Splice isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4: Expression and hypoxic regulation

Using an ex vivo model of isolated–perfused rat hearts and cultured H9c2 cells, the structure–activity relationships of schisandrin B (Sch B), and analogs lacking either the methylendioxy group or cyclooctadiene ring, schisandrin A (Sch A) and dimethyl diphenyl bicarboxylate (DDB), respectively, were investigated. Pretreatment with Sch B, but not with Sch A or DDB, protected against myocardial ischemia–reperfusion (I-R) injury in rats. Although Sch B pretreatment largely prevented H9c2 cells from menadione-induced cytotoxicity, Sch A pretreatment produced only a marginal protection. However, DDB pretreatment did not cause any detectable effect. The myocardial and cellular protection afforded by Sch B pretreatment correlated with increases in mitochondrial ATP generation capacity and/or reduced glutathione level as well as heat shock protein (Hsp)25/70 expression, under both control and oxidative stress conditions. The results indicate that the methylenedioxy group and the cyclooctadiene ring are important structural determinants of Sch B in enhancing mitochondrial functional ability and glutathione status, as well as tissue Hsp25/70 expression, thereby protecting the myocardium against I-R injury.     

Hepatoprotective mechanism of schisandrin B: role of mitochondrial glutathione antioxidant status and heat shock proteins

In this study, the time course of schisandrin B- (Sch B-) induced changes in hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (HSP) 25/70 induction was examined to study their differential roles in the hepatoprotection afforded by Sch B pretreatment against carbon tetrachloride (CCl(4)) toxicity in mice. Dimethyl diphenyl bicarboxylate (DDB), a nonhepatoprotective analog of Sch B, was also included for comparison. The results indicate that Sch B treatment (2 mmol/kg) produced maximum enhancement in hepatic mtGAS and increases in both hepatic HSP 25 and HSP 70 levels at 24 h after dosing. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl(4) was found to correlate inversely with the elapsed time postdosing, the protective effect was associated with the ability to sustain mtGAS and/or HSP 70 levels in a CCl(4)-intoxicated condition. On the other hand, DDB (2 mmol/kg) treatment, which did not sustain mtGAS and HSP 70 level, could not protect against CCl(4) toxicity. Abolition of the Sch B-mediated enhancement of mtGAS by buthionine sulfoximine/phorone did not completely abrogate the hepatoprotective action of Sch B. The results indicate that Sch B pretreatment independently enhances mtGAS and induces HSP 25/70 production, particularly under conditions of oxidative stress, thereby protecting against CCl(4) hepatotoxicity.     

Hepatoprotective action of schisandrin B against carbon tetrachloride toxicity was mediated by both enhancement of mitochondrial glutathione status and induction of heat shock proteins in mice

In the present study, we investigated the differential role of the mitochondrial glutathione status and induction of heat shock proteins (HSPs) 25/70 in protecting against carbon tetrachloride (CCl_4) hepatotoxicity in schisandrin B (Sch B)-pretreated mice. The time-course of Sch B-induced changes in these hepatic parameters were examined. Dimethyl diphenyl bicarboxylate (DDB), a non-hepatoprotective analog of Sch B, was studied for comparison. Sch B treatment (2 mmol/kg) produced maximal enhancement in hepatic mitochondrial glutathione status as well as increases in hepatic HSP 25/70 levels at 24 h post-dosing. The stimulatory effect of Sch B then gradually subsided, but the activities of hepatic mitochondrial glutathione reductase (GR) and glutathione S-transferases (GST) as well as the level of HSP 25 remained relatively high even at 72 h post-dosing. CCl_4 challenge caused significant impairment in mitochondrial glutathione status and a decrease in HSP 70 level, but the HSP 25 level was significantly elevated. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl_4 was found to inversely correlate with the time elapsed after the dosing, the protective effect was associated with the ability of Sch B to maintain the mitochondrial glutathione status and/or induce further production of HSP 25 in CCl_4-intoxicated condition. On the other hand, DDB treatment (2 mmol/kg), which did not increase mitochondrial GSH level and GST activity or induce further production of HSP 25 after CCl_4 challenge, could not protect against CCl_4 toxicity. The results suggest that the enhancement of mitochondrial glutathione status and induction of HSP 25/70 may contribute independently to the hepatoprotection afforded by Sch B pretreatment.     

Cytochrome P450-catalysed reactive oxygen species production mediates the (-)schisandrin B-induced glutathione and heat shock responses in AML12 hepatocytes

Sch B (schisandrin B), the most abundant dibenzocyclooctadiene lignan in Fructus schisandrae, can induce glutathione antioxidant and heat shock responses, as well as protect against oxidant-induced injury in various tissues, including the liver in rodents and AML12 (alpha mouse liver 12) hepatocytes. (-)Sch B is the most potent stereoisomer of Sch B in its cytoprotective action on AML12 hepatocytes. To define the role of ROS (reactive oxygen species) arising from CYP (cytochrome P450)-catalysed metabolism of (-)Sch B in triggering glutathione antioxidant and heat shock responses, the effects of a CYP inhibitor [ABT (aminobenzotriazole)] and antioxidants [DMTU (dimethylthiouracil) and TRX (trolox)] on (-)Sch B-induced ROS production and associated increases in cellular GSH level, as well as Hsp25/70 (heat-shock protein 25/70) production, were investigated in AML12 hepatocytes. The results indicated that (-)Sch B causes a dose dependent and sustained increase in ROS production over 6 h in AML12 hepatocytes, which was completely suppressed by pre-/co-treatment with ABT or DTMU/TRX. Incubation with (-)Sch B for 6 h caused optimal and dose-dependent increases in cellular GSH level and Hsp25/70 production at 16 h post-drug exposure in AML12 hepatocytes. These cellular responses were associated with protection against menadione-induced apoptosis. Pre-/co-treatment with ABT or antioxidants completely abrogated the (-)Sch B-induced glutathione antioxidant and heat shock responses, as well as protection against menadione-induced apoptosis. Experimental evidence obtained thus far supports the causal role of ROS arising from the CYP-catalysed metabolism of (-)Sch B in eliciting glutathione antioxidant and heat shock responses in AML12 hepatocytes.     

Myocardial Hsp70 phosphorylation and PKC-mediated cardioprotection following exercise

Both protein kinase C (PKC) activation and Hsp70 expression have been shown to be key components for exercise-mediated myocardial protection during ischemia-reperfusion injury. Given that Hsp70 has been shown to undergo inducible phosphorylation in striated muscle and liver, we hypothesized that PKC may regulate myocardial Hsp70 function and subsequent exercise-conferred cardioprotection through this phosphorylation. Hence, acute exercise of male Sprague-Dawley rats (30 m/min for 60 min at 2% grade) was employed to assess the role of PKC and its selected isoforms in phosphorylation of Hsp70 and protection of the myocardium during ischemia-reperfusion injury. It was observed that administration of the PKC inhibitor chelerythrine chloride (5 mg/kg) suppressed the activation of three exercise-induced PKC isoforms (PKCalpha, PKCdelta, and PKCepsilon) and attenuated the exercise-mediated reduction of myocardial infarct size during ischemia-reperfusion injury. While this study also demonstrated that exercise led to an alteration in the phosphorylation status of Hsp70, this posttranslational modification appeared to be dissociated from PKC activation, as exercise-induced phosphorylation of Hsp70 was unchanged following inhibition of PKC. Taken together, these results indicate that selected isoforms of PKC play an important role in exercise-mediated protection of the myocardium during ischemia-reperfusion injury. However, exercise-induced phosphorylation of Hsp70 does not appear to be a mechanism by which PKC induces this cardioprotective effect.     

Role of cytochrome P-450 in schisandrin B-induced antioxidant and heat shock responses in mouse liver

In order to explore the role of cytochrome P-450 (P-450) in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effect of 1-aminobenzotriazole (ABT, a broad spectrum inhibitor of P-450) on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression was examined in Sch B-treated mice. The non-specific and partial inhibition of cytochrome P-450 (P-450) by ABT pretreatment significantly caused a protraction in the time-course of Sch B-induced enhancement in hepatic mitGAS and Hsp25/70 expression in mice. Using mouse liver microsomes as a source of P-450, Sch B, but not dimethyl diphenyl bicarboxylate (a non-hepatoprotective analog of Sch B), was found to serve as a co-substrate for the P-450-catalyzed NADPH oxidation reaction, with a concomitant production of oxidant species. Taken together, the results suggest that oxidant species generated from P-450-catalyzed reaction with Sch B may trigger the antioxidant and heat shock responses in mouse liver.     

Effects of pharmacological preconditioning by emodin/oleanolic acid treatment and/or ischemic preconditioning on mitochondrial antioxidant components as well as the susceptibility to ischemia-reperfusion injury in rat hearts

Using an ex vivo rat heart model of ischemia-reperfusion (I-R) injury, we examined the effect of pharmacological preconditioning by chronic treatment with emodin (EMD)/oleanolic acid (OA) at low dose (25 μ mol/kg/day × 15) and/or ischemic preconditioning (IPC) (4 cycles of 5 min ischemia followed by 5 min of reperfusion) on myocardial I-R injury. The results indicated that EMD/OA pretreatment, IPC, or their combinations (EMD+IPC and OA+IPC) protected against myocardial I-R injury, as assessed by lactate dehydrogenase leakage and contractile force recovery. The cardioprotection was associated with a differential enhancement in mitochondrial antioxidant components. The combined EMD/OA and IPC pretreatment produced cardioprotective action in a semi-additive manner. This suggested that EMD/OA pretreatment and IPC protected against myocardial I-R injury via a similar but not identical biochemical mechanism.     

Effects of schisandrin B pretreatment on tumor necrosis factor-alpha induced apoptosis and Hsp70 expression in mouse liver

Tumor necrosis factor-alpha (TNFalpha) could cause apoptosis in hepatic tissue of D-galactosamine sensitized mice, as evidenced by the increase in the extent of DNA fragmentation. The hepatic apoptosis induced by TNFalpha was associated with hepatocellular damage as assessed by plasma alanine aminotransferase activity. Schisandrin B (Sch B) pretreatment at daily doses ranging from 0.5 to 2 mmol/kg for 3 days caused a dose-dependent protection against TNFalpha-induced apoptosis in mice. The hepatoprotection was accompanied by a parallel reduction in the extent of hepatocellular damage. The same Sch B pretreatment regimens increased hepatic Hsp70 level in a dose-dependent manner. The relevance of Sch B-induced increase in Hsp70 expression to the prevention of TNFalpha-triggered hepatic apoptosis remains to be elucidated.     

Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.     

Exercise training improves myocardial tolerance to ischemia in male but not in female rats

Acute exercise increases myocardial tolerance to ischemia-reperfusion (I-R) injury in male but not in female rat hearts, possibly due to a decreased heat shock protein 70 (Hsp70) response in the female hearts. This study examined whether repetitive exercise training would increase Hsp70 and myocardial tolerance to I-R injury in female rat hearts. Adaptations in myocardial manganese superoxide dismutase (MnSOD) and endothelial nitric oxide synthase (eNOS) were also assessed. Ten-week old male (M) and female (F) Sprague-Dawley rats (n = 40 total) exercise-trained for 14 wk; the last 8 wk consisted of running 1 h at 30 m/min (2% incline), 5 days/wk. Following training, left ventricle mechanical function (LVMF) was monitored for 30 min of reperfusion following 30 min of global ischemia (Langendorff procedure). Myocardial Hsp70 content was not different in M and F control groups, while increases were observed in both trained groups (M greater than F; P < 0.05). Although MnSOD content did not differ between groups, endothelial nitric oxide synthase (eNOS) levels were decreased in F, with no change in M, following training (P < 0.05). Hearts from control F demonstrated a greater recuperation of all indices of LVMF following I-R compared with control M hearts (P < 0.05). Hearts of trained M exhibited improved recovery of LVMF (left ventricular diastolic pressure, left ventricular end-diastolic pressure, +dP/dt, -dP/dt) during reperfusion compared with control M hearts (P < 0.05). In contrast, hearts of trained F did not show any change in recovery from I-R. Hence, exercise training is more beneficial to M than F in improving myocardial function following I-R injury.     

Isoproterenol potentiates exercise-induction of Hsp70 in cardiac and skeletal muscle

The response to exercise stress is characterized by an increase in circulating catecholamines and rapid synthesis of the inducible member of the 70 kDa family of heat shock proteins (Hsp70). Cell culture studies indicate that Hsp70 expression is influenced by beta-adrenergic receptor intermediates including cyclic AMP (cAMP) and cAMP dependent protein kinase (PKA). Thus, in the present investigation, the effect of a beta-adrenergic agonist, isoproterenol (ISO; 10 mg/kg) and a beta-adrenergic antagonist, nadolol (NAD; 25 mg/kg), on the in vivo expression of Hsp70 in rodent cardiac and skeletal muscle following moderate (MOD; 17 m/min) and exhaustive (EXH; 30 m/min) exercise was examined. While ISO alone did not induce Hsp70 synthesis, ISO treatment potentiated Hsp70 expression following MOD in the white vastus and heart (395+/-29 and 483+/-29% greater than control respectively, P < 0.05). Furthermore, this effect was reversed with combined beta-adrenergic agonist and antagonist treatment (ISO+NAD) indicating that the isoproterenol induced increase in post-exercise Hsp70 expression was mediated via beta-adrenergic receptor activity. However, there were no differences in Hsp70 levels among treatment groups following EXH. The failure of NAD to attenuate Hsp70 accumulation following EXH suggests that beta-adrenergic receptor activity is not the main signal in the induction of Hsp70 following exercise. Hsp70 induction was dependent on exercise intensity and ISO administration prior to MOD resulted in Hsp70 levels similar to those observed following EXH. The results from the present investigation indicate that beta-adrenergic receptor stimulation does not induce Hsp70 synthesis per se, but may be one factor involved in the complex regulation of the stress response to exercise in vivo.     

Arsenic trioxide induces Hsp70 expression via reactive oxygen species and JNK pathway in MDA231 cells

In the present study, we determined the molecular pathways that induce the heat shock proteins (Hsps) after treatment of cells with arsenic trioxide. Administration of arsenic trioxide to MDA231 cells leads to induce Hsp70, which is accompanied by generation of reactive oxygen species (ROS) and activation of c-Jun N-terminal kinase (JNK). We showed that arsenic trioxide-induced Hsp70 expression was caused by activation of ROS and prevented by the antioxidant N-Acetyl-Cysteine (NAC). SP600125 and dominant-negative SEK suppressed Hsp70 promoter-driven reporter gene expression, suggesting that JNK would be preferentially associated with the protective heat shock response against arsenic trioxide stress. In addition, SP600125, a specific JNK inhibitor, significantly reduced the amount of phosphorylated HSF1 upon administration of arsenic trioxide. It is likely that Hsp70 expression against arsenic trioxide exposure protects cells from oxidative injury and apoptotic cell death by means of JNK activity.     

Geldanamycin induces heat shock protein 70 and protects against MPTP-induced dopaminergic neurotoxicity in mice

As key molecular chaperone proteins, heat shock proteins (HSPs) represent an important cellular protective mechanism against neuronal cell death in various models of neurological disorders. In this study, we investigated the effect as well as the molecular mechanism of geldanamycin (GA), an inhibitor of Hsp90, on 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity, a mouse model of Parkinson disease. Neurochemical analysis showed that pretreatment with GA (via intracerebral ventricular injection 24 h prior to MPTP treatment) increased residual dopamine content and tyrosine hydroxylase immunoreactivity in the striatum 24 h after MPTP treatment. To dissect out the molecular mechanism underlying this neuroprotection, we showed that the GA-mediated protection against MPTP was associated with a reduction of cytosolic Hsp90 and an increase in Hsp70, with no significant changes in Hsp40 and Hsp25 levels. Furthermore, in parallel with the induction of Hsp70, striatal nuclear HSF1 levels and HSF1 binding to heat shock element sites in the Hsp70 promoter were significantly enhanced by the GA pretreatment. Together these results suggested that the molecular cascade leading to the induction of Hsp70 is critical to the neuroprotection afforded by GA against MPTP-induced neurotoxicity in the brain and that pharmacological inhibition of Hsp90 may represent a potential therapeutic strategy for Parkinson disease.     

Loss of exocrine pancreatic stimulation during parenteral feeding suppresses digestive enzyme expression and induces Hsp70 expression

Luminal nutrients are essential for the growth and maintenance of digestive tissue including the pancreas and small intestinal mucosa. Long-term loss of luminal nutrients such as during animal hibernation has been shown to result in mucosal atrophy and a corresponding stress response characterized by the induction of heat shock protein (Hsp)70 expression. This study was conducted to determine if the loss of luminal nutrients during total parenteral nutrition (TPN) would result in atrophy of the exocrine pancreas and small intestinal mucosa as well as an induction of Hsp70 expression in rats. In experiment 1, the treatment groups included an orally fed control, a saline-infused surgical control, or TPN treatment for 7 days. In experiment 2, the treatment groups included an orally fed control and TPN alone or coinfused with varying doses of glucagon-like peptide (GLP)-2, a mucosal proliferation agent, for 7 days. In experiment 1, TPN resulted in a 40% reduction in pancreatic mass that was associated with a dramatic reduction in digestive enzyme expression, enhanced apoptosis, and a 200% increase in Hsp70 expression. Conversely, heat shock cognate 70, Hsp27, and Hsp60 expression was not changed in the pancreas. In experiment 2, TPN resulted in a 30% reduction in jejunal mucosa mass and a similar induction of Hsp70 expression. The inclusion of GLP-2 during TPN attenuated jejunal mucosal atrophy and inhibited Hsp70 expression, suggesting that Hsp70 induction is sensitive to cell growth. These data indicate that pancreatic and intestinal mucosal atrophy caused by a loss of luminal nutrient stimulation is accompanied by a compensatory response involving Hsp70.     

Evidence for a role of Hsp70 in the neuroprotection induced by heat shock pre-treatment against 3,4-methylenedioxymethamphetamine toxicity in rat brain

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22 degrees C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4 degrees C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30 degrees C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22 degrees C. Fluoxetine-induced inhibition of 5-HT release and hydroxyl radical formation did not modify MDMA-induced Hsp70 expression 3 h later. Four- or 8-day heat shock (elevation of basal rectal temperature by 1.5 degrees C for 1 h) or geldanamycin pre-treatment induced Hsp70 expression and protected against MDMA-induced serotoninergic neurotoxicity without affecting drug-induced hyperthermia. Thus, MDMA-induced Hsp70 expression depends on the drug-induced hyperthermic response and not on 5-HT release or hydroxyl radical formation and pre-induction of Hsp70 protects against the long-term serotoninergic damage produced by MDMA.     

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs.