Báo cáo khoa học: ERK and cell death: cadmium toxicity, sustained ERK activation and cell death pot

MINIREVIEW ERK and cell death: cadmium toxicity, sustained ERK activation and cell death Patrick Martin and Philippe Pognonec CNRS FRE3094, Universite ´ de Nice Sophia Antipolis, Nice, France Introduction Extracellular signal-related kinase (ERK) is among the most studied proteins, and is involved in many aspects of cell physiology [1]. Discovered 25 years ago [2], ERK actually consists of two closely related proteins in vertebrates: ERK1 and ERK2. These proteins are encoded by two related, but distinct, genes [3,4]. ERK is commonly considered as a key player in mitogenic signaling following growth factor stimulation [5,6]. The functional specificities of ERK1 and ERK2 have been investigated for many years, and despite earlier results suggesting differences, it now appears that both are functionally indistinguishable [7,8]. The originally reported specificities turned out to be a reflection of their relative abundances [7]. This is also supported by the observation that some species harbor only one ERK gene [7], and by the demonstration that ERK1 and -2 do functionally compensate for one another [9]. Nevertheless, thousands of reports (as of today a Medline search with the term ‘ERK [ti]’ produces 3501 articles) have shown that ERK is a central kinase in signal transduction pathways (reviewed in [10]). Its role in cellular proliferation has been extensively investi- gated, and its ability to protect cells against apoptosis is also widely accepted. The kinetics of ERK activation are biphasic, with a rapid and transient burst of activa- tion (phosphorylation on Tyr and Thr residues) within Keywords cadmium; ERK; PKC; sustained activation; ZIP8 Correspondence P. Pognonec, Transcriptional Regulation and Differentiation, CNRS FRE3094, Universite ´ de Nice, Parc Valrose, 06108 Nice cedex 2, France Tel ⁄ Fax: +33 492 07 64 13 E-mail: pognonec@unice.fr (Received 18 June 2009, revised 30 July 2009, accepted 15 August 2009) doi:10.1111/j.1742-4658.2009.07369.x Extracellular signal-related kinase (ERK) is a key player in cell signaling. After 25 years of investigation, ERK has been associated with every major aspect of cell physiology. Cell proliferation, cell transformation, protection against apoptosis, among others, are influenced by ERK function. Surpris- ingly, ERK has also been associated with two apparently opposing pro- cesses. The involvement of ERK in cell proliferation has been extensively described, as well as its function in postmitotic cells undergoing differentia- tion. The analysis of these apparent discrepancies has led to a more precise understanding of the multiple functions and regulations of ERK. More recently, several groups have identified a new and unexpected role for ERK. Although being accepted as an important player in the protection against cell death by apoptosis, it is now clear that ERK can also be directly linked to cell death signaling. Here, we review the role of ERK in cell response to cadmium and its association with cell toxicity. In this sys- tem, ERK is subjected to a continuous activation that can last for days, which ultimately results in cell death. Cadmium entry into cells is responsi- ble for this sustained ERK activation, probably via reactive oxygen species production, and protein kinase C has a negative action on this cadmium- dependent ERK activation by modulating cadmium entry into cells. Abbreviations ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PMA, 4b-phorbol 12-myristate 13-acetate; ROS, reactive oxygen species. FEBS Journal 277 (2010) 39–46 ª 2009 The Authors Journal compilation ª 2009 FEBS 39 minutes of stimulation, followed by a second and more stable activation that can last for a few hours [11]. The multiple facets of ERK functions have been linked to the kinetics of activation [12], subcellular localization [13] and scaffolding proteins [14]. Cadmium poisoning and sustained ERK activation Cadmium is a nonessential metal, recognized as a potent environmental pollutant. Its very long biologi- cal half life (15–30 years) makes it particularly toxic, as it accumulates in different organs over time, ulti- mately resulting in diseases such as kidney failure and ⁄ or bone injuries. The World Health Organization [15] defines the critical cadmium concentration in the kidney cortex at 200 mgÆkg –1 wet cortex, which approximately corresponds to a 1 mm cadmium con- centration. Above this threshold, kidney dysfunction appears. It was recently discovered that in cells sub- jected to a low-dose cadmium poisoning, ERK dis- plays a very unconventional activation pattern. In all cell types tested (primary mouse and rat fibroblasts, kidney-derived epithelium cell lines, bone marrow- derived macrophages, primary calvaria cells, HeLa cells, HEK293 cells, C2C12 cells), a strong ERK acti- vation was observed 16–24 h after the addition of micromolar concentrations of cadmium. This activa- tion remained high, for up to 6 days after cadmium treatment, until the cells died [16]. ERK activation has been associated with protection against apoptosis when cells are subjected to stressful conditions [17]. Thus, the cadmium-induced sustained activation of ERK could be a response of the cells to survive following cadmium poisoning. However, treatment of intoxicated cells with U0126, a potent and specific mitogen- activated protein kinase ⁄ ERK kinase (MEK) inhibitor that results in the complete inhibition of ERK activa- tion, following cadmium exposure does not increase cell death. Rather, a low level of cadmium is not as toxic to cells in the presence of the MEK inhibitor as it is in its absence. In recent years, a similar observa- tion has been reported by several groups, in experi- ments performed using relatively low concentrations of cadmium (generally between 1 and 10 lm) [16,18–21]. This phenomenon is illustrated in Fig. 1 and has been described in detail elsewhere [16]. It should be noted that the decrease in cell number shown in Fig. 1 fol- lowing cadmium treatment is the result of cell death and not due to an inhibition of cell proliferation, as determined by cell counting as well as quantification of loss of cell integrity [22]. Although the ERK prodeath effect discussed above has now been reported by different groups in multiple cell types, it is intriguing to note that other reports associated ERK with its classical antiapoptotic function following cadmium exposure, particularly in the human nonsmall cell lung carcinoma CL3 cell line [23,24]. Intriguingly, others did not see any effect (neither anti- nor proapoptotic) of ERK activation in response to cadmium, for exam- ple using Clara cells from rat lung [25], mouse brain microvascular endothelial cells (bEnd.3) [26], the human lymphoblastoid Boleth cell line [27] or the human promonocytic U-937 cell line [28]. A key point in explaining these discrepancies could be attributed to the use of different conditions of cadmium treatments. Most of the studies reporting a protective role for A B Fig. 1. (A) Phenotypic illustration of the implication of ERK activa- tion in cell death following cadmium treatment. HEK293 cells were grown for 24 h in control medium (top row) or in the presence of 1 l M CdCl 2 (bottom row). The cells were also treated with 5 lM U0126, a potent MEK inhibitor (middle column), 20 lM U0126 (right column) or no inhibitor (left column). This figure illustrates the toxic- ity of cadmium on cells by comparing the two pictures in the left column. In addition, the protective effect of blocking ERK activation is shown in the lower row, where cadmium toxicity decreased in a dose–response manner following U0126 addition. The top row indi- cates that U0126 treatment alone had a cytostatic effect on cells, as fewer cells were found after 24 h treatment with 20 l M U0126. However, please note that with 20 l M U0126, cell density was similar, independent of the presence of cadmium. This reflects the protective effect of blocking ERK activation. (B) Western blot analy- sis of HEK cell lysates prepared after no treatment (Ø), 24 h 2 l M CdCl 2 treatment (Cd), 24 h 10 lM U0126 treatment (U0), 24 h 2 lM CdCl 2 and 10 lM U0126 cotreatment (U0+Cd). The upper panel displays the activated ERK (P-ERK), which was strongly expressed following cadmium treatment, but almost undetectable when cells were cotreated with cadmium and MEK inhibitor U0126. U0126 alone had no effect on ERK activation (U0). The lower panel repre- sents the total amount of ERK proteins, whose level was not affected by the different treatments. ERK and cell death: cadmium toxicity P. Martin and P. Pognonec 40 FEBS Journal 277 (2010) 39–46 ª 2009 The Authors Journal compilation ª 2009 FEBS ERK used either a higher cadmium concentration and ⁄ or shorter exposure times. Further studies will be required to clarify the reasons for these apparent dis- crepancies, which could also be cell type related. ERK activation ⁄ phosphorylation is the result of the balance between its phosphorylation by MEK and its dephosphorylation by dual-specificity phosphatases [29], among them MKP3. Transiently transfected tetra- cycline-inducible MKP3 phosphatase in HEK293 cells results in diminished ERK activation. Under these con- ditions, we found that HEK293 cells were more resistant to cadmium poisoning as determined by quantifying the number of adherent cells to those with a rounded phenotype (P. Martin et al., unpublished results). Taken together, the sustained activation of ERK can thus be associated with, and is at least partly responsible for, cell death signaling. Reactive oxygen species (ROS) and ERK activation The mechanism responsible for this unusual sustained activation of ERK remains unclear. It is well estab- lished that cadmium results in a time- and dose-depen- dent oxidative response of cells by the production of ROS [30]. Several laboratories have found that pretreat- ment with the N-acetyl cysteine (NAC) antioxidant effi- ciently protects cells against cadmium toxicity and abrogates concomitant ERK activation [31,32]. This implication of ROS in ERK activation is strengthened by a study using differentiated PC12 cells treated with zinc cations. These authors reported a strong activation of ERK leading to apoptosis that could be alleviated by antioxidants [33]. It has also been demonstrated that ROS are responsible for Ras activation, which in turn stimulates its downstream transduction cascade, includ- ing ERK [34–36]. However, the exact mechanisms by which ROS activate mitogen-activated protein kinase (MAPK) are still poorly understood. Taken together, these results suggest that ROS production in response to cadmium does participate in ERK activation. Furthermore, recent data demonstrated that ROS production also promotes MKP3 degradation via the ubiquitination ⁄ proteasome degradation pathway. This MKP3 degradation correlates with a strong activation of ERK [37]. In addition, phosphatases responsible for kinase inactivation are also directly inactivated by oxi- dation of cysteine thiols [38,39]. Therefore, the loss of phosphatase activity is probably at least partly involved in the sustained activation of ERK following cadmium poisoning [33,38]. In conclusion, these data strongly suggest that ROS production following cadmium poisoning results in both the activation of the MAPK cascade leading to ERK activation, and in the inactivation of MKP3, which would stabilize ERK activation. These two mechanisms could act together to induce the delayed and sustained activation of ERK, which is distinct from the canonical ERK activation observed following growth factor stimulation. In this latter case, an immediate response is observed following ligand–receptor interaction, whereas in the case of cadmium intoxication, the progressive accumulation of ROS would result in the delayed and sustained response of ERK. Further studies will be required to validate this model. Cadmium and calcium In addition to the ROS pathway, other mechanisms are known to be affected by cadmium and result in ERK activation. Long-term exposure to 5 l M cadmium leads to a rapid increase in internal calcium concentration ([Ca 2+ ] cyt ) in NIH3T3 cells [40] and in astrocytes. This increase in turn leads to an increase in ROS and to mitochondrial impairment [41]. Pretreatment with a calcium chelator reduced ROS production and increased cell survival, indicating that cadmium-induced cell death resulted from disruption of calcium homeostasis. Similarly, a rapid increase in ([Ca 2+ ] cyt ) is seen in mesangial cells exposed to cadmium. These cells die by autophagy and apoptosis. The use of different calcium inhibitors indicated that the release of calcium from the endoplasmic reticulum plays a crucial role in cadmium-induced cell death. In addition, the cell-permeable calcium chelator 1,2-bis- (o-aminophenoxy)-ethane-N,N,N¢,N¢ tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) eliminated ERK activation as well as mitochondrial depolari- zation and activation of caspases [42]. Together, these results indicate that calcium plays an important role in the cadmium response upstream of ROS production. Sustained ERK activation, apoptosis, autophagy and necrosis How this sustained ERK activation yields to the onset of cell death is still controversial, despite indications pointing towards the activation of caspases-3 and -8 [16]. It should be noted that although caspase activa- tion has been associated with cadmium poisoning, our recent unpublished results show that blocking caspases with P35, a broad spectrum caspase inhibitor that is very effective in protecting cells from a caspase- 8-induced apoptosis [43], does not protect cells from cadmium toxicity, as cell phenotype and cell counts P. Martin and P. Pognonec ERK and cell death: cadmium toxicity FEBS Journal 277 (2010) 39–46 ª 2009 The Authors Journal compilation ª 2009 FEBS 41 remain indistinguishable from control cells. This obser- vation has also been reported by other laboratories showing that complete caspase-3 and -8 inhibition results in blocking poly(ADP-ribose) polymerase cleav- age, but not reducing cell death [27]. Similarly, treat- ment of PC12 cells with zVAD-fmk, a pan-caspase inhibitor, had only a limited effect on cadmium- induced apoptosis [44]. Thus, cadmium, in addition to caspase activation, induces parallel death pathways that are P35-insensitive and that ultimately result in cell death. High doses (350 l M) of cadmium result in mitochondrial membrane depolarization within 1 h and subsequent translocation of apoptosis-inducing factor into the nucleus [27], which could account for such a caspase-independent death pathway. Kim et al. [21] also reported that inhibition of ERK activation results in a better survival of murine J774A.1 macro- phages through the strong attenuation of cadmium- induced necrotic cell death, but not affecting caspase-3 activity and DNA fragmentation. Cell death following cadmium intoxication is therefore not restricted to a single process, but includes apoptosis, necrosis and autophagy, depending upon the context of the intoxication, such as cadmium concentration, as well as the inducing pathway (lipid peroxidation, ROS production, internal calcium concentration) [31,42,45]. Although several lines of evidence indicate that cadmium can result in cell death via multiple path- ways, the best indication that we have to date to support the involvement of ERK sustained activation in cadmium-induced cell death is an engineered cellu- lar system (Raf-1:ER) in which Raf, an upstream ERK kinase, is activated at will by the addition of 4-hydroxytamoxifen [46]. The induced activation of Raf in this system results in sustained activations of MEK and ERK. These activations in turn result in the onset of apoptosis via the death receptor path- way, but are independent of the mitochondrial path- way. In this system, P35 protects cells from apoptosis [46]. Together, these results show that con- stitutive ERK activation per se is sufficient to induce a cellular response that can ultimately lead to cell death in certain systems, but that cadmium, in addi- tion to caspase activation, triggers other parallel pathways that also culminate in cell death. This could explain why ERK inhibitors, although reported by several groups as having a survival effect on cells exposed to cadmium, are usually not sufficient to completely protect cells. In addition, these different death pathways are probably differentially regulated depending upon the cell types considered and the conditions of cadmium exposure [31,32]. Cadmium entry into cells is required for sustained ERK activation and subsequent cell death Cadmium has long been shown to accumulate in cells. However, being a nonessential element, no cadmium- specific transport system exists. Cadmium appears to ‘hijack’ some other transport system(s) in cells. It is reasonable to propose that other divalent metal cation transporters could be used by cadmium for entry into cells, and consequently, these other divalent cations could compete with cadmium for cell entry and subse- quently protect cells against cadmium poisoning. Calcium, copper, magnesium, manganese and zinc divalent metals have been tested as potential competi- tors for cadmium entry. Zinc is considered effective in protecting against cadmium poisoning [47]. In our hands, although zinc has a significant, but somewhat modest, effect on the different cell types tested, manga- nese is by far the most effective competitor. This was demonstrated by intracellular cadmium concentration measurements using 109 Cd. Adding a 10-fold manga- nese molar excess compared with cadmium to the culture medium resulted in a five-fold decrease in intracellular cadmium concentration [48]. Under these conditions, cells are phenotypically protected from cadmium toxicity and ERK is not activated. This is a strong indication that cadmium entry into cells is necessary for sustained ERK activation, and that this activation is tightly associated with cell death. Surpris- ingly, the status of the Ras–Raf–MEK–ERK cascade following cadmium treatment has not yet been pub- lished. Our unpublished data indicate that in HEK293 cells exposed to 2 l M cadmium, c-Raf is maximally phosphorylated after 8 h and then reaches undetect- able levels after 48 h; MEK1 ⁄ 2 also reach a plateau after 8 h and then the level remains unchanged; ERK phosphorylation increases from 8 to 72 h following cadmium addition (P. Martin et al., unpublished results). This suggests that kinases upstream of ERK, at least up to c-Raf, are also triggered by cadmium. Rapid and transient activation of ERK by protein kinase C (PKC) prior to cadmium treatment protects cells from cadmium intoxication Sustained ERK activation following cadmium intoxica- tion is linked to cell death. Could other ERK activa- tors also play a role in the onset of cell toxicity following cadmium treatment? Cells have been pre- treated with the phorbol ester 4b-phorbol 12-myristate ERK and cell death: cadmium toxicity P. Martin and P. Pognonec 42 FEBS Journal 277 (2010) 39–46 ª 2009 The Authors Journal compilation ª 2009 FEBS 13-acetate (PMA), which activates conventional, as well as novel, PKCs. PKCs play a key role in a vast array of cellular signaling, including the activation of ERK [49]. PMA mimics classical cell stimulation by growth factors, and PKC activation results in a strong and transient activation of ERK [50]. Interestingly, when HEK293 cells are pretreated with PMA, their susceptibility to cadmium is substantially decreased. The delayed and sustained cadmium-dependent ERK activation is also strongly reduced under these condi- tions. Prolonged PKC activation by phorbol esters is known to downregulate PKC for at least 24 h [51]. However, it has been shown that the protective effect of PMA on cadmium is not due to its downregulation, but rather to a hit-and-run effect on PKC [22]. Accordingly, when GF 109203X, a broad-spectrum PKC inhibitor, was applied before or together with cadmium, ERK activation increased more than three- fold and cells became more sensitive to cadmium, as evidenced by a decrease in absolute cell number and a more pronounced rounded phenotype [22]. Although earlier work has reported that GF 109203X is also a potent inhibitor of ribosomal S6 kinase (RSK) [52], a kinase downstream of ERK potentially retro-inhibiting the MAPK cascade, it is nevertheless reasonable to assume that this GF 109203X effect, opposed to that of PMA, indicates that PKC activation is not responsible for the delayed and sustained activation of ERK following cadmium treatment. Rather, PKC appears to play a protective role in cells exposed to cadmium, as its early activation by PMA reduces both delayed ERK activation and cadmium toxicity, whereas blocking its activity has the opposite effect (Fig. 2) [22]. In short, PKC early activation has a pro- tective effect on cadmium toxicity in HEK293 cells [22,49,53]. It is intriguing to note that earlier work on cells from rat pulmonary epithelium indicated that pre- treatment with PMA has no significant effect on apop- tosis after a 12 h exposure to 3 and 10 l M cadmium [25]. Surprisingly, similar experiments on rat alveolar epithelial cells showed that exposure to a relatively high level of cadmium (20 l M for 24 h) resulted in PKC activation, and that GF 109203X protected cells from apoptosis [54]. However, in this cell system, PKC activity is already high in control conditions, and cadmium does not activate PKC further [25], or only marginally with high cadmium doses [54]. In contrast, PKC activity is low in HEK293 cells under normal Fig. 2. PKC activity inhibits ERK activation in response to cadmium. Exponentially growing HEK cells were used as a control or treated for 24 h with 2 l M CdCl 2 . As seen by western blotting analysis on the upper panel with an anti-ERK-P IgG, this treatment resulted in the appearance of the activated ⁄ phosphorylated forms of ERK. Cells were also cotreated with 100 ngÆmL –1 PMA and cadmium (PMA ⁄ Cd 24h) or pretreated with PMA for 8 h and then treated with cadmium for 24 h. In both cases, ERK activation was signifi- cantly lower than for cadmium alone. Alternatively, cells were cotreated with 2.5 l M of the PKC inhibitor GF 109203X and 100 ngÆmL –1 PMA and cadmium (GFX ⁄ PMA ⁄ Cd 24h) or pretreated with GF 109203X and PMA for 8 h and then treated with cadmium for 24 h. In both cases, ERK activation was significantly higher than for cadmium alone. The lower panel displays the level of total ERK proteins, which remained unchanged. Fig. 3. Proposition of a schematic model for the ERK response to cadmium poisoning. Cadmium enters cells through the ZIP8 trans- porter, whose activity may be under the negative control of PKC [22]. Once in the cells, cadmium accumulation results in a progres- sive increase in [Ca 2+ ] cyt and subsequently to production of ROS [41]. ROS participate in both the activation of ERK [32] and in the inhibition of dual-specificity phosphatases, including MKP3 [37]. This may participate in the constitutive and long-term activation of ERK [22]. This activation ultimately results in the activation of casp- ases-3 and -8 and in apoptosis [46]. ERK activation has also been involved in necrotic cell death [21] and in autophagy [42]. Other parallel pathways are also present, as ERK inhibition only partially protects from cell death. In these pathways, PKC has been shown to play a prodeath role [25,54]. The dashed lines represent indirect pathways. P. Martin and P. Pognonec ERK and cell death: cadmium toxicity FEBS Journal 277 (2010) 39–46 ª 2009 The Authors Journal compilation ª 2009 FEBS 43 growth conditions, and is strongly activated following a24h2lm cadmium treatment [22]. This could explain the reasons for these discrepancies. Cadmium transport The zinc transporter, ZIP8, is the main transporter hijacked by cadmium to enter cells [55]. Several lines of evidence indicate that PKC activation could inhibit ZIP8 activity. Indeed, PMA treatment of cells exposed to cadmium, in addition to activation of PKC, results in a lower intracellular concentration of cadmium and bet- ter cell survival. An increased cell survival in the pres- ence of cadmium is also obtained by ZIP8 knockdown with specific small interfering RNAs [22]. Finally, three potential sites that could be phosphorylated by PKC have been identified on the ZIP8 amino acid sequence. Together, these results are compatible with a model in which PKC would negatively regulate ZIP8 transporter activity via direct phosphorylation, and limit cadmium entry into cells. Such a direct regulation of transporter activities by PKC has already been reported [56,57]. Fur- ther work will be required to validate this hypothesis. Conclusion Cadmium exposure results in a complex response of cells. It involves internal calcium increases [40], oxida- tive mechanisms [30], gene reprogramming [58], protein degradation [59] and kinase activation [60]. Although no definitive picture is currently available for ERK function in response to cadmium, its sustained activa- tion has been associated with cadmium-induced cell death in several systems [16,18–21]. Particularly, phar- macological inhibition of low-dose cadmium-induced ERK activation diminishes cell death, whereas MKP3 exogenous expression diminishes cell death following cadmium intoxication (P. Martin and P. Pognonec, unpublished data). This sustained ERK activation is probably the result of ROS-dependent inhibition of ERK phosphatases, as well as upstream ERK activa- tion. How ERK sustained activation ultimately results in cell death remains poorly understood. Caspase-3 and -8 activation has been demonstrated following cadmium exposure and sustained ERK activation, reminiscent of the ERK-dependent caspase-3 and -8 activation reported in a synthetic system in which a long-term activation of ERK can be induced [46]. Taken together with the other biological systems pre- sented in this minireview series, cadmium poisoning is a situation in which ERK activation, in addition to its more traditionally described functions, appears to act as a proapoptotic factor. Figure 3 is a schematic repre- sentation of our current understanding of ERK involvement in this process. 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MINIREVIEW ERK and cell death: cadmium toxicity, sustained ERK activation and cell death Patrick Martin and Philippe Pognonec CNRS FRE3094, Universite ´ de. caspase- 8-induced apoptosis [43], does not protect cells from cadmium toxicity, as cell phenotype and cell counts P. Martin and P. Pognonec ERK and cell death: cadmium toxicity FEBS Journal 277 (2010). triggered by cadmium. Rapid and transient activation of ERK by protein kinase C (PKC) prior to cadmium treatment protects cells from cadmium intoxication Sustained ERK activation following cadmium