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Epidemiology and Prevention |
1 Institut National de la Santé et de la Recherche Médicale, INSERM U670, IFR 50, Faculté de Médecine; 2 Centre National de la Recherche Scientifique Unité Mixte de Recherche 6548, Faculté des Sciences; 3 Institut National de la Santé et de la Recherche Médicale, U627, IFR 50; 4 Institut National de la Santé et de la Recherche Médicale/Région Provence Alpes Côte d' Azur, ESPRI 2006/Laboratoire de Pathologie Clinique et Expérimentale, Hôpital Pasteur, Nice, France; and 5 Groupe Appareil Digestif et Environnement (EA3234), Faculté de Médecine, Rouen, France
Requests for reprints: Baharia Mograbi, Institut National de la Santé et de la Recherche Médicale ESPRI 2006, IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 02, France. Phone: 33-4-93-37-77-94; Fax: 33-4-93-37-77-52; E-mail: mograbi{at}hermes.unice.fr.
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Abstract |
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Introduction |
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Physiologically, autophagy ensures in all cell types the turnover of all organelles and most of long-lived proteins by a highly ordered pathway, which begins with the formation of a double-membrane vesicle termed "autophagosome" that engulfs them (for review, see ref. 3). Subsequently, an autophagosome fuses with a lysosome to become an "autolysosome" where the content is finally degraded for the synthesis of new molecules and organelles. As a result, autophagy controls cell modeling throughout development and prevents cell aging during life. In response to environmental stresses (nutrient starvation, hypoxia, infections, etc.), this catabolic process is up-regulated to provide the supply of energy needed for cell survival and repair (3, 4). Not only being a housekeeping process, autophagy protects the cells against the accumulation of damaged organelles and genotoxic substances that would otherwise induce mutations. Consistently, a massive autophagy kills the severely damaged cells as a safeguard mechanism against cancer. Based on these features, it has been proposed that defect in this pathway would confer a selective advantage to cells, with cancer as consequence (5).
Increasing evidence points to an inverse relationship between autophagy and malignant growth. Indeed, autophagy is down-regulated by oncogenic activation of the phosphatidylinositol 3-kinase pathway and inversely up-regulated by tumor suppressors, phosphatase and tensin homologue and p53 (6, 7). Most importantly, it has been shown that the key autophagy protein beclin 1 is an haploinsufficiency tumor suppressor gene in mice (1, 2) and is frequently deleted in human cancers (8). Altogether, these findings have led to the proposals that defects in autophagy may favor carcinogenesis whereas restoration of autophagy may have promising therapeutic implications in cancer. Although critical, there is no cancer therapeutic approach that specifically targets autophagy. A prerequisite for such clinical applications is a better knowledge of the mechanisms by which oncogenes and carcinogens disrupt autophagy. No evidence is indeed available on the autophagic step delayed or stopped during tumorigenesis. Furthermore, the mechanistic basis for these defects remains still elusive. Emerging evidence indicates that the formation of autophagosomes is regulated by multiple signaling pathways as diverse as the GTPase Gi3, the class III phosphatidylinositol 3-kinase, and the serine/threonine kinases mammalian target of rapamycin (mTOR), protein kinase C, and the MAPK p38 (9). However, these signaling pathways are activated by a myriad of stimuli; most of them are not linked to autophagy and carcinogenesis.
Of particular interest, the mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK) pathway, which is commonly activated in cancers, has emerged as a key regulator of autophagy. Indeed, nutrient starvation, a well-known activator of autophagy, enhances ERK activity, and abrogation of this pathway by PD98059 pretreatment inhibits starvation-induced autophagy (10). Moreover, amino acids, physiologic inhibitors of autophagy, were found to impair starvation-induced ERK activation (10, 11). At molecular level, Ogier-Denis et al. (10) have shown that ERK directly phosphorylates and activates G
i3-interacting protein, a positive regulator of autophagy initiation. However, very little is known about the consequences of the constitutive activation of MAPK pathway on autophagy. Overexpression of rasV12 oncogene, the upstream activator of ERK, has been reported to enhance autophagy degradation in HT29 cells (11). Conversely, two other studies have shown that rasV12 reduces autophagy activity and induces autophagic vacuolation in transformed cell lines (12, 13). Part of the difficulty to draw firm conclusion about the role of ERK in autophagy may stem from the observation that rasV12 can activate pathways other than ERK, some of which may regulate autophagy. Consequently, it has never been addressed whether the mere constitutive activation of the MAPK pathway can alter autophagy. Interestingly, expression in transgenic mice of MAPK/ERK kinase 1+ (MEK1+) oncogene that specifically activates ERK results in lens and muscular cell vacuolation (14, 15) but the nature of enlarged compartment remains unknown.
Exposure to environmental carcinogens has long been involved in the development of many common cancers of lung, colon, breast, and testis. As a result, risk assessment of carcinogen chemicals became an important issue of public health policy (16). There is evidence that widely used carcinogens such as Lindane (
-hexachlorocyclohexane) concentrate in fat tissues and testis (17). There, their toxicities persist for years and are able to promote genital malformations, decreased fertility, and several cancers in exposed rodents (18, 19). Strikingly, histologic and biochemical analyses reported in carcinogen-exposed hepatocytes an abnormal accumulation of very large vesicles and a reduced activity of lysosomal enzymes in relation with the possible stimulation of autophagy (20, 21). However, no study has investigated the distribution of autophagic markers in the exposed cells. Within the testis, the earliest testicular damage induced by these carcinogens is the selective appearance of vacuoles in Sertoli cells (19), which support spermatogonial stem cell growth and differentiation. Consequently, alterations of Sertoli cell functions by these environmental carcinogens can contribute to the increased incidence of infertility or testicular cancer, the most common malignancy of young men. Yet, neither the exact nature of enlarged compartments nor the underlying mechanisms have been studied, precluding the establishment of direct "cause-effect" relationship.
Here, we show that Lindane induces cell vacuolation by disrupting the maturation of autophagosomes into functional autolysosomes. This carcinogen-induced vacuolation represents, therefore, a valuable model to identify the signaling events that control the autolysosomal maturation step. We found that activation of MAPK/ERK is essential for Lindane-induced vacuolation. Interestingly, inhibition of this pathway does not impede the autophagy initiation step but rather the maturation step, resulting in the repeated sequestration of autophagosomes into lamellar body. Most importantly, we provide the first evidence that sustained activation of ERK pathway is sufficient to commit cell to autolysosomal vacuolation. Altogether, our data underscore the critical role played by the ERK pathway in maturation step. Implications of these findings in the understanding of how a high constitutive ERK level might lead to carcinogenesis are discussed.
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Materials and Methods |
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Cell culture and treatments. The mouse 42GPA9 Sertoli cell line (25) was maintained in DMEM containing 10% FCS at 33°C. For all the experiments, cells were serum starved for 2 to 16 hours in fresh DMEM supplemented with GMS-A (Invitrogen, Cergy Pontoise, France) or 0.1% bovine serum albumin (BSA A7030, Sigma) and treated with Lindane (50 µmol/L; Sigma) alone or in combination with NH4Cl (5 mmol/L). Lindane at 50 µmol/L induced maximal vacuolation of Sertoli cells without a significant effect on cell viability (26). For these reasons, this dose of Lindane was chosen for all the experiments. When either Park Davis inhibitor PD98059 or U0126 (MEK1-specific inhibitors; 10 µmol/L; Calbiochem) was used, it was added to the starvation medium for 90 minutes before the addition of Lindane. As controls, cells were incubated with vehicle (Me2SO; 1:1,000), chloroquine (membrane-permeant weak base known to induce vacuolation; 500 µmol/L, 24 hours; Sigma), rapamycin (mTOR inhibitor; 50 nmol/L), or insulin (10-7 mol/L) in the presence of an amino acid solution (InsAA, mTOR activator; ref. 27).
The effects of Lindane on the autophagy pathway were studied by transmission electron microscopy, LC3-aggregation, and biochemical assays.
Electron microscopy. The ultrastructural changes caused by Lindane or MEK1+-HA overexpression in Sertoli cells were analyzed by transmission electron microscopy as previously described (28). From each sample were randomly taken 20 to 25 micrographs (primary magnification, x10,000). Then, the volume of autophagic vesicles was estimated by morphometry using Visilog program (Noesis Vision, Inc., France). Autophagic vesicles were classified as early, containing morphologically intact cytoplasm, and late, containing partially disintegrated and electron dense material, according to ref. 29.
Analysis of autophagy by EGFP-LC3 translocation. Cells seeded on glass coverslips were transiently transfected with pEGFP-LC3 using FuGene6 (Roche Diagnostics). After 3 hours of transfection, cells were washed and allowed to recover for 24 hours before Lindane treatment. After indicated times, the cells were fixed and subjected to anti-LAMP1 labeling to detect autolysosomes (LC3+ and LAMP1+). Pictures were taken with a 63x magnification lens using a confocal laser scanning microscope (Leica).
Immunofluorescence staining. Indirect immunofluorescence was done as described (26) using antibodies against specific markers of endoplasmic reticulum (ribosome receptor; ref. 30), medial Golgi apparatus (CTR433; ref. 31), or lysosomes (LAMP1; BD PharMingen). For uptake experiments, cells seeded on glass coverslips were transiently transfected with EGFP-Rab5, EGFP-Rab7, or EGFP-LAMP1 plasmids as described above and treated with Lindane. Twenty-four hours after Lindane addition, rhodamine-dextran (10S dextran, Sigma; 5 mg/mL in DMEM/BSA 0.2%) was added to the vacuolated cells for 90 minutes to label at steady state the endosomal pathway. After washing noninternalized probes, cells were fixed and the slides were mounted in Mowiol medium.
Measurement of degradation of long-lived proteins. Protein degradation was determined according to the method previously reported (32). Briefly, cells were incubated for 72 hours at 33°C in fresh DMEM/F12 medium containing 2% dialyzed FCS and 0.15 µCi of L-[14C]valine (Amersham). Unincorporated radioisotopes were removed by rinsing with DMEM thrice. Cells were then chased with the culture medium containing 10 mmol/L cold valine in the presence or the absence of Lindane to allow vacuolation. After overnight incubation, at which time short-lived proteins were being degraded, the chase medium was replaced with the fresh serum-free and amino acid–free medium (HBSS) containing Lindane and, when required, 3-methyladenine (10 mmol/L) was added to specifically inhibit autophagic degradation. Five hours later, total protein degradation was measured by counting the trichloroacetic acid–soluble radioactivity recovered from both cells and medium. The contribution of autophagy was calculated by subtracting the radioactivity remaining after inhibition with 3-methyladenine from the total radioactivity. All experiments were done at least six times with duplicate samples.
Neutral red uptake assay. To determine the pH of Lindane-induced vacuoles, living cells were stained for 5 minutes with neutral red (0.05% in phenol red–free DMEM/BSA 0.1%), a membrane-permeable dye that accumulates in acidic compartments. Five minutes later, cells were washed and directly observed under phase contrast.
Lysosomal enzyme activities. Sertoli cells were treated with Lindane. After 24 hours, cells were washed, scraped, and homogenized by sonication in water. Lysosomal enzyme activities were detected using fluorometric and colorimetric assays as described (33). In each experiment, the enzyme activities were measured from two parallel culture dishes. Statistical significance was evaluated using Student's t test.
Western blotting. Cell lysates were prepared in NP4O/Brij lysis buffer and analyzed by Western blotting as previously described (26) with antibodies that recognize the phosphorylated active forms of ERK, S6K, MAPK-activated protein kinase 2 (MAPKAP kinase 2), or p38 (Cell Signaling Technologies). After stripping, equal loadings of proteins were verified by reprobing the same blots with all these total forms of ERK or S6K (data not shown).
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Results |
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8 to 9 hours after Lindane addition and increased in number and size, filling up the cell cytoplasm as exposure continued (Fig. 1A, inset
). Interestingly, this vacuolation was not associated with cell death or apoptosis as evidenced by its reversion on Lindane removal and the absence of procaspase 3 and poly(ADP-ribose) polymerase cleavages (data not shown). Altogether, these findings show that Lindane did promote in vitro reversible vacuole formation independently of apoptosis.
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Lindane-induced vacuoles are inaccessible to external probes. To test this possibility, we next determined whether the Lindane-induced vacuoles communicate with the endocytic pathway. To this end, cells were transiently transfected with pEGFP-Rab5, pEGFP-Rab7, or pEGFP-LAMP1, treated with Lindane, and ultimately incubated with rhodamine-dextran, a fluid-phase endocytic marker. Along the endocytic pathway, the internalized molecules are transiently present in early endosomes, then transported to late endosomes, before being degraded in lysosomes. After incubation for 90 minutes with control cells, all endocytic vesicles from early endosomes, late endosomes to lysosomes were labeled at steady state by rhodamine-dextran (Fig. 1C). In Lindane-treated cells, this fluid-phase marker was equally distributed within Rab5, Rab7, and LAMP1 punctuate structures, indicating that the general trafficking from early endosomes to lysosomes was not affected by Lindane. However, the large Rab7+/LAMP1+ compartments were not reached by endocytosed material after 90 minutes. Similar results were obtained when Lindane-treated cells were allowed to internalize rhodamine-dextran for 8 hours (data not shown). Altogether, these data indicate that although the Lindane-induced vacuoles contained the endocytic markers Rab7 and LAMP1, they were distinct from the endocytic compartments.
Lindane promotes the development of giant autolysosomal compartment. In addition to endocytosis, Rab7 and LAMP1 proteins are involved in autophagy, a process recently shown to be deregulated in tumors (5, 35–37). Electron and immunofluorescence microscopy analyses reveal that these structures were enlarged autophagic compartments (Figs. 2 and 3 ). At ultrastructural level, numerous large autophagic vesicles were observed within Lindane-treated cells but not in untreated cells. Despite the presence of these vesicles, the nuclei and the organelles exhibited morphologies indistinguishable from that of control cells (Supplementary Fig. S2), consistently with the above shown absence of cell death. Strikingly, a high number of small autophagic vesicles accompanied the Lindane-induced vacuolation from initial double membrane–enclosed autophagosomes sequestrating organelles (Fig. 2A-C) to single membrane–bound autolysosomes containing electron-dense materiel (Fig. 2D-F). At high magnification, many autophagosomes were found in close vicinity or in contact with large vesicles, likely in the initial stage of fusion (Fig. 2F).
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Disruption of autophagy by Lindane is correlated with a decline in lysosomal activity of aryl sulfatase A. Given that mature autolysosomes ensure by acid hydrolases the degradation of their content (3), it was of interest to address the autophagic activity of Lindane-treated cells. To investigate this, cells were metabolically labeled with [14C]valine, chased with unlabeled valine for 16 hours in the presence of Lindane to allow vacuolation, before being incubated under either normal or nutrient starvation conditions for 5 hours. Protein degradation was then measured by trichloroacetic acid–soluble counts in both cell lysates and media. Surprisingly, Lindane failed by itself to enhance degradation of long-lived proteins but instead significantly decreased starvation-induced proteolysis. Using 3-methyladenine (10 mmol/L), an inhibitor of macroautophagy, we found that the diminution of total proteolysis in Lindane-treated cells was mainly due to a decrease in autophagy. These data suggested that the giant autolysosomes could result from defective autophagic degradation (Fig. 4A ).
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The mTOR kinase pathway is not involved in Lindane-induced autophagy. Signal transduction pathways allow cells to sense and respond to their environment. In response to nutrient starvation, autophagy is up-regulated to allow cell survival. The serine-threonine protein kinase mTOR acts as a "nutrient-sensor" and treatment with rapamycin, which inactivates mTOR, induces autophagy (9). To identify the mechanism(s) through which Lindane affects autophagy, we initially investigated the effect of Lindane on mTOR pathway. To this end, Sertoli cells were serum starved for 4 hours, treated with Lindane for various times, and lysed. Activation of mTOR was then assessed by Western blotting with antibodies directed against the phosphorylated form of p70S6K, known to be under the control of mTOR. As presented in Fig. 5A , the combination of insulin and amino acids (InsAA), used as positive control, induced a strong S6K phosphorylation whereas Lindane had no effect. Pretreatment with rapamycin (50 nmol/L) completely blocked the InsAA-induced S6K phosphorylations, confirming that this response required mTOR (27). By contrast, a 5-hour Lindane pretreatment did not modify the phosphorylation level of S6K compared with controls (Fig. 5B). Similar results were obtained by analyzing the phosphorylations of S6K by gel mobility shift. Altogether, this indicated that inhibition of mTOR signaling pathway did not account for Lindane-induced autophagy.
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Lindane-induced disruption of autophagy is dependent of ERK activation. Given the abilities of Lindane to induce ERK and the formation of giant autolysosomes, we sought to determine whether this MAPK might contribute to this cellular response. Of particular interest, Lindane induced the sustained activation of ERK1/2 and abrogation of this activation by pretreatment with the specific MEK1/2 inhibitor PD98059 (10 µmol/L) suppressed Lindane-induced vacuolation (Fig. 6A and Supplementary Fig. S3). At that stage, it was of interest to address whether the abrogation of MAPK pathway impairs the formation of autophagosomes or their maturation into autolysosomes. When analyzed by fluorescence microscopy, we found that the giant LAMP1+ perinuclear vesicles in Lindane-treated cells were replaced by smaller more scattered vesicles in the presence of PD98059 and Lindane. Strikingly, this was accompanied by the accumulation of ring-like LC3+ structures, some of which contained an LC3+ dot (inset) without any LAMP1-colocalization, establishing the persistence of autophagosome formation but the disappearance of autolysosomal compartments. This prompted us to investigate the nature of these structures by electron microscopy. High magnification evidenced the dramatic appearance of lamellar structures that contained a material indistinguishable from the cytosol and a recognizable isolation membrane, highly suggesting that they were formed by the repeated sequestration of autophagosomes. As expected, similar results were obtained with U0126, another MEK1 inhibitor (Fig. 6 and Supplementary Fig. S3). These results provide the first evidence that inhibition of the ERK pathway did not impede the formation of autophagosomes but rather blocked the maturation of autophagosomes into autolysosomes.
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Discussion |
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To address this issue, we focused our attention on environmental carcinogens, such as Lindane, which display adverse effects on human health and give increase to cell vacuolation. Within the testis, one of the earliest and most documented events following Lindane exposure is the selective vacuolation of Sertoli cells, strongly suggesting that these cells are the direct targets of these chemicals (19). However, this hypothesis still lacks experimental support, given the complex interdependence of testicular cells. Moreover, neither the nature of enlarged compartments nor the underlying molecular mechanisms have been studied. The present study provides the first evidence that the Sertoli cells responded in vitro directly, within hours, to this carcinogen by promoting extensive vacuolation. By electron and immunofluorescence microscopy analyses, we identified that the Lindane-induced vacuoles were derived from autophagic pathway as (a) they were double membrane–enclosed vesicles, (b) containing organelles, (c) inaccessible to external probe, and (d) positive for LC3, four hallmarks of autophagic vesicles.
The presence of autophagic vesicles has long been blamed in dying cells as a second form of programmed cell death, in addition to the type I apoptosis. Whereas considerable progress has been made in defining the machinery of apoptosis, very little information is currently available about the control of autophagy. This is in part because autophagy coexists with apoptosis in many pathologic tissues and in response to various death signals (such as starvation, ischemia, pathogens, heat, or oxidation; ref. 5). Along this line, Lindane-induced vacuolation was proposed to result primarily from its cytotoxic potential. However, little, if any, dead cells, together with the observation of reversibility, showed that Lindane did not induce cell vacuolation by promoting cell death. Importantly, the absence of several apoptotic features such as caspase activation, chromatin condensation, nuclear pyknosis, and mitochondria degeneration indicates that Lindane-induced autophagy did not use common machinery with apoptosis. The characterization of Lindane-induced autophagic vacuolation therefore supports the recent notion that autophagy is an adaptive response that protects rather than commits cell to death (43).
Autophagy is a dynamic process, which begins with the formation of the double-membrane autophagosome that engulfs a part of cytoplasm. Subsequently, the outer membrane of the autophagosome fuses with late endosomes and lysosomes to become an autolysosome where the content is finally degraded for the synthesis of new molecules and organelles. As a consequence of this rapid turnover, only a few autophagosomes and autolysosomes are observed under starvation (38). By contrast, the Lindane-treated cells displayed the aberrant accumulation of large autophagic vesicles that acquired LC3, LAMP1, and an acidic pH. This established that the initial step of autophagic maturation (i.e., fusion of autophagosomes with lysosomes) did take place. However, the maintenance of enlarged hybrid compartments suggests delayed/arrested autolysosomal maturation. Consistent with this possibility, Lindane-induced vacuolation did not reflect enhanced but rather reduced starvation-induced autophagic degradation in relation with a decline of the activity of lysosomal hydrolases.
A large body of evidence suggests an inverse relationship between autophagy and aberrant cell growth. Indeed, autophagy plays a critical role in tumor suppression and autophagy degradation is suppressed by conditions that promote cell growth (5), such as oncogene expressions and exposure to carcinogens, as we evidenced herein with Lindane. Likewise, many antitumor drugs commit cancer cells to death by restoring autophagy [such as tamoxifen (44), radiation (45), and histone deacetylase inhibitors (46)]. However, natures of autophagic step disrupted by cell transformation and of underlying mechanism(s) remain unknown. The present study shows that carcinogen Lindane selectively disrupted the autolysosomal maturation step. Therefore, Lindane-induced vacuolation provides a valuable model not only for unraveling the fundamental process that controls autolysosomal maturation but also for designing new anticancer drugs capable of restoring full autophagy maturation and degradation.
Autophagy is regulated by multiple signaling pathways as diverse as the GTPase Gi3, the class III phosphatidylinositol 3-kinase, and the protein kinases mTOR, ERK, and p38 (9). However, the molecular control of autophagy maturation remains elusive because most of these pathways function during the early steps of autophagosome formation. Of particular interest, we found that Lindane mimicked many effects of a good inducer of autophagy, rapamycin, which inactivates mTOR. Indeed, Lindane, like rapamycin, induced autophagy even in the presence of nutrients. Strikingly, mTOR inhibition shares with Lindane the feature to increase the flux of autophagosomes into autolysosomes and thereby the size of autolysosomes (47). However, at odds with these observations, we found that Lindane did not induce mTOR nor did it impede its activation.
Other kinases that can be considered in Lindane-induced autolysosomal vacuolation are the MAPKs ERK. Indeed, we have shown that (a) Lindane induced the rapid and sustained activation of the ERK pathway; (b) pretreatment with the MEK1/2 inhibitors PD98059 or U0126 abrogated Lindane-induced autophagic vacuolation and concomitantly induced the repetitive sequestration of autophagosomes into lamellar bodies; and (c) by the use of a dominant MEK1+ construct, we showed that activation of the ERK pathway was sufficient to arrest the maturation autophagic step with the characteristic accumulation of autophagosomes and swelling of the Rab7/LAMP1 compartment. This is the first demonstration of a link between ERK and the autophagy maturation step. Previously, ERK activation was also required for starvation-induced autophagic proteolysis (10) but the precise role of ERK in autophagy initiation or maturation has not been defined. Consistently with our findings, the oncogenic activation of ras (rasV12), the upstream activator of ERK, has been reported to induce autophagic vacuolation and to reduce autophagic degradation in transformed cell lines (11, 12). However, these studies have not addressed whether rasV12-induced autophagy is dependent on ERK. In addition, expression of MEK1+ in transgenic mice has been reported to result in lens and muscular cell vacuolation (14, 15) but the nature of enlarged compartment remains unknown.
These data created an apparent paradox in that starvation, Lindane, and growth factors all stimulate ERK and yet have opposite effects on autophagy (11). An attractive possibility to explain this discrepancy might be to consider that the cell response depends critically of the nature, strength, and duration of MAPK pathways activated (48). Interestingly, whereas starvation induces a barely detectable ERK activity (11) and growth factors trigger transient and strong ERK activity with multiple pathways, we have shown that Lindane, as did MEK1+, induced a sustained and specific ERK activation for >24 hours. Altogether, our results point to the new notion that autophagic vacuolation (i.e., defect at autophagy maturation step) is not merely an adaptive cellular response to an environmental carcinogen but rather the exaggerated consequence of sustained and specific ERK activation. In this regard, evidence of the involvement of a high constitutive ERK activation in 50% to 90% of colorectal, melanoma, pancreas, and other common cancers is of great interest (49). Therefore, the present study provides the rationale for developing anticancer therapy aimed at restoring autophagy maturation through modulation of ERK pathway. Elucidating precisely how ERK may control the autophagosome maturation step is a major challenge. One possibility would be that ERK, once activated, may modulate by phosphorylation a key regulator of autophagosome maturation. Alternatively, ERK may be a general component of the autophagic machinery, present and activated on autophagosomes to modulate their maturation. Recently, activated ERK1/2 was localized to autophagic vesicles (50). Defining the autophagic target of ERK involved in autolysosomal maturation should not only provide important insights into the role of autophagy dysfunction in tumorigenesis but also identify potential targets for specific therapeutic interventions. This work is currently in progress in our laboratory.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Mireille Mari, Nadir Djerbi, Anne Doye, Elodie Cordero, and Pascale Monzo for technical assistance; Isabelle Bourget, Isabelle Mothe-Satney, Philippe Gual, and Mireille Cormont for helpful discussions; Tamotsu Yoshimori and Noboru Mizushima for EGFP-LC3; Michel Bornens for anti CTR433; and Gilles Pages and Philippe Lenormand for MEK1+-HA construct.
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Footnotes |
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Received 10/ 3/05. Revised 4/ 7/06. Accepted 5/ 5/06.
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References |
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) and autolysosomes (autoLy) to the total cellular area; bars, SD.
) migrated more slowly than nonphosphorylated forms (
). C, Lindane pretreatment (50 µmol/L, 5 or 24 hours) was unable to affect anisomycin-induced p38 activity, as evaluated by Western blotting with antibodies against phosphorylated active forms of p38 (Pp38; top) and MAPKAP kinase 2 (PMk2; bottom). As controls, cells that were stimulated by anisomycin (A; 10 µg/mL, 10 minutes) showed high p38 and MAPKAP kinase 2 phosphorylations that were completely inhibited by SB203580. Representative Western blots. Arrowheads, positions of the phosphorylated and unphosphorylated p70S6K, p38, and MAPKAP kinase 2.