Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

Hsp105 (Hsp105alpha and Hsp105beta), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105alpha regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105alpha or Hsp105beta by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105alpha or Hsp105beta. In addition, we found that overexpression of Hsp105alpha or Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105alpha or Hsp105beta. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells.     

Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases.

The major heat shock protein, Hsp70, is an effective inhibitor of apoptosis. To study its mechanism of action, we created tumor cell lines with altered Hsp70 levels. The expression levels of Hsp70 in the cells obtained correlated well with their survival following treatments with tumor necrosis factor, staurosporine and doxorubicin. Surprisingly, the surviving Hsp70-expressing cells responded to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3-like proteases in a manner essentially similar to that of the dying cells with low Hsp70 levels. However, Hsp70 inhibited late caspase-dependent events such as activation of cytosolic phospholipase A2 and changes in nuclear morphology. Furthermore, Hsp70 conferred significant protection against cell death induced by enforced expression of caspase-3. Thus, Hsp70 rescues cells from apoptosis later in the death signaling pathway than any known anti-apoptotic protein, making it a tempting target for therapeutic interventions.     

Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation

Hsp70 overexpression can protect cells from stress-induced apoptosis. Our previous observation that Hsp70 inhibits cytochrome c release in heat-stressed cells led us to examine events occurring upstream of mitochondrial disruption. In this study we examined the effects of heat shock on the proapoptotic Bcl-2 family member Bax because of its central role in regulating cytochrome c release in stressed cells. We found that heat shock caused a conformational change in Bax that leads to its translocation to mitochondria, stable membrane association, and oligomerization. All of these events were inhibited in cells that had elevated levels of Hsp70. Hsp70 did not physically interact with Bax in control or heat-shocked cells, indicating that Hsp70 acts to suppress signals leading to Bax activation. Hsp70 inhibited stress-induced JNK activation and inhibition of JNK with SP600125 or by expression of a dominant negative mutant of JNK-blocked Bax translocation as effectively as Hsp70 overexpression. Hsp70 did not protect cells expressing a mutant form of Bax that has constitutive membrane insertion capability or cells treated with a small molecule activator of apoptosome formation, indicating that it is unable to prevent cell death after mitochondrial disruption and caspase activation have occurred. These results indicate that Hsp70 blocks heat-induced apoptosis primarily by inhibiting Bax activation and thereby preventing the release of proapoptotic factors from mitochondria. Hsp70, therefore, inhibits events leading up to mitochondrial membrane permeabilization in heat-stressed cells and thereby controls the decision to die but does not interfere with cell death after this event has occurred.     

Hsp70 may protect cardiomyocytes from stress-induced injury by inhibiting Fas-mediated apoptosis

Expression of Hsp70 is an endogenous mechanism by which living cells adapt to stress and the protection of Hsp70 may interfere with the apoptotic machinery in a variety of ways. Here, we observed the change of Hsp70 expression in rat myocardium under stress and explored the protective effect of Hsp70 on the Fas-mediated pathway to cardiomyocyte apoptosis. The results showed that restraint stress led to cardiac dysfunction and structural damage of the myocardium, as well as activation of the Fas pathway. A similar increase in the Fas expression level, caspase-8/3 activity, and the apoptotic rate of the cardiomyocyte also were found, which indicated that Fas-mediated apoptosis of cardiomyocytes might be one of the mechanisms of cardiomyocyte injury induced by stress. Changes in Hsp70 levels and distribution occurred during the stress process, which correlated with the severity of myocardium injury. Heat preconditioning induced the upregulation of Hsp70 synthesis, which in turn may have mitigated subsequent restraint stress-induced damage, including electrocardiography (ECG) abnormality, myocardium damage, and cell death. Moreover, Hsp70 overexpression induced by heat preconditioning had no effect on Fas expression in the cardiomyocyte, but could inhibit activation of caspase-8/3 induced by the Fas signaling pathway and, as a result, prevent cell apoptosis. These results suggest that Hsp70 is capable of protecting the cardiomyocyte from stress-induced injury by inhibiting Fas-mediated apoptosis, and Hsp70 could be considered a target in future drugs to prevent cardiovascular injury caused by stress.     

A Jurkat T cell variant resistant to death receptor-induced apoptosis. Correlation with heat shock protein (Hsp) 27 and 70 levels

Ligation of Fas induces an apoptotic program in Jurkat cells (Jd). We describe a Jurkat T cell variant (Jr) which shows total resistance to Fas-mediated apoptosis but which exhibits sensitivity to non-death-receptor pro-apoptotic stimuli such as staurosporine. Resistance to Fas-induced apoptosis in Jr cells is correlated with high expression of Hsps. A prior heat-shock increases Hsp27 and 70 expression and protects Jd and Jr cells from Fas- and staurosporine-induced apoptosis. Staurosporine, but not the anti-Fas antibody CH11, abrogates constitutive Hsp70 expression at 37 degrees C and staurosporine also inhibit Hsp27 expression in Jd and Jr cells at 42 degrees C. These data suggest that constitutive expression of Hsp27 inhibits Fas-mediated apoptosis, but only induced expression of Hsp70 can protect T cells from staurosporine-induced apoptosis. Thus, Hsp27 could play a role in the regulation of death receptor-mediated apoptosis, while Hsp70 could regulate mitochondrial-dependent cell death.     

Hsp72 inhibits Fas-mediated apoptosis upstream of the mitochondria in type II cells

Heat shock protein 72 (Hsp72) inhibits apoptosis induced by some stresses that trigger the intrinsic apoptosis pathway. However, with the exception of TNFalpha-induced apoptosis, a role for Hsp72 in modulating the extrinsic pathway of apoptosis has not been clearly established. In this study, it was demonstrated that Hsp72 could inhibit Fas-mediated apoptosis of type II CCRF-CEM cells, but not type I SW480 or CH1 cells. Similar results were obtained when Fas ligand or an agonistic Fas antibody initiated the Fas apoptosis pathway. In CCRF-CEM cells, Hsp72 inhibited mitochondrial membrane depolarization and cytochrome c release but did not alter surface Fas expression or processing of caspase-8 and Bid, indicating that Hsp72 acts upstream of the mitochondria to inhibit Fas-mediated apoptosis. Thus, the ability of Hsp72 to inhibit Fas-mediated apoptosis is limited to type II cells where involvement of the intrinsic pathway is required for efficient effector caspase activation.     

Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism

Hsp27 inhibits mitochondrial injury and apoptosis in both normal and cancer cells by an unknown mechanism. To test the hypothesis that Hsp27 decreases apoptosis by inhibiting Bax, Hsp27 expression was manipulated in renal epithelial cells before transient metabolic stress, an insult that activates Bax, induces mitochondrial injury, and causes apoptosis. Compared with control, enhanced Hsp27 expression inhibited conformational Bax activation, oligomerization, and translocation to mitochondria, reduced the leakage of both cytochrome c and apoptosis-inducing factor, and significantly improved cell survival by >50% after stress. In contrast, Hsp27 down-regulation using RNA-mediated interference promoted Bax activation, increased Bax translocation, and reduced cell survival after stress. Immunoprecipitation did not detect Hsp27-Bax interaction before, during, or after stress, suggesting that Hsp27 indirectly inhibits Bax. During stress, Hsp27 expression prevented the inactivation of Akt, a pro-survival kinase, and increased the interaction between Akt and Bax, an Akt substrate. In contrast, Hsp27 RNA-mediated interference promoted Akt inactivation during stress. Hsp27 up- or down-regulation markedly altered the activity of phosphatidylinositol 3-kinase (PI3-kinase), a major regulator of Akt. Furthermore, distinct PI3-kinase inhibitors completely abrogated the protective effect of Hsp27 expression on Akt activation, Bax inactivation, and cell survival. These data show that Hsp27 antagonizes Bax-mediated mitochondrial injury and apoptosis by promoting Akt activation via a PI3-kinase-dependent mechanism.     

The protonophore CCCP induces mitochondrial permeability transition without cytochrome c release in human osteosarcoma cells

Mitochondrial permeability transition (MPT) and cytochrome c redistribution from mitochondria are two events associated with apoptosis. We investigated whether an MPT event obligatorily leads to cytochrome c release in vivo. We have previously shown that treatment of human osteosarcoma cells with the protonophore m-chlorophenylhydrazone (CCCP) for 6 h induces MPT and mitochondrial swelling without significant cell death. Here we demonstrate that release of cytochrome c does not occur and the cells remain viable even after 72 h of treatment with CCCP. Bax is not mobilized to mitochondria under these conditions. However, subsequent exposure of CCCP-treated cells to etoposide or staurosporine for 48 h results in rapid cell death and cytochrome c release that is accompanied by Bax association with mitochondria, demonstrating competency of these mitochondria to release cytochrome c with additional triggers. Our findings suggest that MPT is not a sufficient condition, in itself, to effect cytochrome c release.     

Selective inhibition of apoptosis by TPA-induced differentiation of U937 leukemic cells.

U937 leukemic cells treated for 24 h with 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), that induces their macrophagic terminal differentiation, become resistant to etoposide-induced apoptosis. Exposure of undifferentiated U937 cells to 50 microM etoposide for 6 h, that triggers apoptosis in 80% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Mcl-1 expression without modifying Bcl-2, Bcl-xL and Bax protein levels. All these events are inhibited in TPA-differentiated U937 cells that are also resistant to vinblastine-induced and Fas-mediated cell death. Interestingly, these cells are not inherently resistant to apoptosis induction. Exposure of TPA-differentiated U937 cells to 0.8 microg/ml cycloheximide for 24 h, that triggers apoptosis in 50% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Bcl-xL expression without modifying Bcl-2, Mcl-1 and Bax protein levels. All these events are not observed in undifferentiated cells treated in similar conditions. These results indicate that the apoptotic pathway that involves the release of cytochrome c from mitochondria and the cleavage of procaspases remains functional in TPA-differentiated cells.     

Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli

Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c-mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

E-cadherin decreased human breast cancer cells sensitivity to staurosporine by up-regulating Bcl-2 expression

E-cadherin, a well-characterized cell-cell adhesion molecule, executes multifunction roles on cell behaviors. However, its effect on chemo-resistance remains controversial. In this study, we found that E-cadherin positive breast cell lines were less sensitive to staurosporine compared to E-cadherin negative ones. Next, we substantiated that the expression of E-cadherin in MDA-MB-435 cells could partly counteract the cytotoxic effect induced by staurosporine through a series of apoptosis assay. The resistance of E-cadherin over-expressing cells to staurosporine may due to the up-regulation of Bcl-2/Bax ratio. When E-cadherin interference plasmids were transfected into MCF-7 cells, Bcl-2 expression was down-regulated. Moreover, perturbation of E-cadherin function by blocking the cell-cell contact resulted in decreased cellular levels of Bcl-2 protein expression. All these results demonstrated the chemo-resistance function of E-cadherin in the condition of staurosporine treatment, therefore, might contribute effective therapeutic strategies in breast carcinoma.     

Endogenous bax translocation in SH-SY5Y human neuroblastoma cells and cerebellar granule neurons undergoing apoptosis

Changes at the mitochondria are an early, required step in apoptosis in various cell types. We used western blot analysis to demonstrate that the proapoptotic protein Bax translocated from the cytosolic to the mitochondrial fraction in SH-SY5Y human neuroblastoma cells undergoing staurosporine- or EGTA-mediated apoptosis. Levels of mitochondrial Bax increased 15 min after staurosporine treatment. In EGTA-treated cells, increased levels of mitochondrial Bax were seen at 4 h, consistent with a slower onset of apoptosis in EGTA versus staurosporine treatments. We also demonstrate the concomitant translocation of cytochrome c from the mitochondrial to the cytosolic fractions. We correlated these translocations with changes in caspase-3-like activity. An increase in caspase-3-like activity was evident 2 h after staurosporine treatment. Inhibition of the mitochondrial permeability transition had no effect on Bax translocation or caspase-3-like activity in staurosporine-treated SH-SY5Y cells. In primary cultures of cerebellar granule neurons undergoing low K(+)-mediated apoptosis, Bax translocation to the mitochondrial fraction was evident at 3 h. Cytochrome c release into the cytosol was not significant until 8 h after treatment. These data support a model of apoptosis in which Bax acts directly at the mitochondria to allow the release of cytochrome c.     

Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of Bcl-2 protein family.

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.     

PIG11与热休克蛋白60的相互作用介导肝癌HepG2细胞凋亡

为了探讨候选肝癌抑癌蛋白PIG11(p53-induced gene 11,PIG11)诱导细胞凋亡的机制,首次在HepG2细胞株中鉴定了11个PIG11结合蛋白,热休克蛋白60(heat shock protein 60,Hsp60)为其中之一．采用免疫共沉淀联合Western blot 技术对Hsp60进行了验证．用Western blot检测其蛋白质表达,结果显示：pLXSN-PIG11-HepG2细胞中Hsp60蛋白表达较pLXSN-HepG2、HepG2细胞组下调(n=3,P < 0.01)．选取与Hsp60关系密切的Bax蛋白进行研究,Western blot结果显示PIG11高表达可引起胞浆Bax向线粒体转位．以上结果表明,PIG11蛋白能与HepG2细胞中的Hsp60结合,促进Hsp60-Bax的分离,引起Bax从胞液到线粒体转位,激活线粒体凋亡途径,这可能是其诱导HepG2细胞凋亡的主要机制之一．     

Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways.

Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been isolated from a variety of tumorigenic and nontransformed cell lines. Because of the importance of blood vessel growth to cell and tissue development, we have examined VEGF gene expression in a variety of mouse tissues and rodent models of cellular differentiation. Using a cloned murine VEGF cDNA we show that VEGF mRNA is expressed at relatively low levels in many adult mouse tissues examined. However, this message is dramatically induced in two models of cell differentiation: 3T3-adipose conversion and C2C12 myogenic differentiation. VEGF protein secretion is also induced in adipocyte differentiation. VEGF mRNA is markedly regulated in a pheochromocytoma (PC12) cell model of transformation and differentiation. The transformed undifferentiated cells express moderate levels of VEGF mRNA and this expression is virtually extinguished when cells differentiate into non-malignant neuron-like cells. Experiments employing phorbol esters and cAMP analogues indicate that VEGF mRNA expression is stimulated in preadipocytes by both protein kinase C and protein kinase A-mediated pathways. These results suggest that VEGF mRNA levels are closely linked to the process of cellular differentiation; they also clearly demonstrate that expression of this angiogenic factor is specifically regulated in a transformed cell line, possibly via aberrant activation of cellular second messenger pathways.     

Mouse 3T3-L1 cells acquire resistance against oxidative stress as the adipocytes differentiate via the transcription factor FoxO

Repression of excessive increase and enlargement of adipocytes that is closely associated with obesity is effective in the prevention and treatment of metabolic syndrome. Generally, apoptosis is induced in cells via a wide variety of intracellular or extracellular substances, and recently, it has been suggested that the FoxO subfamily is involved in the induction of apoptosis. We aimed to elucidate the mechanism of FoxO-mediated apoptosis-induction in the adipocytes under the reactive oxygen species (ROS) stimulus. The treatment of differentiated and undifferentiated 3T3-L1 cells with glucose oxidase (GOD), an enzyme that generates H₂O₂, induced apoptosis and led to the accumulation of 8-OHdG. Apoptosis analysis revealed that GOD treatment induced apoptosis in differentiated 3T3-L1 cells less efficiently than in undifferentiated preadipocytes. GOD remarkably increased the levels of Bad, Bax, and Bim—the genes that are actively involved in cell apoptosis. GOD treatment also increased the expression of FoxO3a mRNA and protein. The introduction of FoxO3a-siRNA into 3T3-L1 cells suppressed the oxidative stress-induced expression of Bim mRNA, as well as the GOD-induced apoptosis. Furthermore, the expression of MnSOD, Cu/ZnSOD, and catalase, as well as of FoxO, increased significantly along with the progression of adipocyte differentiation. These results indicated that ROS-induced apoptosis in undifferentiated 3T3-L1 cells via the expression of FoxO3a, whereas FoxO expression suppressed the ROS-induced apoptosis in differentiated 3T3-L1 cells via the expression of ROS-scavenging enzymes.     

The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation

The mitochondrial cell death pathway is known for its role in signaling apoptosis. Here, we describe a novel function for the mitochondrial cell death pathway in signaling initiation of differentiation in the developing lens. Most remarkably, we induced lens cell differentiation by short-term exposure of lens epithelial cells to the apoptogen staurosporine. Activation of apoptosis-related pathways induced lens epithelial cells to express differentiation-specific markers and to undergo morphogenetic changes that led to formation of the lens-like structures known as lentoids. The fact that multiple stages of differentiation are expressed at a single stage of development in the embryonic lens made it possible to precisely determine the timing of expression of proteins associated with the apoptotic pathway. We discovered that there was high expression in the lens equatorial epithelium (the region of the lens in which differentiation is initiated) of pro-apoptotic molecules such as Bax and Bcl-x(S) and release of cytochrome c from mitochondria. Furthermore, we found significant caspase-3-like activity in the equatorial epithelium, yet this activity was far lower than that associated with lens cell apoptosis. These apoptotic pathways are likely regulated by the concurrent expression of prosurvival molecules, including Bcl-2 and Bcl-x(L); phosphorylation of Bad; and high expression of inhibitor of apoptosis proteins chicken IAP1, IAP3, and survivin. This finding suggests that prosurvival pathways allow pro-apoptotic molecules to function as molecular switches in the differentiation process without tipping the balance toward apoptosis. We call this process apoptosis-related Bcl-2- and caspase-dependent (ABC) differentiation.     

Differentiated Paju cells have increased resistance to toxic effects of potassium ionophores

In this study we have investigated the impact of differentiation of neuronal cells on their sensitivity to microbial toxins. We used the human neural crest-derived tumor cell line Paju, which can be induced to differentiation in vitro by treatment with phorbol 12-myristate 13-acetate. Addition of the highly toxic potassium ionophores cereulide (4.5 and 9.0 ng/ml) or valinomycin (20 ng/ml), to cultures of undifferentiated Paju cells caused collapse of the mitochondrial membrane potential - measured with the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazole carbocyanine iodide (JC-1) followed by detachment of the cells and their apoptotic death. After induced differentiation of the Paju cells, their mitochondria retained the membrane potential upon exposure to the toxins and the cells displayed increased resistance to apoptosis as compared with undifferentiated cells. This effect may be caused by an elevated expression of the anti-apoptotic protein Bcl-2 and of the neuroprotective factor, stanniocalcin, in differentiated cells.