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The small intestine is highly sensitive to ischaemia–reperfusion (I/R) induced injury which is associated with high morbidity and mortality. Apoptosis, or programmed cell death, is a major mode of cell death occurring during I/R induced injury. However, the mechanisms by which I/R cause apoptosis in the small intestine are poorly understood. p53 upregulated modulator of apoptosis (PUMA) is a p53 downstream target and a member of the BH3‐only group of Bcl‐2 family proteins. It has been shown that PUMA plays an essential role in apoptosis induced by a variety of stimuli in different tissues through a mitochondrial pathway.
The role of PUMA in I/R induced injury and apoptosis in the small intestine was investigated. The mechanisms by which PUMA is regulated in I/R induced intestinal apoptosis were also studied.
Ischaemia was induced by superior mesenteric artery occlusion in the mouse small intestine. Induction of PUMA in response to ischaemia alone, or ischaemia followed by reperfusion (I/R), was examined. I/R induced intestinal apoptosis and injury were compared between PUMA knockout and wild‐type mice. The mechanisms of I/R induced and PUMA mediated apoptosis were investigated through analysis of caspase activation, cytosolic release of mitochondrial cytochrome c and alterations of the proapoptotic Bcl‐2 family proteins Bax and Bak. To determine whether PUMA is induced by reactive oxygen species and/or reactive nitrogen species generated by I/R, superoxide dismutase (SOD) and N‐nitro‐L‐arginine methyl ester (L‐NAME) were used to treat animals before I/R. To determine whether p53 is involved in regulating PUMA during I/R induced apoptosis, PUMA induction and apoptosis in response to I/R were examined in p53 knockout mice.
PUMA was markedly induced following I/R in the mucosa of the mouse small intestine. I/R induced intestinal apoptosis was significantly attenuated in PUMA knockout mice compared with that in wild‐type mice. I/R induced caspase 3 activation, cytochrome c release, Bax mitochondrial translocation and Bak multimerisation were also inhibited in PUMA knockout mice. SOD or L‐NAME significantly blunted I/R induced PUMA expression and apoptosis. Furthermore, I/R induced PUMA expression and apoptosis in the small intestine were not affected in the p53 knockout mice.
Our data demonstrated that PUMA is activated by oxidative stress in response to I/R to promote p53 independent apoptosis in the small intestine through the mitochondrial pathway. Inhibition of PUMA is potentially useful for protecting against I/R induced intestinal injury and apoptosis.
Small intestinal epithelium normally renews every three and a half days with differentiated cells at the tip of villi removed by apoptosis, a physiological process for eliminating unwanted or damaged cells.1,2 This rapid and relatively well characterised renewal process makes the small intestinal epithelium an excellent system to study how tissue homeostasis is achieved in vivo through the balance of cell birth and cell death.3,4,5 Apoptosis has also been implicated in tissue damage under a number of pathological conditions, such as ischaemia–reperfusion (I/R) induced injury in the small intestine, brain, myocardium and liver.6,7 Recent evidence suggests that apoptosis is a major mode of cell death caused by I/R in these tissues.7,8,9,10,11 The small intestine is one of the internal organs that are most sensitive to I/R induced apoptosis.12 However, the molecular determinants of this sensitivity remain largely unknown.
It is thought that the initial ischaemic insult induces tissue injury through decreased oxygen and nutrients. Reperfusion of the ischaemic tissues exacerbates tissue damage, in part due to oxidative stress and free radicals. After intestinal I/R, oxygen free radicals are generated both in the mucosa and in the lumen. Two candidates for early mediators of I/R injury include reactive oxygen species (ROS) and inducible nitric oxide synthase derived reactive nitrogen species (RNS).13,14 It has been suggested that ROS and RNS are involved in I/R induced apoptosis which can be suppressed by antioxidants.15,16,17,18
Apoptosis in mammalian cells is regulated by the Bcl‐2 family members which are evolutionally conserved proteins that mediate apoptosis through the mitochondria.19,20,21,22 p53 upregulated modulator of apoptosis (PUMA) was identified as a BH3‐only Bcl‐2 family protein that plays an essential role in p53 dependent and independent apoptosis in several tissues and cell types.23,24,25,26,27,28,29PUMA functions through other Bcl‐2 family members, such as Bax, Bcl‐2 and Bcl‐xL, to induce mitochondrial dysfunction and caspase activation.23,25,27 Deletion of PUMA in human cancer cells attenuated the apoptotic response to p53, DNA damaging agents and hypoxia.25PUMA knockout mice recapitulated several major apoptotic phenotypes observed in p53 knockout mice, including deficiencies in apoptosis induced by gamma irradiation in thymocytes and developing neurons, that were induced by oncogenes in embryonic fibroblasts, and that were induced by cytokine withdrawal in haematopoietic cells.26,28PUMA also mediates p53 independent apoptosis induced by the glucocorticoid dexamethasone, the kinase inhibitor staurosporine or phorbol esters in lymphocytes.26,28 Although the function of PUMA in p53 dependent apoptosis has been extensively studied, the mechanisms of p53 independent apoptosis mediated by PUMA are poorly understood.23,24,27,30 Furthermore, the role of PUMA in regulating in vivo apoptosis in the small intestine has not been established.
In this study, we used an acute I/R model to study the role of PUMA in I/R induced apoptosis in the mouse small intestine as well as the underlying mechanisms. We found that PUMA was induced by I/R through oxidative stress in a p53 independent manner. Targeted deletion of PUMA attenuated I/R induced apoptosis and tissue injury in the small intestine. These results revealed an important role of PUMA in I/R induced apoptosis and suggested that inhibition of PUMA might be useful for protecting the small intestine against I/R associated injury.
The procedures for all animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh, Pennsylvania, USA. Eight to 10 week‐old male mice (20–25 g) were used for all experiments. PUMA+/− mice in a mixed background of 129 and C57BL/6 were backcrossed to C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine, USA) for three generations (F3) and then used to generate PUMA+/+ and PUMA−/− littermates. p53−/− and p53+/+ mice on the C57BL/6 background were purchased from Jackson Laboratory. The mice were housed in micro isolator cages in a room illuminated from 7:00am to 7:00pm (12:12 h light–dark cycle), and allowed free access to water and food. The mice were genotyped by polymerase chain reaction (PCR) using genomic DNA extracted from tail snips. The wild‐type PUMA allele was identified using the sense primer 5′‐TTA TAG CCG CTG AGT CAG CA‐3′ and the antisense primer 5′‐CAG GCA GTT GTC AGC TGG G‐3′. The mutant PUMA allele was detected using the sense primer 5′‐TTG ACG ATT TCT TCT GAG GG‐3′ and the same antisense primer for the wild‐type allele.26 The wild‐type p53 allele was identified using the sense primer 5′‐CCC GAG TAT CTG GAA GAC AG‐3′ and the antisense primer 5′‐ATA GGT CGG CGG TTC AT‐3′. The mutant p53 allele was detected using the sense primer 5′‐AGG TGA GAT GAC AGG AGA TC‐3′ and the antisense primer specific for the neomycin resistant gene 5′‐CTT GGG TGG AGA GGC TAT TC‐3′.
All animal surgeries were performed in the morning (9:00–12:00am). Before surgery, the mice were allowed access to water and chow ad libitum. For surgery, the mice were anaesthetised for 3 h by intraperitoneal injection of 4% chloral hydrate (200 mg/kg). A laparotomy was performed under anaesthesia. The superior mesenteric artery was occluded for 60 min with a micro‐bulldog clamp, as previously described.17,18,31 Intestinal ischaemia was confirmed by the paleness of the jejunum and ileum. After 60 min of ischaemia, the clamp was released and three drops of lidocaine were applied directly onto the superior mesenteric artery to facilitate 60 min of reperfusion.17,18,31 In sham operated mice, the superior mesenteric artery was isolated but not occluded. To inhibit the generation of total ROS, intraperitoneal injection of 15000 U/kg bovine liver superoxide dismutase (SOD; Sigma, St Louis, Missouri, USA) was performed 30 min prior to ischaemia, as previously described.32 To inhibit RNS, intraperitoneal injection of 30 mg/kg of N‐nitro‐L‐arginine methyl ester (L‐NAME; Sigma) was performed 30 min prior to ischaemia, as previously described.17
Immediately after the treatment, the entire small intestines from randomly selected animals from each group were carefully isolated and placed on ice. The oral 5 cm segments (duodenum) were removed, and the rest of the intestines were divided into two equal segments, representing the proximal (jejunum) and distal (ileum) segments. The jejunal segments were rinsed thoroughly with ice cold physiological saline and subjected to one of the following treatments, as described previously17,18: (1) to prepare cell scraping, the jejunal segments of six mice from each group were opened longitudinally on the antimesenteric border to expose the intestinal mucosa. The mucosal layers were harvested by gentle scraping with a glass slide. The scraping samples were stored at −80°C before the analysis; (2) to prepare paraffin sections, the middle one third sections of jejunum of three mice from each group were carefully isolated and immediately fixed in 10% neutral buffered formalin before embedding; (3) to prepare frozen sections, the middle one third sections of jejunum of three mice from each group were isolated, immediately embedded in OCT compound (Tissue‐Tek, Torrance, California, USA) and then stored at −80°C before the analysis.
The amount of fragmented DNA was determined as previously described.17,18 In brief, intestinal mucosal scrapings were homogenised in a lysis buffer consisting of 5 mM Tris‐HCl (pH 8.0), 20 mM EDTA (Sigma) and 0.5% (w/vol) Triton X‐100 (Sigma). Aliquots of homogenate (1 ml each) from each sample were centrifuged at 18000 g for 20 min at 4°C to separate the intact chromatin (pellet) from the fragmented DNA (supernatant). The supernatant was decanted and saved, and the pellet was resuspended in 1 ml of buffer consisting of 10 mM Tris‐HCl (pH 8.0) and 1 mM EDTA. The DNA contents of the pellet and supernatant fractions were assayed by diphenylamine reaction. Absorbance at 595 nm was measured by a Victor III (Perkin Elmer/Wallace, Boston, Massachusetts, USA) plate reader. Each experiment was performed in triplicate and the results are expressed as the percentage of fragmented DNA divided by total DNA. Six mice in each group were studied.
Total DNA from 0.4 ml of the homogenate was extracted by a phenol/chloroform/isoamyl alcohol mixture (25:24:1, vol:vol:vol) to remove proteins, and then purified as previously described.17,18 Total DNA (10 μg) was resolved by electrophoresis on 1.5% agarose gels containing 1.0 μg/ml ethidium bromide. Electrophoresis was performed for 2 h at 70 V, and the DNA was visualised under ultraviolet light illumination.
For histological and terminal deoxynucleotidyl transferase mediated deoxyuridinetriphosphate nick end labelling (TUNEL) analysis, formalin fixed tissues were embedded in paraffin and sectioned. The 4 μm sections were stained by haematoxylin and eosin. TUNEL staining was performed using an Apop Tag Kit (Chemicon International, Temecula, California, USA) according to the manufacturer's instructions. The specimens were counterstained by nuclear fast red (Vector, Burlingame, California, USA). The apoptotic index (%) was determined by dividing the number of apoptotic cells by the total number of cells in the epithelium of at least 20 randomly selected villi and crypts. Three mice from each group were studied.
The frozen tissues from three randomly selected animals from each group were used to prepare sections. For PUMA immunohistochemical staining, the 10 μm frozen sections were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min and then permeabilised with 1% Triton X‐100 for 30 min, followed by three PBS washes. PUMA was detected using a rabbit polyclonal anti‐PUMA antibody (1:100; Abcam Cambridge, Massachusetts, USA) followed by detection using the ABC staining system (Vector), and sections were counterstained with nuclear fast red (Vector). For caspase 3 immunofluorescence staining, active caspase 3 was detected by a rabbit polyclonal antibody (1:200; BD Biosciences, Franklin Lakes, New Jersey, USA). Antibody–antigen complexes were visualised by incubation with biotin conjugated secondary antibody and streptavidin Alexa 488 (Molecular Probes, Eugene, Oregon, USA), with the nuclei counterstained with 2 μg/ml of 4',6‐diamidino‐2‐phenylindole dihydrochloride (Molecular Probes).
For analysis of caspase 3 activity, an aliquot of each mucosal scraping sample was placed in 5 ml of ice cold PBS (pH 7.4). After centrifugation (500 g for 10 min), the supernatant was removed and the cell pellet was homogenised in 2 ml of lysis buffer consisting of 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 1 mM DTT and 0.1 mM EDTA. Cell lysates were centrifuged at 10000 g for 10 min at 4°C. Aliquots of the supernatant were used to determine protein concentration and caspase 3 activity. Protein concentration was measured using a kit (Bio‐Rad, Hercules, California, USA). Caspase 3 activity was measured using a colorimetric assay kit (Calbiochem, La Jolla, California, USA) according to the manufacturer's instructions. In brief, 100 μg of the protein extracts were incubated in a caspase 3 assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 0.1 mM EDTA and 10% glycerol) containing 0.2 mM of caspase 3 substrate. The absorbance at 405 nm was measured by a Victor III (Perkin Elmer/Wallace) plate reader. Six mice in each group were studied. Each experiment was performed in triplicate and repeated at least twice. The results were expressed as absorbance/mg of protein.
To analyse Bax/Bak translocation, an aliquot of each intestinal mucosal scraping sample was used to isolate mitochondrial and cytosolic fractions by the differential centrifugation method previously described.25 Briefly, the samples were washed with ice cold PBS and resuspended in homogenisation buffer (0.25 M sucrose, 10 mM HEPES, pH 7.4, and 1 mM EGTA) and subjected to 40 strokes of homogenisation in a Dounce homogeniser. The homogenate was subjected to centrifugation at 1000 g for 15 min at 4°C to pellet the nuclei and unbroken cells. The supernatant was subsequently centrifuged at 10000 g for 15 min at 4°C to obtain the cytosolic fraction (supernatant) and the mitochondrial fraction (pellet). The mitochondrial fraction was resuspended in homogenisation buffer following one wash. Aliquots of both fractions were mixed with equal volumes of 2×Laemmli sample buffer and analysed by western blotting for Bax and Bak.
To detect the formation of Bak multimeric complexes, aliquots of isolated mitochondrial fractions were cross linked with 1 mM dithiobis (Pierce, Rockford, Illinois, USA) at 37°C for 30 min. DMSO alone was used as the control. The cross linked samples were then centrifuged at 10000 g for 15 min at 4°C. After the supernatant was removed, the pellet was washed once with homogenisation buffer and lysed with 2×Laemmli sample buffer. Samples were subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis under non‐denaturing conditions as previously described,25 followed by immunoblotting for Bak.
The intestinal mucosal scraping samples were centrifuged at 500 g for 10 min. The cell pellet was homogenised in 3 ml of PBS (pH 7.4). Aliquots of the tissue homogenate were used to determine the concentration of total proteins and for the ROS and RNS assays. ROS (hydrogen peroxide) was measured using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instructions. The Griess reaction was used to detect nitrite (RNS) formed by the spontaneous oxidation of nitric oxide using a Griess reagent kit (Invitrogen) according to the manufacturer's instructions. Absorbance at 560 nm and the fluorescence emission at 590 nm were measured to determine the levels of hydrogen peroxide and nitrite, respectively, in a Victor III (Perkin Elmer/Wallace) plate reader. Six mice from each group were analysed. Each measurement was performed in triplicate and repeated at least twice. The results of ROS were expressed as μM hydrogen peroxide/mg total proteins. The results of RNS were expressed as μM nitrite/mg total proteins.
Total RNA was isolated from the intestinal mucosal scraping samples using the RNAgents Total RNA Isolation System (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. First strand cDNA was synthesised using Superscript Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Real time PCR was performed on a Chromo 4 Detector System (MJ Research, Sierra Point, California, USA) using gene specific primers and DyNAmo SYBR Green Master Mix (Finnzymes, Finland). PUMA were amplified using primers PUMA‐exon 3F, 5′‐ATG GCG GAC GAC CTC AAC‐3′ and PUMA‐exon 4R, 5′‐AGT CCC ATG AAG AGA TTG TAC ATG AC‐3′ to yield the 103 bp product.26 As the internal control, expression of β‐actin in each sample was also quantified using the sense primers 5′‐GTG GGC CGC TCT AGG CAC CA‐3′ and the antisense primer 5′‐CGG TTG GCC TTA GGG TTC AGG GGG G‐3′ (242 bp product). Three mice were used in each group. PCR products were analysed by agarose gel electrophoresis.
Total protein extract, and mitochondrial and cytosolic fractions were analysed by NuPage gel (Invitrogen) electrophoresis, as previously described.17,33 Antibodies used for western blotting included those for PUMA (Abcam), p53, α‐tubulin (EMD Biosciences, San Diego, California, USA), Bax, cytochrome c (Santa Cruz Biotech, Santa Cruz, California), Bak (Upstate USA, Charlottesville, Virginia, USA), active caspase 3 (BD Biosciences), Cox IV (Invitrogen) and β‐actin (Sigma). Appropriate horseradish peroxidase conjugated secondary antibodies were used to detect the primary antibody/antigen complexes. The signal was detected by Western Lightning Western Blot Chemiluminescence Reagent Plus (PerkinElmer). After quantifying the signals by densitometry, the results were normalised to the signals of the loading controls.
Data were analysed by ANOVA in which multiple comparisons were performed using the method of least significant difference. Differences were considered significant if the probability of the difference occurring by chance was less than 5 in 100 (p<0.05). Results are expressed as mean (SD).
To study the role of PUMA in I/R induced apoptosis in the small intestine, PUMA+/+ (wild‐type) and PUMA−/− (knockout) littermates were produced from PUMA+/− (heterozygote) mating. PCR was performed to identify the animals of desired genotypes (fig 1A1A).). Western blotting analysis confirmed that PUMA is only expressed in PUMA wild‐type mice but not in the knockout mice (fig 1B1B).). Real time PCR and western blotting were used to examine PUMA expression in the intestinal mucosa after I/R. Sixty minutes of ischaemia (60 I) significantly induced PUMA expression compared with the control sham operated (SO) mice (fig 1C, 1D1D).). Sixty minutes of ischaemia followed by 60 min of reperfusion (60 I/60 R) further increased the expression of PUMA in the small intestinal mucosa (fig 1C, 1D1D).). Quantitation by real time PCR revealed a striking 9‐fold increase in PUMA mRNA in the mucosa after I/R compared with a 1‐fold increase after 60 min of ischaemia alone (60 I) (fig 1C1C).). Immunohistochemical staining showed that PUMA was expressed only in the crypt of the sham operated animals (fig 1E1E).). Ischaemia for 60 min alone enhanced PUMA expression in the crypt and the bottom of villi (data not shown). Expression of PUMA was significantly increased in the crypts and, more importantly, in the intestinal epithelial cells of the villi following 60 I/60 R whereas PUMA was not detected in knockout mice (fig 1E1E).). These results indicate that I/R induced PUMA expression at both the mRNA and protein levels in the intestinal mucosa.
Previous studies have demonstrated that I/R caused injury and apoptosis in the jejunum and ileum.17,18,31 Although PUMA is known to be essential for p53 dependent and independent apoptosis in several cell types, it was unclear whether this is the case in intestinal epithelial cells.25,26,28 We therefore determined whether PUMA plays a role in mediating apoptosis induced by I/R in the small intestine. PUMA wild‐type and knockout littermates were divided into three groups: (1) sham operated; (2) 60 min of ischaemia (60 I); and (3) 60 min of ischaemia followed by 60 min of reperfusion (60 I/60 R). The percentage of the fragmented DNA relative to total DNA, which is an indicator of apoptosis, was determined using a diphenylamine assay. Ischaemia significantly induced DNA fragmentation in the jejunum of the PUMA wild‐type animals compared with the sham operated controls, with 16% and 3% DNA fragmentation, respectively. Furthermore, ischaemia followed by reperfusion dramatically increased DNA fragmentation to 30% (fig 2A2A).). However, apoptosis was significantly inhibited in the PUMA knockout mice, with 11% and 18% DNA fragmentation in the 60 I and 60 I/R groups, respectively (fig 2A2A)) (p<0.01). DNA laddering was analysed by gel electrophoresis to visualise the fragmented DNA in the intestinal mucosa (fig 2B2B).). The 60 I and 60 I/60 R groups showed progressively increased DNA laddering compared with the sham operated group in both PUMA wild‐type and knockout mice. However, the increase in DNA laddering in the PUMA knockout mice was lower than that in the wild‐type mice (fig 2B2B),), suggesting that I/R induced apoptosis is inhibited in the PUMA knockout mice.
To confirm these results, TUNEL staining was performed to determine the location and percentage of the apoptotic cells in situ (fig 2C2C).). Few TUNEL positive cells were detected at the villus tips in the sham operated mice, consistent with the physiological apoptosis during the renewal of intestinal epithelium.4 Ischaemia markedly increased the number of TUNEL positive cells on the tip of the villi of the wild‐type mice, with slight erosion at the villus tips. I/R led to further increases in apoptosis and destruction of the normal intestinal structure, with obvious signs of mucosal erosion and oedema (fig 2C2C).). However, apoptosis and tissue damage were significantly attenuated in the PUMA knockout mice (fig 2C2C),), with the apoptotic index reduced by 48% and 36% following 60I and 60 I/60 R, respectively, in the PUMA knockout mice (fig 2D2D)) (p<0.01). These results demonstrated that PUMA is an important mediator of I/R induced intestinal injury and apoptosis.
During mitochondrial mediated apoptosis, several mitochondrial apoptogenic proteins, such as cytochrome c, are released into the cytosol to facilitate the formation of the apoptosome and subsequent activation of caspase cascade.22 To investigate the mechanisms of I/R induced and PUMA mediated apoptosis, mitochondrial and cytosolic fractions were purified through differential centrifugation from the mucosa of the PUMA wild‐type and knockout animals. The release of cytochrome c was examined by western blotting (fig 3A3A).). In the sham operated mice, cytochrome c was detected in the mitochondrial fractions but not in the cytosolic fractions (fig 3A3A).). The amount of cytochrome c was markedly increased in the cytosolic fractions of the intestinal mucosa after ischaemia or I/R. However, cytochrome c release after ischaemia and I/R was decreased by 57% and 83%, respectively, in the PUMA deficient mice (fig 3A3A).). Both ischaemia and I/R induced caspase 3 activities were significantly reduced in the PUMA knockout mice compared with those in the wild‐type mice (fig 3B3B)) (p<0.01). Using an active caspase 3 specific antibody, we analysed the expression of active caspase 3 in the purified cytosolic fractions and tissue sections. Western blotting showed that the activation of caspase 3 induced by ischaemia and I/R was suppressed by 56% and 74%, respectively, in the PUMA knockout mice (fig 3C3C).). The immunofluorescence staining confirmed the decrease in active caspase 3 in the PUMA knockout animals (fig 3D3D).). Furthermore, I/R induced Bax mitochondrial translocation and formation of Bak multimeric complexes were found to be reduced in the PUMA knockout mice (fig 3E, 3F3F).). These results suggest that cytochrome c release, caspase 3 activation and alterations in Bax/Bak are involved in PUMA mediated intestinal apoptosis after I/R.
Oxidative stress has been implicated in I/R induced tissue injury.17,18,31 High concentrations of ROS and RNS have also been shown to promote apoptosis in the small intestine which can be suppressed by administration of antioxidants.7,13,14,34,35,36 We therefore examined whether oxidative stress is involved in the activation of PUMA during I/R induced apoptosis. As expected, I/R significantly induced intestinal hydrogen peroxide and nitrite in the PUMA wild‐type mice (fig 4A, 4B4B).). Treating the mice with the superoxide scavenger SOD or nitric oxide synthase inhibitor L‐NAME prior to I/R reduced hydrogen peroxide and nitrite production by 60–70% (fig 4A, 4B4B).). Apoptosis was also found to be significantly reduced in mice pretreated with SOD or L‐NAME by three different assays, including DNA fragmentation (fig 4C4C),), activation of caspase 3 (fig 4D4D)) and TUNEL staining (data not shown). Importantly, pretreatment with SOD or L‐NAME also reduced PUMA expression at both the mRNA and protein levels (fig 4E, 4F4F).). Quantitation by real time PCR revealed that pretreatment with SOD and L‐NAME suppressed PUMA induction by 50–60% following I/R (fig 4E4E).). These results suggest that PUMA contributes to I/R induced apoptosis mediated by oxidative stresses.
PUMA was initially identified as a gene activated in cells undergoing p53 induced apoptosis.23 However, it plays an important role in both p53 dependent and independent apoptosis.25,26,28 To determine whether p53 is involved in the activation of PUMA by I/R, we compared p53 wild‐type (p53+/+) and null (p53−/−) mice for I/R induced PUMA expression and intestinal apoptosis. I/R did not increase the level of p53 in p53+/+ mice (fig 5A5A).). PUMA was induced by I/R at a similar level in p53−/− mice compared with p53+/+ mice (fig 5A5A).). Analysis of DNA fragmentation and laddering revealed that p53 deficiency did not protect mice from the I/R induced intestinal apoptosis (fig 5B, 5C5C).). TUNEL staining confirmed that the I/R induced intestinal structural destruction and apoptosis were not improved in p53−/− mice (fig 5D, 5E5E).). Similarly, I/R induced caspase 3 activation was also unchanged in p53−/− mice compared with that in p53+/+ mice (fig 5F, 5G5G).). These results indicate that I/R induced PUMA expression and apoptosis in the small intestine are p53 independent.
I/R injury of the small intestine is associated with high morbidity and mortality. It can occur following abdominal aortic aneurysm surgery, small bowel transplantation, sepsis, neonatal necrotising enterocolitis and mesenteric arterial thrombotic or embolic disease.5,7 Among all internal organs, the small intestine is probably the most sensitive to I/R induced injury. Oxidative stress and free radicals have been shown to contribute to the I/R induced intestinal injury.5,7 Our results demonstrated that the BH3‐only Bcl‐2 family protein PUMA contributes to I/R induced injury and apoptosis in the small intestine ((figsfigs 1–3). However, only about 30% of I/R induced apoptosis and DNA fragmentation are inhibited in PUMA knockout mice (fig 22),), suggesting that other proteins are involved in apoptosis induction. In addition to PUMA, several other BH3‐only Bcl‐2 family members, such as Noxa, Bid and Bim, can regulate apoptosis through multiple redundant and parallel pathways.37 It is possible that several BH3‐only proteins are collectively responsible for I/R induced apoptosis in the small intestine.
Our data showed that antioxidants, such as SOD and L‐NAME, inhibit I/R induced PUMA induction and apoptosis (fig 44),), suggesting that the induction of PUMA during ischaemia and I/R is mediated by ROS and RNS. Administration of several antioxidants prior to ischaemia, such as N‐acetyl cystine and SOD, has been shown to reduce intestinal injury induced by I/R by affecting ROS generation and inflammatory responses in animal models.38,39 Tumour necrosis factor α inhibitors and interleukin 11 were also reported to have protective effects.40 Although various treatment modalities can prevent I/R induced injury in experimental models, the current understanding of the molecular nature of I/R induced injury and the mechanisms by which these agents exert their protective effects are still rather limited. It is possible that the inhibitory effects of the antioxidants we observed are solely the result of a reduction in oxidative stress itself. Additional experiments will be necessary to answer whether the influence of oxidative stress on PUMA expression contributes to I/R induced apoptosis. Previously, we and others found that PUMA overexpression resulted in apoptosis in colon cancer cells in a Bax dependent manner, possibly through generation of ROS. Antioxidants, such as diphenyleneiodonium, can protect against PUMA induced apoptosis.23,25,41 Deletion of PUMA or Bax in HCT 116 colon cancer cells attenuated apoptosis and ROS generation following p53 expression.42 These data suggest that PUMA induction and ROS generation may form a positive feedback control loop to induce apoptosis.
PUMA appears to mediate I/R induced intestinal apoptosis through the mitochondrial pathway. Mitochondria play a key role in cell life and death. Several mitochondrial apoptogenic proteins, including cytochrome c, can be released into the cytosol to initiate apoptosis through the formation of the apoptosome and subsequent activation of caspase cascade. BH3‐only proteins act through multidomain proapoptotic proteins Bax and/or Bak to induce apoptosis by either directly activating Bax or Bak or indirectly by antagonising Bcl‐2‐like proteins.22 Previous studies in cultured cells indicated that PUMA promotes apoptosis in a Bax/Bak dependent manner through mitochondrial membrane depolarisation, release of cytochrome c and activation of caspases, and that PUMA induced apoptosis can be suppressed by Bcl‐2 overexpression.25,27,43 Bcl‐2 overexpression was previously reported to have protective effects on I/R induced intestinal apoptosis.44 In agreement with these findings, our data indicated that PUMA mediates I/R induced intestinal apoptosis via its regulation of Bax/Bak, cytochrome c release and caspase activation (fig 33).
BH3‐only proteins are the apical sensors of apoptotic stimuli and are activated by a number of mechanisms, including transcriptional activation and post‐translational modifications. At least 10 mammalian BH3‐only proteins have been identified. These proteins appear to mediate apoptosis in a tissue and stimuli specific manner.22,45 For example, Bid mediates apoptosis induced by death receptors in hepatocytes whereas Bim is required for apoptosis induced by cytokines deprivation in haematopoietic cells.46,47PUMA is a major mediator of DNA damage induced apoptosis in epithelial cells, fibroblasts, neurons and haematopoietic cells.22,29,45,48,49 It has been demonstrated that p53 binds to the PUMA promoter to activate PUMA transcription following DNA damage to initiate apoptosis. In contrast, it is not well understood how PUMA is activated by non‐genotoxic stresses independent of p53.24,29,30 We found that PUMA is induced by I/R at both the mRNA and protein levels in the small intestine through p53 independent mechanisms ((figsfigs 1, 55).). In addition, it was reported that cardiomyocyte apoptosis during acute myocardial infarction occurs in a p53 independent manner.50 Targeted deletion of PUMA was recently shown to attenuate cardiomyocyte death and improve cardiac function during I/R.51 It is possible that the induction of PUMA in response to I/R is mediated by pathways previously shown to activate PUMA independent of p53. For example, the p53 homologue p73 and transcription factor FOX3a have been shown to active PUMA independent of p53.43,52 Future work is clearly needed to further understand the precise mechanisms of p53 independent induction of PUMA by I/R.
Taken together, our results showed that I/R stimulated PUMA expression to promote intestinal apoptosis via the mitochondrial pathway independent of p53. Our results also suggest that apoptosis is an important mechanism underlying the pathophysiology of I/R induced tissue injury, and that inhibition of PUMA might be particularly useful in protecting against I/R induced intestinal injury and apoptosis.
This work was supported in part by the National Institutes of Health (CAI06348), the General Motors (GM) Cancer Research Foundation, the U Foundation for Cancer Research (LZ), the Flight Attendent Medical Research Institute (FAMR) and the Alliance for Cancer Gene Therapy (ACGT) (JY).
I/R - ischaemia–reperfusion
L‐NAME - N‐nitro‐L‐arginine methyl ester
PBS - phosphate buffered saline
PCR - polymerase chain reaction
PUMA - p53 upregulated modulator of apoptosis
RNS - reactive nitrogen species
ROS - reactive oxygen species
SOD - superoxide dismutase
TUNEL - terminal deoxynucleotidyl transferase mediated deoxyuridinetriphosphate nick end labelling
Funding: This work was supported in part by the General Motors (GM) Cancer Research Foundation, the V Foundation for Cancer Research (LZ), the Flight Attendant Medical Research Institute (FAMRI) and the Alliance for Cancer Gene Therapy (ACGT) (JY).
Competing interests: None.