André-Patrick Arrigo

Hsp27 negatively regulates cell death by interacting with cytochrome c

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

SMALL STRESS PROTEINS AS NOVEL REGULATORS OF APOPTOSIS : HEAT SHOCK PROTEIN 27 BLOCKS FAS/APO-1- AND STAUROSPORINE-INDUCED CELL DEATH

Small stress protein expression enhances the survival of mammalian cells exposed to numerous injuries that induce necrotic cell death. The cell surface receptor Fas/APO-1 and its ligand have been recently identified as important mediators of apoptosis. Here, we show that constitutive expression of human heat shock protein (hsp)27 in murine L929 cells blocks Fas/APO-1-mediated cell death. Expression of human hsp27 prevented anti-APO-1-induced DNA fragmentation and morphological changes. These results strongly suggest that human hsp27 acts as a cellular inhibitor of Fas/APO-1-induced apoptosis. We also report that the expression of small stress proteins from different species, such as human hsp27, Drosophila Dhsp27, or human αB-crystallin, confers resistance to apoptotic cell death induced by staurosporine, a protein kinase C inhibitor. Hence, small stress proteins are novel regulators that are able to block apoptosis induced by different pathways.

Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB‐crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha‐mediated ROS increment, such as NF‐kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3‐ras cells. This phenomenon was essential for the shsp‐mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB‐crystallin against TNFalpha‐mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.

GENE EXPRESSION AND THE THIOL REDOX STATE

The intracellular redox status is a tightly regulated parameter which provides the cell with an optimal ability to counteract the highly oxidizing extracellular environment. Intracellular redox homeostasis is regulated by thiol-containing molecules, such as glutathione and thioredoxin. Essential cellular functions, such as gene expression, are influenced by the balance between pro- and antioxidant conditions. The mechanism by which the transcription of specific eukaryotic genes is redox regulated is complex, however, recent findings suggest that redox-sensitive transcription factors play an essential role in this process. This review is focused on the recent knowledge concerning some eukaryotic transcription factors, whose activation and DNA binding is controlled by the thiol redox status of the cell.

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.

Actin cytoskeleton and small heat shock proteins: how do they interact?

Abstract Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin–sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers.

hsp27 as a Switch between Differentiation and Apoptosis in Murine Embryonic Stem Cells

Small stress proteins are developmentally regulated and linked to cell growth and differentiation. The early phase of murine embryonic stem (ES) cell differentiation, characterized by a gradual growth arrest, is accompanied with hsp27 transient accumulation. This differentiation process also correlated with changes in hsp27 phosphorylation and oligomerization. The role of hsp27 was investigated in ES clones stably transfected with murine or human hsp27 genes, placed in sense or antisense orientation. Several clones were obtained that either underexpressed endogenous murine hsp27 or overexpressed murine or human hsp27. Maintained undifferentiated, these clones showed similar growth rates. We report here that hsp27 constitutive overexpression enhanced the differentiation-mediated decreased rate of ES cell proliferation but did not alter morphological changes. In contrast, hsp27 underexpression, which attenuated cell growth arrest, induced differentiation abortion because of an overall cell death by apoptosis. Recently, we showed that hsp27 interfered with cell death probably because of its ability to modulate intracellular glutathione. hsp27 accumulation during ES cell differentiation was also correlated with an increase in glutathione, which was attenuated by hsp27 down-expression. Hence, hsp27 transient expression seems essential for preventing differentiating ES cells from undergoing apoptosis, a switch that may be redox regulated.

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.

Autophagy activation by NFκB is essential for cell survival after heat shock

The heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFκB transcription factor is activated during the recovery period after heat shock. In this study, we analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent (p65/RelA-depleted) cells. We demonstrate for the first time that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery and cell death is strongly increased in NFκB incompetent cells. Moreover, if autophagy is artificially induced in these cells, the cytotoxicity of heat shock is turned back to normal. We show that despite a post-heat shock increase of Beclin 1 level in NFκB competent cells, neither Beclin 1/class III PI3K complex, Bcl2/BclXL nor mTOR kinase are NFκB targets whose modulation of expression could be responsible for NFκB activation of autophagy during heat shock recovery. In contrast, we demonstrate that aberrantly folded/aggregated proteins are prime events in the signaling pathway leading to NFκB mediated autophagy after heat shock. Hence, our findings demonstrate that NFκB-induced autophagy during heat shock recovery is an additional cell response to HS-induced protein denaturation/aggregation; this mechanism increases cell survival, probably through clearance of irreversibly damaged proteins.