Our data demonstrate that HuR plays a key role in the enhancement of caspase-dependent apoptosis induced by extreme stress conditions. Although it is well established that HuR affects cell fate through its role as an RNA-binding protein (Brennan and Steitz, 2001
; Abdelmohsen et al., 2007
), evaluating the possibility that HuR also functions through an alternative pathway has been elusive. In this study, we identify a novel regulatory mechanism through which HuR participates in the apoptotic pathway. We show that HuR potentiates the apoptotic response by undergoing a caspase-mediated cleavage at a specific aspartate residue located in its HNS region. This cleavage activity occurs only after HuR enters the cytoplasmic compartment in a pp32/PHAP-I–dependent manner. HuR exerts its proapoptotic effects through its association with pp32/PHAP-I, a known enhancer of the apoptosome (Jiang et al., 2003
; Hill et al., 2004
). Thus, we propose a model whereby in response to a lethal stress, HuR accumulates in the cytoplasm, where it undergoes caspase-mediated cleavage. This cleavage appears to be important for pp32/PHAP-I–mediated enhancement of the caspase-dependent apoptosis ().
Figure 9. Model of how HuR protein participates in the pp32/PHAP-I–mediated enhancement of apoptosome activity. Upon induction of stress-induced apoptosis, HuR and its partner pp32/PHAP-I translocate together to the cytoplasm. In the cytoplasm, HuR is targeted (more ...)
Previous studies had shown that HuR regulates the stability and/or the translation of mRNAs encoding for pro- (p21Waf1
, and p53) and antiapoptotic (proTα) proteins (Schiavone et al., 2000
; Wang et al., 2000
; Kullmann et al., 2002
; Mazan-Mamczarz et al., 2003
; Lal et al., 2005
). However, no explanation has been given as to how HuR may be active in these two opposite processes. In response to different extracellular stimuli, HuR associates with mRNAs encoding for proteins that are involved in different mechanisms such as cell cycle, cell differentiation, and stress response (van der Giessen et al., 2003
; Abdelmohsen et al., 2007
). Thus, the cellular function of HuR could vary depending on the nature of the stimuli applied. Consistent with this idea, we noted that knocking down the expression of HuR in cells treated with a lethal dose of STS causes a significant delay in caspase-dependent apoptosis (). This observation implies that HuR promotes cell death under conditions in which the integrity of the cell is altered beyond repair. In contrast, it has been seen that upon mild stress, HuR promotes death resistance by affecting the translation of the mRNA encoding for proTα (Lal et al., 2005
). Together, these and our findings argue that HuR can function during the two characteristic steps of the cell stress response. First, it participates in processes that activate prosurvival pathways to facilitate cell recovery (proTα). Then, if the stress becomes persistent, HuR promotes cell death.
To our surprise, while delineating the cellular mechanism by which HuR promotes apoptosis, we observed that HuR itself is cleaved in a caspase-dependent manner. We found that although only caspase-7 is able to cleave HuR in vitro (), in HeLa cells, this proteolytic activity involves both caspase-7 and -3 (). This caspase-mediated cleavage generates fragments of 24 kD (HuR-CP1) and 9 kD (HuR-CP2; and ). These data are consistent with the well-established notion that the cleavage cascade requiring a series of caspases represents a key process needed to generate active proapoptotic proteins. Our results argue that cytoplasmic HuR becomes a member of this family of cell death effectors that are induced by caspase-mediated cleavage. HuR has features of a caspase target protein, such as a specific aspartate residue (D226) cleavage site as well as the generation of active CPs ( and ). Surprisingly, mutating this residue to alanine not only protected HuR protein from proteolysis () but also generated a noncleavable isoform that is able to interfere with its role in apoptosis ( and ). Together, these data suggest that as soon as a lethal stress is applied, HuR partially migrates to the cytoplasm and participates in activation of the apoptotic machinery to promote cell death ().
Activation of the apoptosome is a cytoplasmic event that involves Apaf-1 oligomerization upon cyt c
release from the mitochondria (Cain et al., 2002
). It has been shown that HuR associates with the apoptosome activator pp32/PHAP-I through its HNS and RRM3 motifs (Brennan et al., 2000
). Here, we observed that HuR diffuses and colocalizes in the cytoplasm with pp32/PHAP-I upon STS treatment (). Knocking down the expression of pp32/PHAP-I protein significantly reduces the cytoplasmic accumulation of HuR. This is consistent with previously published data showing that pp32/PHAP-I is part of the HuR complex that translocates to the cytoplasm under different stress conditions (Gallouzi et al., 2000
, 2001). Likewise, we found that the percentage of total HuR that is cleaved in the cytoplasm () corresponds to the same amount that associates with pp32/PHAP-I (Brennan et al., 2000
). Thus, it is possible that by cleaving HuR within its HNS motif, caspase-7/-3 induce the release of an active part of HuR that participates in pp32/PHAP-I–mediated apoptosome activation. This is likely the case because overexpressing the noncleavable isoform of HuR, HuRD226
A, in HeLa cells delays the STS-induced cell death and stabilizes the pp32/PHAP-I–HuR complex in the cytoplasm (). Moreover, interfering with apoptosome activation by depleting the expression of pp32/PHAP-I using RNAi significantly reduces the cleavage of both HuR and PARP proteins (). These and our in vitro depletion/rescue experiments () argue that HuR plays a critical role in activating the apoptosome complex via its association with pp32/PHAP-I. The results described herein shed light on a new player involved in the stimulatory effect of pp32/PHAP-I on the apoptosome. However, pp32/PHAP-I does not directly interact with any of the apoptosome components (Hill et al., 2004
), and an apoptosome-activating mechanism remains unknown. It was demonstrated that the same C-terminal acidic domain of pp32/PHAP-I that is critical for its proapoptotic activity (Hill et al., 2004
) is also involved in its association with HuR (Brennan et al., 2000
). Our data suggest that the cleavage of HuR plays an important role in the proapoptotic function of pp32/PHAP-I.
Our data showing that RNAi-mediated depletion of pp32/PHAP-I leads to a significant reduction in PARP and HuR cleavage () argue that an active apoptosome is required for HuR proteolysis. These observations suggest two possibilities: (1) at early stages of the cell stress response, a basal apoptosome activity could be present in HeLa cells, resulting in caspase-7/-3 activation before the apoptosome reaches its full capacity; and (2) caspase-7/-3 could be activated in an apoptosome-independent manner. Our time-course experiments, in which we followed caspase-3 processing during cell response to STS treatment, favor the first possibility. We observed that at early stages (<100 min) of apoptotic response, when HuR localizes to the cytoplasm, the apoptosome harbors a basal level of activity (). However, at later stages (>100 min), we observed a significant increase in caspase-3 activation (), correlating with the appearance of HuR-CP1 (). These observations imply that the small amount of activated caspase-7/-3 at early stages of apoptosis could be sufficient to cleave HuR, generating the HuR-proapoptotic active forms. This form will then participate in the amplification loop that enhances the apoptosome activity, which feeds down on more HuR processing during apoptosis.
Although our data argue that HuR promotes apoptosis through a mechanism that is independent of its RNA-binding activity, we cannot rule out the possibility that the HuR CPs enhance apoptosis by also posttranscriptionally affecting the expression of some proapoptotic mRNAs. Indeed, recent observations indicated that the depletion of HuR results in a significant decrease in the expression of caspase-9 mRNA and protein (unpublished data). This suggested that HuR, through its RNA-binding activity, could also affect apoptosome formation. Thus, through their RNA-binding motifs, HuR-CP1 (RRM1–RRM2) or HuR-CP2 (RRM3; ) could affect the stability and/or the translation of proapoptotic mRNAs. This suggests a model in which the caspase-mediated HuR cleavage represents the regulatory step during which HuR switches from an antiapoptotic function at early steps of the stress response to a proapoptotic role when cell death is unavoidable. This raises the possibility that in cancer cells, the cleavage of HuR could be altered, such as by a mutation to yield a noncleavable isoform and/or a defect in caspase-7/-3 activity, interfering with its RNA- and protein-mediated proapoptotic function. Therefore, investigating the effect of cell transformation on HuR functions as a proapoptotic factor will open the door to the possibility of defining posttranscriptional regulators as potential targets for cancer therapy.