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Results for “Caspase 12”

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At least 19 recordsLinked to original sources

PubMed2002

Human caspase 12 has acquired deleterious mutations.

Caspase 12 has been cloned from rodent cells, in which it mediated apoptosis in response to endoplasmic reticulum stress. Based on experiments with murine cells it was suggested that this caspase plays a central role in the pathogenesis of Alzheimer's disease. By alignment of the murine caspase 12 cDNA with the human genome sequence we localized the human caspase 12 gene at a single locus within the caspase 1/ICE gene cluster on chromosome 11q22.3. RT-PCR and molecular cloning revealed that nine alternatively spliced transcripts of this gene are expressed. A frame shift mutation and a premature stop codon which is present in all splice variants preclude the expression of a full length protein. An additional loss-of-function mutation within the SHG box, a critical site in caspases, prohibits any proteins, if they are produced, from acting catalytically. Based on our data we conclude that functional caspase 12 is lost in humans and that it can therefore not play a role in Alzheimer's disease.

Alternative Splicing↗

PubMed2006

Caspase 12 in calnexin-deficient cells.

We investigated a role for calnexin, caspase 12, and Bap31 in endoplasmic reticulum stress-induced apoptosis in calnexin-deficient mouse embryonic fibroblasts and a calnexin-deficient human T cell line (NKR). We showed that calnexin-deficient mouse embryonic fibroblasts are relatively resistant to endoplasmic reticulum stress-induced apoptosis. Western blot analysis demonstrated that both wild-type and calnexin-deficient cells contained a caspase 12 protein. Caspase 12 expression was slightly inhibited in calnexin-deficient cells, and the protein carried out specific cleavage in the presence of thapsigargin. Immunoprecipitation experiments revealed that in the endoplasmic reticulum, caspase 12 forms complexes with Bap31 and calnexin. Treatment of wild-type cells with thapsigargin induced apoptosis and cleavage of Bap31. However, in the absence of calnexin, there was no significant cleavage of Bap31. There was also a negligible processing of caspase 8 in these cells. This work indicates that calnexin may play a role in modulating the sensitivity of a cell to apoptosis induced by endoplasmic reticulum stress, in conjunction with caspase 12 and Bap31.

Animals↗

PubMed2005

Endoplasmic reticulum stress-associated caspase 12 mediates cisplatin-induced LLC-PK1 cell apoptosis.

Reactive oxygen metabolites are important mediators in cisplatin-induced apoptosis in renal tubular epithelial cells (LLC-PK1). Mitochondria have been implicated to play a principal role in cisplatin-induced apoptosis. Caspase 12, an endoplasmic reticulum (ER)-specific caspase, participates in apoptosis under ER stress. Cytochrome P450 system is crucial to the generation of reactive oxygen metabolites and is present at high concentration in the ER. The direct role of caspase 12 in any model of renal injury has not previously been described. In this study, cleavage of procaspase 12 preceded that of caspases 3 and 9 after cisplatin treatment of LLC-PK1 cells. The active form of caspase 8 was not detected throughout the course of study. Preincubation of the LLC-PK1 cells with the caspase 9 inhibitor did not attenuate caspase 3 activation and provided no significant protection. Caspase 3 inhibitor provided only modest protection against cisplatin-induced apoptosis. LLC-PK1 cells that were transfected with anti-caspase 12 antibody significantly attenuated cisplatin-induced apoptosis. Taken together, these data indicate that caspase 12 plays a pivotal role in cisplatin-induced apoptosis. It is proposed that the oxidative stress that results from the interaction of cisplatin with the ER cytochrome P450 leads to activation of procaspase 12, resulting in apoptosis.

Antineoplastic Agents↗

PubMed2005

Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism.

The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.

Animals↗

PubMed2005

[Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA].

OBJECTIVES: To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12. METHODS: The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT. RESULTS: All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01). CONCLUSION: siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.

Animals↗

PubMed2005

AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress.

Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.

AMP-Activated Protein Kinases↗

PubMed2005

Toxicant-induced ER-stress and caspase activation in the olfactory mucosa.

The potent olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO(2)) induces rapid cell death and long-term metaplastic changes in the olfactory regions of rodents. The damage is related to a tissue-specific and extensive cytochrome P450 (CYP)-mediated metabolic activation of the compound to reactive intermediates. The aim of the present study was to examine the early, cell-specific changes leading to cell death in the olfactory mucosa of mice exposed to 2,6-diClPh-MeSO(2). We have examined the expression of the ER-specific stress protein GRP78, the presence of secretory glycoproteins, and the cellular activation of the initiator caspase 12 and the downstream effector caspase 3. 2,6-DiClPh-MeSO(2) induced rapid and cell-specific expression of GRP78, and activation of caspases 12 and 3 in the Bowman's glands. No similar early onset changes in the neuroepithelium were observed. Based on these results, we propose that extensive lesions are initiated in the Bowman's glands and that the metabolic activation of 2,6-diClPh-MeSO(2) elicits ER-stress response and subsequent apoptotic signaling at this site. Since most of the Bowman's glands had oncotic morphology, the results suggest that the terminal phase of apoptosis was blocked and that these glands finally succumb to other routes of cell death.

Animals↗

PubMed2003

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis.

Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

Animals↗

PubMed2002

Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells.

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2 alpha, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

Animals↗

PubMed2003

Interferon-gamma induces reactive oxygen species and endoplasmic reticulum stress at the hepatic apoptosis.

Interferon-gamma (IFN-gamma) induces cell-cycle arrest and p53-independent apoptosis in primary cultured hepatocytes. However, the detailed mechanism, including regulating molecules, is still unclear. In this study, we found that IFN-gamma induced generation of reactive oxygen species (ROS) in primary hepatocytes and that pyrrolidinedithiocarbamate (PDTC), an anti-oxidant reagent, completely suppressed IFN-gamma-induced hepatic apoptosis. PDTC blocked apoptosis downstream from IRF-1 and upstream from caspase activation, suggesting that the generation of ROS occurred between these stages. However, IFN-gamma also induced the generation of ROS in IRF-1-deficient hepatocytes, cells insensitive to IFN-gamma-induced apoptosis. Moreover, a general cyclooxygenase (COX) inhibitor, indomethacin (but not the cyclooxygenase 2-specific inhibitor, NS-398) also inhibited the apoptosis without blocking the generation of ROS. Both PDTC and indomethacin also blocked IFN-gamma-induced release of cytochrome c from mitochondria. These results suggest that ROS are not the only or sufficient mediators of IFN-gamma-induced hepatic apoptosis. In contrast, we also found that IFN-gamma induced endoplasmic reticulum (ER) stress proteins, CHOP/GADD153 and caspase 12, in wild-type primary hepatocytes, but induced only caspase 12 and not CHOP/GADD153 protein in IRF-1-deficient hepatocytes. These results suggest that IFN-gamma induces ER stress in primary hepatocytes. Both the ROS and ER stress induced by IFN-gamma may be complementary mediators that induce apoptosis in primary hepatocytes.

Apoptosis↗

PubMed2004

Conserved cysteine-rich domain of paramyxovirus simian virus 5 V protein plays an important role in blocking apoptosis.

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5VDeltaC) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5VDeltaC induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5VDeltaC, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5VDeltaC induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5VDeltaC was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5VDeltaC was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5VDeltaC-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5VDeltaC infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5VDeltaC can trigger cell death by inducing ER stress.

Animals↗

PubMed2006

The testes-specific bZip type transcription factor Tisp40 plays a role in ER stress responses and chromatin packaging during spermiogenesis.

We previously reported that the spermatid-specific transcription factor Tisp40 functions through UPRE and CRE. To investigate Tisp40 function in vivo, we generated TISP40(-/-) mice. TISP40(-/-) mice were born at expected ratios, were healthy, and mutant males bred normally. However, the ER stress-response protein Grp78/BiP accumulated in the TISP40(-/-) testis and RAMP4 (Ribosome-associated membrane protein 4) mRNA level was up-regulated. Disruption of TISP40 caused ER stress and activation of caspase 12 but not caspase 9, leading to apoptosis of meiotic/postmeiotic germ cells. On the other hand, DAPI staining and electron microscopy revealed that epididymal sperm nuclei were abnormally relaxed in the TISP40(-/-) testis, a phenotype that was independent of the expression and maturation of transition proteins and protamines but due to abnormally retained histones. Histones localized to the cytoplasm as well as to the nucleus and were also retained in epididymal sperm. Histones H2A and H4 were dramatically up-regulated and the acetylation of H2A, H2B and H4 was also enhanced in the TISP40(-/-) testis. Taken together, we conclude that Tisp40 plays an important role in the unfolded protein response of the testis and in regulating the maturation of sperm head nuclei.

Animals↗

PubMed2005

Norepinephrine-induced oxidative stress causes PC-12 cell apoptosis by both endoplasmic reticulum stress and mitochondrial intrinsic pathway: inhibition of phosphatidylinositol 3-kinase survival pathway.

Norepinephrine (NE) induces endoplasmic reticulum (ER) unfolded protein response and reduces maturation and translocation of NE transporter to cell membrane via enhanced formation of reactive oxygen species in PC-12 cells. In the present study, we investigated whether ER stress is also implicated in the proapoptotic effect of NE. We found that the apoptotic effect of NE was associated with increased processing of ER-resident pro-caspase-12, cleavage of caspase-9 and -3, and mitochondrial release of cytochrome c. ER stress was evidenced by upregulation of ER chaperone GRP78 and transcription factor CHOP and the translocation of XBP-1 from the ER to the nucleus by NE. NE also reduced phospho-Akt (Ser473), indicating suppression of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt survival pathway. Similar results were produced by thapsigargin. NGF, which promotes the PI3-kinase/Akt activity, reduced the effects of NE and thapsigargin on apoptosis and activation of caspase-12 and -3. However, the effects of NE, but not of thapsigargin, were abolished by pretreatment with SOD and catalase. In contrast, the PI3-kinase inhibitors LY-294002 and wortmannin abolished the protective effects of both SOD/catalase and NGF on NE-induced apoptosis. The functional importance of caspase-12 activation was supported by the use of Z-ATAD-FMK, which reduced the NE-induced processing of caspase-12 and cell apoptosis, but the caspase-12, -9, and -3 inhibitors had no effects on the increase in cytosolic cytochrome c produced by NE. In contrast, the release of mitochondrial cytochrome c was abolished by SOD/catalase and NGF. These results indicate that NE induced cell apoptosis by both ER stress and a mitochondrial death pathway and that the effects of NE were mediated via oxidative stress and inhibition of the PI3-kinase/Akt survival pathway.

Animals↗

PubMed2004

Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells exposed to diindolylmethane in vitro.

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical cancer in animal model studies and in human clinical trials. I3C and its physiologic condensation product diindolylmethane (DIM) also induce apoptosis of tumor cells in vitro and in vivo, suggesting that these phytochemicals might be useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic acid microarray studies on transformed keratinocytes and tumor cell lines exposed to pharmacologic concentrations of DIM in vitro are consistent with a cellular response to nutritional deprivation or disruptions in protein homeostasis such as endoplasmic reticulum (ER) stress. In this report we investigate whether specific stress response pathways are activated in tumor cells exposed to DIM and whether the ER stress response might contribute to DIM's cytotoxicity. Induction of the stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78, GRP94, and asparagine synthase was documented by Western blot and real-time reverse transcriptase-polymerase chain reaction in C33A cervical cancer cells, and induction of a subset of these was also observed in cancer cell lines from breast (MCF-7) and prostate (DU145). The results are consistent with activation of more than 1 stress response pathway in C33A cells exposed to 75 microM DIM. Phosphorylation elF2alpha was rapidly and transiently increased, followed by elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional activation dependent on an ATF6-XBP-1 binding site was detected by transient expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT); induction of the GADD153 (CHOP) promoter was also confirmed by transient expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved at later times, coinciding with the onset of apoptosis. The results support the hypothesis that cytotoxic concentrations of DIM can activate cellular stress response pathways in vitro, including the ER stress response. Conversely, DIM was especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing degrees; nutrient limitation had a similar, but even more pronounced, effect. Because DIM toxicity in vitro is enhanced in cells undergoing nutritional deprivation and ER stress, it is possible that stressed cells in vivo, such as those within developing solid tumors, also have increased sensitivity to killing by DIM.

Activating Transcription Factor 4↗

PubMed2006

Endoplasmic and vascular surface activation during organ preservation: refining upon the benefits of machine perfusion.

The endoplasmic reticulum (ER) represents a subcellular target reactive to various cytosolic impairments. The involvement of ER-stress in organ preservation was investigated, comparing machine preservation, cold storage (CS) and a novel concept of only temporary perfusion after procurement. Rat livers were retrieved 30 min after cardiac arrest and preserved for 18 h by CS, oxygenated machine perfusion for 18 h (18 h MP) or for 2 h with subsequent CS for 16 h (2 h MP + 16 h CS). Upon reperfusion, 18 h MP significantly improved enzyme leakage (ALT, LDH) and promoted a 2-fold increase of metabolic recovery compared to CS. However, vascular stress, evaluated by endothelin-release, was significantly elevated after 18 h MP. Interestingly, better viability was obtained using the short-term perfusion protocol (2 h MP + 16 h CS), which further reduced enzyme leakage, maintained energetic recovery and mitigated endothelin-release compared to 18 h MP. Caspase 12-mRNA was upregulated in the 18 h MP-group but unchanged after CS or 2 h MP + 16 h CS. Activation/cleavage of caspase 12 protein was significantly enhanced after 18 h MP and very low in the 2 h MP + 16 h CS-group. Correspondingly, electron microscopy showed ultrastructural alterations of ER after CS and especially after 18 h MP but not after 2 h MP + 16 h CS. At this time mitochondrial appearance was unaffected in all groups, suggesting the ER to be an early subcellular target of preservation injury. In our model, ER and vascular endothelium were best protected by only temporary machine perfusion, which also maintained overall graft viability.

Animals↗

PubMed2005

Role of calreticulin in the sensitivity of myocardiac H9c2 cells to oxidative stress caused by hydrogen peroxide.

Calreticulin (CRT), a Ca2+-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H2O2, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca2+ ([Ca2+]i) were significantly increased by H2O2, whereas in controls, only a slight increase was observed. The H2O2-induced apoptosis was enhanced by the increase in [Ca2+]i caused by thapsigargin in control cells but was suppressed by BAPTA-AM, a cell-permeable Ca2+ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca2+]i in the sensitivity to H2O2-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared with controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca2+]i in the CRT-overexpressing cells treated with H2O2 compared with controls. Thus we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H2O2 through a change in Ca2+ homeostasis and the regulation of the Ca2+-calpain-caspase-12 pathway in myocardiac cells.

Animals↗

PubMed2005

Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs.

Previously, we showed that feeding micropigs ethanol with a folate-deficient diet promoted the development of hepatic injury while increasing hepatic levels of homocysteine and S-adenosylhomocysteine (SAH) and reducing the level of S-adenosylmethionine (SAM) and the SAM-to-SAH ratio. Our present goals were to evaluate mechanisms for hepatic injury using liver specimens from the same micropigs. The effects of ethanol feeding or folate-deficient diets, singly or in combination, on cytochrome P-450 2E1 (CYP2E1) and signal pathways for apoptosis and steatosis were analyzed using microarray, real-time PCR, and immunoblotting techniques. Apoptosis was increased maximally by the combination of ethanol feeding and folate deficiency and was correlated positively to liver homocysteine and SAH. Liver CYP2E1 and the endoplasmic reticulum stress signals glucose-regulated protein 78 (GRP78), caspase 12, and sterol regulatory element binding protein-1c (SREBP-1c) were each activated in pigs fed folate-deficient or ethanol diets singly or in combination. Liver mRNA levels of CYP2E1, GRP78, and SREBP-1c, and protein levels of CYP2E1, GRP78, nuclear SREBP, and activated caspase 12 each correlated positively to liver levels of SAH and/or homocysteine and negatively to the SAM-to-SAH ratio. The transcripts of the lipogenic enzymes fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase were elevated in the ethanol-fed groups, and each was positively correlated to liver homocysteine levels. The induction of abnormal hepatic methionine metabolism through the combination of ethanol feeding with folate deficiency is associated with the activation of CYP2E1 and enhances endoplasmic reticulum stress signals that promote steatosis and apoptosis.

Animals↗

PubMed2006

Possibility of conditioning predamaged grafts after cold storage: influences of oxygen and nutritive stimulation.

The potential of short-term oxygenated perfusion after cold storage (CS) to reverse deleterious priming of nonheart beating donors grafts should be investigated, addressing the respective role of oxygenation and nutrients or metabolic charge. Livers were retrieved 30 min after cardiac arrest of male Wistar rats and preserved with histidine tryptophan ketoglutarate (HTK)-solution for 18 h by CS. After 16 h, some livers were put on an oxygenated machine-preservation-circuit for the last 2 h and conditioned by cold perfusion with either HTK (conHTK), HTK supplemented with adenosine, phosphate and glucose (conHTK+) or Williams-E solution (conWE). Upon warm reperfusion, postconditioning with any of the solutions led to a significant (three- to fivefold) reduction of parenchymal damage (ALT, GLDH-release) compared with CS. Metabolic recovery (bile production) was also significantly enhanced compared with CS, with best results found after conHTK. The beneficial effect of postconditioning with HTK was associated with a significantly mitigated cleavage of caspase 12 and 3. We conclude from these data that conditioning of predamaged livers is possible even after CS by short-term oxygenated perfusion in the cold and, under these conditions, not depending on energetic support or nutritive stimulation.

Animals↗