Catherine Paul

Hsp27 as a Negative Regulator of Cytochrome c Release

ABSTRACT We previously showed that Hsp27 protects against apoptosis through its interaction with cytosolic cytochrome c. We have revisited this protective activity in murine cell lines expressing different levels of Hsp27. We report that Hsp27 also interferes, in a manner dependent on level of expression, with the release of cytochrome c from mitochondria. Moreover, a decreased level of endogenous Hsp27, which sensitized HeLa cells to apoptosis, reduced the delay required for cytochrome c release and procaspase 3 activation. The molecular mechanism regulating this function of Hsp27 is unknown. In our cell systems, Hsp27 is mainly cytosolic and only a small fraction of this protein colocalized with mitochondria. Moreover, we show that only a very small fraction of cytochrome c interacts with Hsp27, hence excluding a role of this interaction in the retention of cytochrome c in mitochondria. We also report that Bid intracellular relocalization was altered by changes in Hsp27 level of expression, suggesting that Hsp27 interferes with apoptotic signals upstream of mitochondria. We therefore investigated if the ability of Hsp27 to act as an expression-dependent modulator of F-actin microfilaments integrity was linked to the retention of cytochrome c in mitochondria. We show here that the F-actin depolymerizing agent cytochalasin D rapidly induced the release of cytochrome c from mitochondria and caspase activation. This phenomenon was delayed in cells pretreated with the F-actin stabilizer phalloidin and in cells expressing a high level of Hsp27. This suggests the existence of an apoptotic signaling pathway linking cytoskeleton damages to mitochondria. This pathway, which induces Bid intracellular redistribution, is negatively regulated by the ability of Hsp27 to protect F-actin network integrity. However, this upstream pathway is probably not the only one to be regulated by Hsp27 since, in staurosporine-treated cells, phalloidin only partially inhibited cytochrome c release and caspase activation. Moreover, in etoposide-treated cells, Hsp27 still delayed the release of cytochrome c from mitochondria and Bid intracellular redistribution in conditions where F-actin was not altered.

Hsp27 protects mitochondria of thermotolerant cells against apoptotic stimuli

Abstract 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.

Differential regulation of HSP27 oligomerization in tumor cells grown in vitro and in vivo.

HSP27 form oligomeric structures up to 800 Kda. In cultured cells, the equilibrium between small and large oligomers shifted towards smaller oligomers when phosphorylated on serine residues. To further explore HSP27 structural organization and its repercussion in HSP27 antiapoptotic and tumorigenic properties, we transfected colon cancer REG cells with wild type HSP27 and two mutants in which the phosphorylatable serine residues have been replaced by alanine (to mimic the non phosphorylated protein) or aspartate (to mimic the phosphorylated protein). In growing cells, wild type and alanine mutant formed small and large oligomers and demonstrated antiapoptotic activity while aspartate mutant only formed small multimers and had no antiapoptotic activity. In a cell-free system, only large oligomeric structures interfered with cytochrome c-induced caspase activation, thereby inhibiting apoptosis. The inability of the aspartate mutant to form large oligomers and to protect tumor cells from apoptosis was overcome by growing the cells in vivo, either in syngeneic animals or nude mice. These observations were reproduced by culturing the cells at confluence in vitro. In conclusion (1) large oligomers are the structural organization of HSP27 required for its antiapoptotic activity and (2) cell-cell contacts induce the formation of large oligomers, whatever the status of phosphorylatable serines, thereby increasing cell tumorigenicity.

Dynamic processes that reflect anti-apoptotic strategies set up by HspB1 (Hsp27).

Human HspB1 (also denoted Hsp27) is an oligomeric anti-apoptotic protein that has tumorigenic and metastatic roles. To approach the structural organizations of HspB1 that are active in response to apoptosis inducers acting through different pathways, we have analyzed the relative protective efficiency induced by this protein as well its localization, oligomerization and phosphorylation. HeLa cells, that constitutively express high levels of HspB1 were treated with either etoposide, Fas agonist antibody, staurosporine or cytochalasin D. Variability in HspB1 efficiency to interfere with the different apoptotic transduction pathways induced by these agents were detected. Moreover, inducer-specific dynamic changes in HspB1 localization, native size and phosphorylation were observed, that differed from those observed after heat shock. Etoposide and Fas treatments gradually shifted HspB1 towards large but differently phosphorylated oligomeric structures. In contrast, staurosporine and cytochalasin D induced the rapid but transient formation of small oligomers before large structures were formed. These events correlated with inducer-specific phosphorylations of HspB1. Of interest, the formation of small oligomers in response to staurosporine and cytochalasin D was time correlated with the rapid disruption of F-actin. The subsequent, or gradual in the case of etoposide and Fas, formation of large oligomeric structures was a later event concomitant with the early phase of caspase activation. These observations support the hypothesis that HspB1 has the ability, through specific changes in its structural organization, to adapt and interfere at several levels with challenges triggered by different signal transduction pathways upstream of the execution phase of apoptosis.

Small stress proteins: novel negative modulators of apoptosis induced independently of reactive oxygen species.

The execution phase of the apoptotic cell death process occurs throughout the proteolytic activation of proteolytic enzymes called caspases (Nicholson and Thornberry 1997; Thornberry and Lazebnik 1998). Several different pathways can lead to the activation of caspases, among them, one can cite the death receptors (i.e. Fas) (Scaffidi et al. 1998) and mitochondria pathways (Reed 1997; Green and Reed 1998). When activated by ligand binding, death receptors (i.e. Fas) recruit adapter polypeptides (i.e. FADD) that interact with and subsequently activate pro-caspases (i.e. pro-caspase 8) or trigger a signal transduction pathway that activates specific genes (i.e. the DAXX/ASK1/JNK pathway). In contrast, in the mitochondria pathway, different inducers have the ability to induce the release in the cytoplasm of different proteins present in mitochondria, such as cytochrome c, apoptosis-inducing factor (AIP), Hsp60, HsplO, adenylate kinase, Smac/Diablo as well as the fraction of pro-caspase 2,3,8, and 9 present in mitochondria (Kluck et al. 1997; Reed 1997; Yang et al. 1997; Kohler et al. 1999; Samali et al. 1999a; Susin et al. 1999a,1999b; Xanthoudakis et al. 1999; Du et al. 2000; Verhagen et al. 2000). Once it has been released from the mitochondria, cytochrome c interacts with Apaf-1 in the presence of ATP/dATP. This results in the formation of the apoptosome complex which recruits and activates pro-caspase 9 which subsequently activates pro-caspase 3 (Li et al. 1997; Saleh et al. 1999). The release of cytochrome c from the mitochondria is a caspase-independent early phenomenon that precedes mitochondrial membrane potential loss (Bossy-Wetzel et al. 1998). This phenomenon may be induced by conformational changes of Bax (Desagher et al. 1999) or by BAK oligomerization induced by tBID, a membrane-targeted death ligand (Wei et al. 2000). Stress-induced apoptosis usually occurs through the activation of the mitochondria pathway. This is particularly the case when mild oxidative or heat stresses are considered (Samali et al. 2000).

Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress–mediated apoptosis: possible implication in asthma

Abstract Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27.

S-Nitrosylation of the Death Receptor Fas Promotes Fas Ligand-Mediated Apoptosis in Cancer Cells

BACKGROUND & AIMS Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fas-mediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS Colon and mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NO-mediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate-Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CONCLUSIONS We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.

Effect of Plasma Phospholipid Transfer Protein Deficiency on Lethal Endotoxemia in Mice

Lipopolysaccharides (LPS) are components of Gram-negative bacteria. The cellular response from the host to LPS is mediated through stepwise interactions involving the lipopolysaccharide-binding protein (LBP), CD14, and MD-2, which produces the rearrangement of TLR4. In addition to LBP, the lipid transfer/lipopolysaccharide-binding protein gene family includes the phospholipid transfer protein (PLTP). Here we show that the intravascular redistribution of LPS from the plasma lipoprotein-free fraction toward circulating lipoproteins is delayed in PLTP-deficient mice. In agreement with earlier in vitro studies, which predicted the neutralization of the endotoxic properties of LPS when associated with lipoproteins, significant increases in the plasma concentration of proinflammatory cytokines were found in PLTP-deficient as compared with wild type mice. Similar inflammatory damage occurred in tissues from wild type and PLTP-deficient mice 24 h after one single intraperitoneal injection of LPS but with a more severe accumulation of red blood cells in glomeruli of LPS-injected PLTP-deficient mice. Complementary ex vivo experiments on isolated splenocytes from wild type and PLTP-deficient mice further supported the ability of cell-derived PLTP to prevent LPS-mediated inflammation and cytotoxicity when combined with lipoprotein acceptors. Finally, PLTP deficiency in mice led to a significant increase in LPS-induced mortality. It is concluded that increasing circulating levels of PLTP may constitute a new and promising strategy in preventing endotoxic shock.

Comparison of the protective activities generated by two survival proteins: Bcl-2 and Hsp27 in L929 murine fibroblasts exposed to menadione or staurosporine.

Hsp27 and Bcl-2 are survival proteins that protect against cell death. We have compared the specific protective activity (protection per number of molecules expressed) mediated by these proteins when they are expressed in L929 murine fibroblasts. We found that Hsp27 and Bcl-2 efficiently delayed the cytotoxicity generated by menadione. Both proteins interfered with the mitochondria membrane potential collapse, the reactive oxygen species (ROS) burst and the decrease in glutathione level induced by this oxidant. In untreated cells, both proteins decreased the ROS levels and raised the glutathione cellular content. Taking their levels of expression into account, we concluded that Bcl-2 was much more active than Hsp27 for counteracting the above-mentioned menadione effects, and for modulating the ROS and glutathione levels in untreated cells. Both Hsp27 and Bcl-2 also conferred cellular resistance to staurosporine, a kinase inhibitor that induces apoptosis without generating an oxidative stress. In this case, Bcl-2 was again much more active than Hsp27. Fractionation studies indicated that, in L929 cells, Hsp27 is essentially present in the cytosol while Bcl-2 is membrane and mitochondria-associated. Hence, despite some similar cellular effects resulting from their expression, Bcl2 and Hsp27 polypeptides protect against oxidative stress and apoptosis with different efficiencies and by using different mechanisms.

Small stress proteins: modulation of intracellular redox state and protection against oxidative stress.

Small stress proteins (also denoted small heat shock proteins: sHsp) are oligomeric phospho-polypeptides (Arrigo and Welch 1987; Arrigo et al. 1988; de Jong et al. 1993; Buchner et al. 1998) which increase the cell resistance to different types of stress, including heat shock and oxidative stress (reviewed in Arrigo and Landry 1994; Arrigo 1998, 2000; Arrigo and Preville 1999). In vitro, these proteins have been described as ATP-independent chaperones which counteract protein denaturation and help in the refolding of misfolded polypeptides (Jakob et al. 1993; Jakob and Buchner 1994; Ehrnsperger et al. 2000). Except for a role in maintaining cytoskeletal architecture, little information was available concerning the mode of action of these proteins in vivo. Recently, it has been proposed that large sHsp oligomers bind to misfolded polypeptides (Ehrnsperger et al. 1997; Lee et al. 1997) and present them to ATP-dependent protein chaperones (Hsp70, Hsp40, Hsp90 and co-chaperones) (see Haslbeck and Buchner, Chap. 3, this Vol.).