttle L, Zhang XJ, Endre ZH, Winterford C, and Gobe GC. Bcl-xL translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress. Kidney Int 59: 1779-1788, 2001.9 P6 m. Q0 B7 _" \1 l7 f
# W2 l$ ]: b3 z6 p, M, s2 G
  N- e6 c6 {9 R/ G9 P0 O

8 ~. r, p7 G: M3 z8 n3 l+ [/ z0 LDaemen MA, van 't Veer C, Denecker G, Heemskerk VH, Wolfs TG, Clauss M, Vandenabeele P, and Buurman WA. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin Invest 104: 541-549, 1999.1 b+ B$ e" E% D- N  q/ Q. Z& y. n( J# I

, |6 M! ^6 ^/ t6 c9 w# u, G
; `0 `! `8 t6 w1 w
" I+ {) |8 H% e0 g) v2 bDatta SR, Brunet A, and Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 13: 2905-2927, 1999.5 R& N" x5 ]# m+ C
- B" n3 _  J: `' ?7 U6 Q
) W: Q5 f4 x& p. z3 h

. K9 F2 m6 }: A" M6 y3 TDesagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, and Martinou JC. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 144: 891-901, 1999.
! D) V2 l8 B9 c9 F# D9 W
' I1 {7 A) H4 _! o6 o/ W  g* e! U
8 u2 I/ U! A6 |$ J
Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Lammers JW, Koenderman L, and Coffer PJ. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. J Cell Biol 156: 531-542, 2002.. _: ]3 x6 `9 ?0 v
3 \: b+ ?- F) l, g5 n7 ^
5 I) s( W) d, G! C. [, c$ H- W

0 e& j: a* L5 g" Y. dDi Mari JF, Davis R, and Safirstein RL. MAPK activation determines renal epithelial cell survival during oxidative injury. Am J Physiol Renal Physiol 277: F195-F203, 1999.
- ]  c$ @& |* ^0 s4 z# k' `
" ], x7 n/ P, n" G  k
8 w" y3 `7 m, K: K9 d) l; j/ {# C; d1 z7 x& f( `
Earnshaw WC, Martins LM, and Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383-424, 1999.0 U1 i% X1 H5 d- o& U3 O; t! v1 K

- w3 z1 \& G; q+ p! \! {8 e( }; p" _, e! J- ^0 I+ h$ I# i

6 \  {% y' o5 GEdelstein CL, Shi Y, and Schrier RW. Role of caspases in hypoxia-induced necrosis of rat renal proximal tubules. J Am Soc Nephrol 10: 1940-1949, 1999.5 U' z9 D. N# B9 t: A' L

* [2 H! D2 ^9 v% H, B" Y
, U  S6 R; H# T; E7 P
4 R' Q) |1 s. CFaleiro L, Kobayashi R, Fearnhead H, and Lazebnik Y. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J 16: 2271-2281, 1997.9 i4 Q1 F" v, c! X  J1 H
( U( }7 H3 N0 m  K' w; g- E; O

. Z8 V/ W( k1 b2 s2 ^3 I' T# W$ ?; y0 V/ K- X# p* |; _6 f
Feldenberg LR, Thevananther S, del Rio M, de Leon M, and Devarajan P. Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells. Am J Physiol Renal Physiol 276: F837-F846, 1999.
: x  f  c2 v2 m* l% o
+ u3 S' P( c- E  B
* o4 H" q* ^8 L, j2 X5 [( |- D' U( \  U
Filipovic DM, Meng X, and Reeves WB. Inhibition of PARP prevents oxidant-induced necrosis but not apoptosis in LLC-PK 1 cells. Am J Physiol Renal Physiol 277: F428-F436, 1999.0 T+ F  G' \( s4 T! q: K

7 k; ?% W; A6 v7 v% a
0 f! V* y. x8 A% e3 o0 X& ~
6 Y/ S- Q& ^1 [: H5 \. R+ hFleury C, Mignotte B, and Vayssiere JL. Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84: 131-141, 2002.
* ^. {8 b" [! P% {: G. E1 ^) G7 Y, Q2 W
. E& ]6 _8 `, J

6 D" a6 n' g7 d: {  x. i" a1 TFranke TF, Kaplan DR, and Cantley LC. PI3K: downstream AKTion blocks apoptosis. Cell 88: 435-437, 1997.5 h3 {' X9 U! f, i0 a- M* s

! V, R: t: v6 P) M, O5 |
) D- f7 [7 M, c2 q4 O
1 Z4 {3 _4 \6 B& c$ G- VFuruyama T, Nakazawa T, Nakano I, and Mori N. Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. Biochem J 349: 629-634, 2000.
) a( X; ~, H5 H2 t; M5 D
5 x6 c( j8 p7 A( i1 s, E9 O3 W+ F4 R# \! J8 U7 ^1 Q

2 z$ A1 V" ?( G" S, yGilley J, Coffer PJ, and Ham J. FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons. J Cell Biol 162: 613-622, 2003.7 Q7 d3 C% [7 I, [

$ N! g( w- j- \4 v
+ c) ^5 b: @" A' z7 \
) m' h3 h$ K7 S5 l) d* y2 @Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1312, 1998.
9 O: j1 X8 Z  S* ^# F) a3 U) V" Y( n1 B5 A3 ^5 B" j( {- V3 I% p4 _
, e/ w+ h" h7 {5 e

* P1 }2 Y, y- nGross A, McDonnell JM, and Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13: 1899-1911, 1999.
, S; N% j' a8 Q
: ~/ `2 q9 l* l1 p9 e; G
: H0 V' J* D, U) V* l; o+ s& p+ T$ H
& x* `6 x' a) i2 pHagar H, Ueda N, and Shah SV. Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK 1 cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F209-F215, 1996.5 b/ T/ G5 O8 L9 M- a3 @
+ O, z- u- v, X' U; Z

. Y' N0 Z. `& v0 u* e8 ], y1 z1 m, z7 E. }1 w1 h1 i
Hill-Kapturczak N, Chang SH, and Agarwal A. Heme oxygenase and the kidney. DNA Cell Biol 21: 307-321, 2002.
1 \# |# d  h6 v! h4 ~9 i
/ d' L( P" |$ O/ O8 [( L' z" i8 H, E' S& e! a+ i

) _0 [2 \) [; z& U4 VKaushal GP. Role of caspases in renal tubular epithelial cell injury. Semin Nephrol 23: 425-431, 2003.1 M" Y* S. ]0 |/ [

4 Q/ _/ M+ [6 x+ |3 b. |
% ~0 z1 \* k! E6 P* [2 {7 S
/ H5 p0 h9 P" J6 C$ g* ~Kaushal GP, Kaushal V, Hong X, and Shah SV. Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells. Kidney Int 60: 1726-1736, 2001.* x' S7 @7 L6 B6 H! J& m: G) z

4 x4 L6 @* O, a5 A0 c2 v4 e6 i% j" n. S( D: d2 Q

6 `4 @  P, t8 v% E/ XKaushal GP, Singh AB, and Shah SV. Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury. Am J Physiol Renal Physiol 274: F587-F595, 1998.
. d: p, g' X1 C. o2 ]! _, K& h$ \- L- m' _+ [; U

& |, s& ~$ W: A" C8 i% k2 M. N- V' ?
Kaushal GP, Ueda N, and Shah SV. Role of caspases (ICE/CED 3 proteases) in DNA damage and cell death in response to a mitochondrial inhibitor, antimycin A. Kidney Int 52: 438-445, 1997.9 v- T' {* o9 p7 p$ h% h" K

/ W+ L, R2 f' v  A" t6 ~8 r" W8 h% }9 y$ k  F

  I5 Y' n' j, u0 VKuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R, Green DR, and Newmeyer DD. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111: 331-342, 2002.
4 ^* M0 c8 L0 R0 T" s
( j7 y; o# |0 q! k9 i6 V! L
( \% i4 {( U' K8 m6 H- h8 m8 {( M/ c8 n# L
Lieberthal W, Triaca V, Koh JS, Pagano PJ, and Levine JS. Role of superoxide in apoptosis induced by growth factor withdrawal. Am J Physiol Renal Physiol 275: F691-F702, 1998./ ]' r# ]8 Y1 F" t
5 a; X" y. R( j6 k& @; T

# N) w: m3 a7 S" s( ~9 Y/ F! p8 J- N+ _
Macip S, Igarashi M, Berggren P, Yu J, Lee SW, and Aaronson SA. Influence of induced reactive oxygen species in p53-mediated cell fate decisions. Mol Cell Biol 23: 8576-8585, 2003.
, Y1 T6 D1 z5 x1 Q. S4 L/ W' j  X& Y2 m, k9 K% r) N2 S

) `% o! @* Q7 B+ ?/ t# R( Q( o0 B; z! O9 T8 p# o+ x7 s
Martindale JL and Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 192: 1-15, 2002.; V1 r* X* g( [4 l  _, x% c* E
# x7 X3 p6 N' l9 h" W) e. C

) ^  v6 q' ^1 M. y( k% ~: x7 ?) I/ f% |  d, ]
Melnikov VY, Faubel S, Siegmund B, Lucia MS, Ljubanovic D, and Edelstein CL. Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice. J Clin Invest 110: 1083-1091, 2002.- ^0 N5 A+ {9 |
0 D5 G5 t+ @& a) a, [. n

$ ?1 ]7 T! m" l8 p' D6 e3 J* G" M6 X. j
Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, and Moll UM. p53 Has a direct apoptogenic role at the mitochondria. Mol Cell 11: 577-590, 2003.2 e- N# T; j9 H9 r% m
8 a2 V6 g1 e0 y) _. C! @

/ G8 v! m) \; R$ U+ j. S, h6 g6 {0 j) l3 w. i
Nath KA and Norby SM. Reactive oxygen species and acute renal failure. Am J Med 109: 665-678, 2000.
* `# K2 g) F7 R, y/ N# b$ ~- J
' U5 Y! N: t0 L# ^5 [- w
9 [3 c8 u: P+ @
1 V- {( e: C( q$ f" T' C" @* |Ortiz A, Lorz C, Justo P, Catalan MP, and Egido J. Contribution of apoptotic cell death to renal injury. J Cell Mol Med 5: 18-32, 2001.& t% T+ M# f/ i# I) _
1 ?# G9 a( {' S2 l5 N  ?5 _) I/ {- u
  o* Y3 j" ~( Q! q5 [
6 @3 }& t" O- u8 Q  D- |, Q! ]' o: m
Platt JL and Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med 4: 1364-1365, 1998.
& Z% o2 x* d" R: U9 t. O$ H; @) t  K

/ B% F2 k  I/ [5 a& w: f# T: A: u* D8 i( K
Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM, and Venkatachalam MA. Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17: 3401-3415, 1998.) g: ?, M9 K5 o9 v: ~4 q. L

. k  G% y$ L! @. A/ I( l7 f2 E( l1 I# I( U' D$ j

  t  }# ?+ d% L2 l# w% ISaito M, Korsmeyer SJ, and Schlesinger PH. BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol 2: 553-555, 2000.; a( w! I) x1 d0 H! W# u( S' T  ?
9 Z; N3 }4 M8 X! T/ F* }

$ o5 m! H; d4 g7 Q* {+ N9 h0 @4 b2 j! f7 M  z. c  Q+ O2 r. |3 J
Scorrano L and Korsmeyer SJ. Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Biophys Res Commun 304: 437-444, 2003.( K0 P  m+ d; [5 X& s- Q

5 J- N5 z- K% H: [; j3 e
; y/ j  Y( o' f) n' p* B- s, B! k; V
Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9: 459-470, 2002.
! X) @) h. @+ F- N* e$ G  [' Z( T# s

: ^: Y6 U. D1 ^) ^
4 W1 f4 L6 R3 HShimizu S, Narita M, and Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399: 483-487, 1999.
& y$ G3 k% {! S, A; n- Q
* e) B. X: {: C  u: j; x( k; d4 f* M* K" N/ w0 {0 k' ?( a* W5 C( A
5 {2 D# B, n  d
Stahl M, Dijkers PF, Kops GJ, Lens SM, Coffer PJ, Burgering BM, and Medema RH. The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2. J Immunol 168: 5024-5031, 2002.% ^' U8 l" W, @! l
" w0 O2 D/ u" g* _1 S. v: n3 f  x

+ \. @# Q( I% C
6 ?: L% m4 l& @3 [+ _4 rStennicke HR and Salvesen GS. Caspase assays. Methods Enzymol 322: 91-100, 2000.0 M, y8 k9 h9 x

  o9 d/ W& x5 @8 r0 H! c5 b3 w5 O& C: m  C& [

/ _' ]: K' G' J. O1 y' R9 I2 O- VStrasser A, O'Connor L, and Dixit VM. Apoptosis signaling. Annu Rev Biochem 69: 217-245, 2000.
) f: c4 j3 E3 W  F
5 S! f+ o4 |) s7 l. S. z0 U7 |! ?* h- y
% n+ S% J% I8 B. k5 s
Stridh H, Kimland M, Jones DP, Orrenius S, and Hampton MB. Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis. FEBS Lett 429: 351-355, 1998.$ q! \2 O7 {, ^

0 _- e- o1 F1 d0 l$ b- U% M& i$ H2 z0 u: e1 P* _5 Z$ a
3 Y' C" q, y2 }& T- U7 _
Takeyama N, Miki S, Hirakawa A, and Tanaka T. Role of the mitochondrial permeability transition and cytochrome c release in hydrogen peroxide-induced apoptosis. Exp Cell Res 274: 16-24, 2002., B4 n/ e3 E4 q3 [8 Z

% K& O5 F; Q  o6 v
( X' p5 Y# `) t9 V3 @# g; L. [% u8 `5 ^* X0 P
Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979.
0 T9 h& q' h7 i- ~% O, m- w3 Z7 V; f- r% T- q

' l. X1 T4 e) c+ {- ]+ w7 a. {
! j: ?( \  W/ M5 W* ~2 Z3 M0 GUeda N, Kaushal GP, and Shah SV. Apoptotic mechanisms in acute renal failure. Am J Med 108: 403-415, 2000.4 W! P  P5 \7 f

" u6 A; @: N. L" E+ h# A4 [& t7 {% T4 j, ?5 j7 q3 b3 {

; }- N+ Q0 p: r7 HUeda N and Shah SV. Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J Clin Invest 90: 2593-2597, 1992.
; F* r$ w' S( y- c! I
8 L. ]2 G% Y& t5 Z$ Z6 J
* e0 a6 s! q; l4 t, H9 o
, V$ X8 v& v) u8 D" Q8 o: TVon Harsdorf R, Li PF, and Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation 99: 2934-2941, 1999.
& A) v7 X/ ]( J+ H7 y3 K/ R$ H& K* d) ?: H% |# _) `, i6 |

* }- \6 r: m# ~) h9 s1 z6 V) g
( ^! r; y( A& _' H* O2 ?Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, and Reed JC. Ca 2  -induced apoptosis through calcineurin dephosphorylation of BAD. Science 284: 339-343, 1999.' i  C% ]' |) Z3 s) _

* X) V1 S0 q. g" e; W  E  o6 Y# r5 w0 [

( r* {# n" y! XWang X, McCullough KD, Franke TF, and Holbrook NJ. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 275: 14624-14631, 2000.
: q) q$ q, D* F7 L( N4 P" S  K9 K
4 ^( q+ [. i- X: N  z6 e
- |0 R7 M) W1 Q6 {" F" Q& {; m
" Y% d5 H; {2 e) x9 @& BWolf BB and Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 274: 20049-20052, 1999.' t5 g4 L! Z3 J$ F0 l9 K/ K

. x( {6 a" c" K% W. ^5 w$ u! P- s8 U

2 W! z2 ^% O2 Q# n  X1 S: K2 sYang E, Zha J, Jockel J, Boise LH, Thompson CB, and Korsmeyer SJ. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80: 285-291, 1995.( V% M9 D! p8 q

, K* J, p' X' `. V* T( C
/ h" u5 u" z3 T; H2 L# i2 ~! l/ Y5 Q, h, K" U* M
Zha J, Harada H, Yang E, Jockel J, and Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL. Cell 87: 619-628, 1996.

Rank: 2

积分
116 
威望
116  
包包
1263  
发表于 2015-6-1 18:00 |显示全部帖子
真是汗啊  我的家财好少啊  加油  

Rank: 2

积分
72 
威望
72  
包包
1291  
发表于 2015-6-23 12:13 |显示全部帖子
希瑞干细胞
呵呵,明白了  
佰通生物

Rank: 2

积分
97 
威望
97  
包包
1170  
发表于 2015-7-1 13:27 |显示全部帖子
干细胞之家微信公众号
加油啊!!!!顶哦!!!!!  

Rank: 2

积分
69 
威望
69  
包包
1219  
发表于 2015-7-29 11:28 |显示全部帖子
谢谢干细胞之家提供资料

Rank: 2

积分
161 
威望
161  
包包
1292  
发表于 2015-8-2 15:39 |显示全部帖子
世界上那些最容易的事情中,拖延时间最不费力。  

Rank: 2

积分
162 
威望
162  
包包
1154  
发表于 2015-8-5 13:01 |显示全部帖子
胚胎干细胞

Rank: 2

积分
64 
威望
64  
包包
1200  
发表于 2015-8-13 14:27 |显示全部帖子
羊水干细胞

Rank: 2

积分
162 
威望
162  
包包
1178  
发表于 2015-8-23 04:58 |显示全部帖子
今天临床的资料更新很多呀

Rank: 2

积分
64 
威望
64  
包包
1165  
发表于 2015-8-25 21:35 |显示全部帖子
我卷了~~~~~~~  
你需要登录后才可以回帖 登录 | 注册
验证问答 换一个

Archiver|干细胞之家 ( 吉ICP备13001605号 )

GMT+8, 2018-9-25 20:07

Powered by Discuz! X1.5

© 2001-2010 Comsenz Inc.