Bimei Jiang

Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells

Abstract Oxidative stress may cause apoptosis of cardiomyocytes in ischemia-reperfused myocardium, and heat shock pretreatment is thought to be protective against ischemic injury when cardiac myocytes are subjected to ischemia or simulated ischemia. However, the detailed mechanisms responsible for the protective effect of heat shock pretreatment are currently unclear. The aim of this study was to determine whether heat shock pretreatment exerts a protective effect against hydrogen peroxide(H2O2)–induced apoptotic cell death in neonatal rat cardiomyocytes and C2C12 myogenic cells and whether such protection is associated with decreased release of second mitochondria-derived activator of caspase–direct IAP binding protein with low pI (where IAP is inhibitor of apoptosis protein) (Smac/DIABLO) from mitochondria and the activation of caspase-9 and caspase-3. After heat shock pretreatment (42 ± 0.3°C for 1 hour, recovery for 12 hours), cardiomyocytes and C2C12 myogenic cells were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 hours. Apoptosis was evaluated by Hoechst 33258 staining and DNA laddering. Caspase-9 and caspase-3 activities were assayed by caspase colorimetric assay kit and Western analysis. Inducible heat shock proteins (Hsp) were detected using Western analysis. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blot and indirect immunofluorescence analysis. (1) H2O2 (0.5 mmol/L) exposure induced apoptosis in neonatal rat cardiomyocytes and C2C12 myogenic cells, with a marked release of Smac/DIABLO from mitochondria into cytoplasm and activation of caspase-9 and caspase-3, (2) heat shock pretreatment induced expression of Hsp70, Hsp90, and αB-crystallin and inhibited H2O2-mediated Smac/DIABLO release from mitochondria, the activation of caspase-9, caspase-3, and subsequent apoptosis. H2O2 can induce the release of Smac/DIABLO from mitochondria and apoptosis in cardiomyocytes and C2C12 myogenic cells. Heat shock pretreatment protects the cells against H2O2-induced apoptosis, and its mechanism appears to involve the inhibition of Smac release from mitochondria.

Increased stability of Bcl-2 in HSP70-mediated protection against apoptosis induced by oxidative stress.

We have previously shown that heat shock protein 70 (HSP70) markedly inhibits H2O2-induced apoptosis in mouse C2C12 myogenic cells by reducing the release of Smac. However, the molecular mechanism by which HSP70 interferes with Smac release during oxidative stress-induced apoptosis is not understood. In the current study, we showed that HSP70 increased the stability of Bcl-2 during oxidative stress. An antisense phosphorothioate oligonucleotide against Bcl-2 caused selective inhibition of Bcl-2 protein expression induced by HSP70 and significantly attenuated HSP70-mediated cell protection against H2O2-induced release of Smac and apoptosis. Taken together, our results indicate that there are important relationships among HSP70, Bcl-2, release of Smac, and induction of apoptosis by oxidative stress.

Nucleolin/C23 mediates the antiapoptotic effect of heat shock protein 70 during oxidative stress

Although heat shock protein 70 (Hsp70) has been shown to markedly inhibit H2O2‐induced apoptosis in C2C12 cells, and nucleolin/C23 has also been implicated in apoptosis, the relationship of these two molecules is still largely unknown. The aim of the current study was to investigate the potential involvement of nucleolin/C23 in the antiapoptotic mechanism of Hsp70. We found that primary cultures of neonatal rat cardiomyocytes underwent apoptosis upon H2O2 treatment, and in these cells nucleolin/C23 protein was highly unstable and had a half‐life of less than 4 h. However, transfection with Hsp70 greatly stabilized nucleolin/C23 and also protected the cells from H2O2‐induced apoptosis. When nucleolin/C23 was knocked down with an antisense oligomer, H2O2‐induced apoptosis became more severe, even in Hsp70‐overexpressed cells, demonstrating an essential role of nucleolin/C23 in the antiapoptotic effects of Hsp70. Similar results were obtained by both nuclear morphology observation and caspase‐3 activity assay. Therefore, these data provide evidence that nucleolin/C23 is an essential downstream effecter of Hsp70 in the protection of cardiomyocytes against oxidative stress‐induced apoptosis.

ATP‐binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria‐derived activator of caspase and apoptosis in C2C12 cells

Hydrogen peroxide (H2O2) is a well known oxidative stress inducer causing apoptosis of many cells. Previously, we have shown that heat shock pretreatment blocked the release of the second mitochondria‐derived activator of caspase (Smac) to the cytosol and inhibited apoptosis of C2C12 myoblast cells in response to H2O2. The present study aimed to elucidate the underlying mechanism by over‐expressing a major stress‐inducible protein, heat shock protein (HSP) 70, and characterizing the resulting cellular changes. We demonstrate that HSP70 over‐expression markedly inhibited the release of Smac and prevented the activation of caspases‐9 and ‐3 and apoptosis in C2C12 cells under H2O2 treatment. However, no direct interaction between HSP70 and Smac was observed by co‐immunoprecipitation. Mutational analysis demonstrated that the ATP‐binding domain of HSP70, rather than the peptide‐binding domain, was essential for these observed HSP functions. Taken together, our results provide evidence supporting the role of HSP70 in the protection of C2C12 cells from H2O2‐induced and Smac‐promoted apoptosis by preventing the release of Smac from mitochondria, thereby inhibiting activation of caspases‐9 and ‐3. This mechanism of HSP70 action is dependent on its ATP‐binding domain but independent of its interaction with Smac protein.

Enhancement of PPAR-β activity by repetitive low-grade H2O2 stress protects human umbilical vein endothelial cells from subsequent oxidative stress-induced apoptosis

Repetitive stress has been shown to up-regulate antioxidant defense and increase survival after subsequent oxidative injury. The up-regulation of antioxidant defense has been identified as an underlying cause of the apoptosis-inhibitory effects exerted by repetitive stress. However, it remains unclear what the important signaling mechanisms are by which cells preexposed to low-grade stress deal with apoptosis-inducing stress. In this study, we repetitively stressed human umbilical vein endothelial cells (HUVECs) through multiple exposures to a low dose (30 microM) of H(2)O(2) in culture for 4 weeks. We then examined the effects of repetitive stress on PPAR-beta expression and activity as well as the role of PPAR-beta in the protective potency of repetitive stress. Our results show that repetitive stress enhances PPAR-beta expression and activity, thereby inhibiting oxidative stress-induced apoptosis. Further, PPAR-beta-directed antisense oligonucleotides reduced the PPAR-beta protein content, enhanced the H(2)O(2)-mediated apoptosis, and ablated the protective effect of repetitive low-grade H(2)O(2) stress. The specific PPAR-beta agonist L-165041 significantly potentiated the apoptosis induced by H(2)O(2) (p<0.05) and increased the protective effect of repetitive stress. These findings indicate that repetitive low-grade H(2)O(2) stress protects HUVECs from subsequent oxidative stress-induced apoptosis by enhancing PPAR-beta expression and activity.

MiR-21 Protected Cardiomyocytes against Doxorubicin-Induced Apoptosis by Targeting BTG2.

Doxorubicin (DOX) is an anthracycline drug with a wide spectrum of antineoplastic activities. However, it causes cardiac cytotoxicity, and this limits its clinical applications. MicroRNA-21 (miR-21) plays a vital role in regulating cell proliferation and apoptosis. While miR-21 is preferentially expressed in adult cardiomyocytes and involved in cardiac development and heart disease, little is known regarding its biological functions in responding to DOX-induced cardiac cytotoxicity. In this study, the effects of DOX on mouse cardiac function and the expression of miR-21 were examined in both mouse heart tissues and rat H9C2 cardiomyocytes. The results showed that the cardiac functions were more aggravated in chronic DOX injury mice compared with acute DOX-injury mice; DOX treatment significantly increased miR-21 expression in both mouse heart tissue and H9C2 cells. Over-expression of miR-21 attenuated DOX-induced apoptosis in cardiamyocytes whereas knocking down its expression increased DOX-induced apoptosis. These gain- and loss- of function experiments showed that B cell translocation gene 2 (BTG2) was a target of miR-21. The expression of BTG2 was significantly decreased both in myocardium and H9C2 cells treated with DOX. The present study has revealed that miR-21 protects mouse myocardium and H9C2 cells against DOX-induced cardiotoxicity probably by targeting BTG2.

Nucleolin/C23 is a negative regulator of hydrogen peroxide-induced apoptosis in HUVECs

Nucleolin plays important roles in chromatin structure, rDNA transcription, rRNA maturation, nucleocytoplasmic transport, and ribosome assembly. Although it has been shown to be anti-apoptotic, the underlying mechanisms remain unclear. In the current study, we first examined endogenous nucleolin expression in response to oxidative stress-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). Flow cytometry and caspase activity assays showed that H2O2 treatment caused apoptosis of the cells; reverse-transcription polymerase chain reaction and Western blotting revealed the downregulation of nucleolin expression and increased protein cleavage during this process. Overexpression of nucleolin protein by transfecting cells with the full-length nucleolin cDNA inhibited apoptosis, but nucleolin deficiency brought about by transfection with antisense oligonucleotide increased apoptosis of HUVECs. Concurrently, the expression of the apoptotic protein gene Bax was also downregulated following nucleolin overexpression. All these results indicate an important negative regulatory role for nucleolin in the apoptosis of endothelial cells, likely involving the Bax pathway.

Role of PPAR-beta in hydrogen peroxide-induced apoptosis in human umbilical vein endothelial cells.

Peroxisome proliferator-activated receptor beta (PPAR-beta) is a ligand activated transcription factor belonging to the nuclear receptor superfamily. Recent evidence suggests that PPAR-beta has clearly defined roles in skin wound healing, inflammation and cell proliferation. However, little is known about the role of PPAR-beta in oxidative stress-induced apoptosis in human umbilical vein endothelial cells (HUVECs). In this study, a specific PPAR-beta ligand, L-165041, and antisense phosphorothioate oligodeoxynucleotides (asODNs) against PPAR-beta were used to reveal the role of PPAR-beta in oxidative stress-induced apoptosis. The results showed that H(2)O(2) at 0.5mM resulted in a marked increase of apoptosis and a significant down-regulation of PPAR-beta expression and activation in HUVECs. Moreover, L-165041 significantly inhibited H(2)O(2)-induced apoptosis (P<0.05) and asODNs against PPAR-beta markedly inhibited the de novo synthesis of PPAR-beta, which was accompanied by enhanced apoptosis induced by H(2)O(2) (P<0.05). These data demonstrated that H(2)O(2) down-regulated the expression and activation of PPAR-beta, which played important roles in H(2)O(2)-induced apoptosis in HUVECs.

HSF-1 is involved in attenuating the release of inflammatory cytokines induced by LPS through regulating autophagy.

ABSTRACT Autophagy plays a protective role in endotoxemic mice. Heat shock factor 1 (HSF-1) also plays a crucial protective role in endotoxemic mice by decreasing inflammatory cytokines. The purpose of this study was to determine whether HSF-1 is involved in attenuating the release of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated mice and peritoneal macrophages (PMs) through regulating autophagy activity. Autophagosome formation in HSF-1+/+ and HSF-1-/- mice and PMs stimulated by LPS was examined by Western blotting and immunofluorescence. Lipopolysaccharide-induced autophagy and inflammatory cytokines were examined in HSF-1+/+ and HSF-1-/- PMs treated with 3-methyladenine (3-MA) or rapamycin. Results showed that LPS-induced autophagy was elevated transiently at 12 h but declined at 24 h in the livers and lungs of mice. Higher levels of inflammatory cytokines and lower autophagy activity were detected in HSF-1-/- mice and PMs compared with HSF-1+/+ mice and PMs. Interestingly, LPS-induced release of inflammatory cytokines did not further increase in HSF-1-/- PMs treated with 3-MA but aggravated in HSF-1+/+ PMs. Lipopolysaccharide-induced autophagy did not decrease in HSF-1-/- PMs treated with 3-MA but decreased in HSF-1+/+ PMs. Taken together, our results suggested that HSF-1 attenuated the release of inflammatory cytokines induced by LPS by regulating autophagy activity.

Nucleolin protects the heart from ischaemia–reperfusion injury by up-regulating heat shock protein 32

AIMS Nucleolin plays important roles in a variety of cellular processes. In this study, we aimed to investigate the role of nucleolin in cardiac ischaemia-reperfusion (I-R) injury. METHODS AND RESULTS We investigated the expression pattern of nucleolin in hearts subjected to I-R, or neonatal rat cardiomyocytes subjected to hypoxia-re-oxygenation. We found that nucleolin expression was significantly down-regulated and the cleaved protein was present, both in vivo and in vitro. Gene transfection and RNA interference approaches were employed in cardiomyocytes to investigate the function of nucleolin. Over-expression of nucleolin was cytoprotective, whereas nucleolin ablation enhanced both hypoxia- and H₂O₂-induced cardiomyocyte death. Furthermore, transgenic mice with cardiac-specific over-expression of nucleolin were resistant to I-R injury as indicated by decreased cellular necrosis and decreased infarct size. The cardio-protective roles of nucleolin in cardiomyocytes, are attributable to the interaction of nucleolin with the mRNA of heat shock protein 32 (Hsp32), resulting in an increase of Hsp32 mRNA stability, and subsequent up-regulation of Hsp32 expression. The selective Hsp32 inhibitor, zinc protoporphyrin-IX, abrograted the cardiac protection mediated by nucleolin. CONCLUSION This study has demonstrated that nucleolin is involved in the regulation of I-R-induced cardiac injury and dysfunction via the regulation of Hsp32, and may be a novel therapeutic target for ischaemic heart diseases.