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The drs tumor suppressor is involved in the maturation process of autophagy induced by low serum
Cancer Letters 283 (2009) 74–83
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The drs tumor suppressor is involved in the maturation process of autophagy induced by low serum Yukihiro Tambe a, Akitsugu Yamamoto b, Takahiro Isono c, Tokuhiro Chano d, Mitsunori Fukuda e, Hirokazu Inoue a,* a
Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan Department of Cell Biology and Bioscience, The Nagahama Institute of Bioscience and Technology, Nagahama, Shiga 526-0829, Japan c Central Research Laboratory, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan d Clinical Laboratory Medicine, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan e Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Miyagi 980-8578, Japan b
a r t i c l e
i n f o
Article history: Received 2 September 2008 Received in revised form 3 February 2009 Accepted 17 March 2009
Keywords: drs Autophagy Knockout mouse Mouse embryonic fibroblasts Rab24
a b s t r a c t The drs gene is an apoptosis-inducing tumor suppressor. Previously, we showed that drs contributes to the suppression of tumor formation by generating drs-knockout mice. In this study, by using drs KO mouse embryonic fibroblasts, we found that drs is involved in the autophagy regulation under low serum culture conditions. Both electron microscopy and GFP-LC3 analyses demonstrated that drs is involved in the maturation process of autophagy from autophagosomes to autolysosomes. In addition, drs could associate with Rab24 and the association between drs and Rab24 was enhanced during autophagy. drs was also co-localized with Rab24 on punctuated structures during autophagy. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In determining whether tumor cells survive or die under stress conditions such as depletion of growth factors and nutrients, macroautophagy (hereafter referred to as autophagy) as well as apoptosis play crucial roles. Autophagy is a genetically regulated bulk degradation/recycling system conserved in eukaryotic cells [1,2]. During autophagy, parts of the cytoplasm, including macromolecules and organelles, are sequestered within autophagosomes, which are characteristic double-membraned vacuoles, and finally are degraded in the mature autolysosomes, which are formed by the fusion of autophagosomes and lysosomes. Since autophagy regenerates metabolic intermediates, it plays a critical role as a cell survival pathway in response to nutrient starvation. However, when autophagy is intensively induced and prolonged, it causes autophagic cell * Corresponding author. Tel.: +81 77 548 2177; fax: +81 77 548 2404. E-mail address: [email protected] (H. Inoue). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.03.028
death (type II programmed cell death). Recent studies suggested that autophagy is involved either in tumor development or regression together with apoptosis [1,3,4]. The drs gene was originally isolated as a transformation suppressor for the v-src oncogene [5,6], and the expression of drs mRNA has been shown to be markedly downregulated in a variety of human cancer cell lines and malignant tumor tissues [7–9]. Furthermore, ectopic expression of the drs protein induced apoptosis in human cancer cell lines via the novel pathway initiated from the endoplasmic reticulum (ER), involving the binding to ASY/Nogo-B/RTN-XS, apoptosis-inducing proteins localized in ER, and activation of caspase-12, -9, and -3 [10]. In previous study, we showed that malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of drsknockout (KO) mice [11]. Reintroduction of drs into a tumor cell line, LC-T1, derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis. Introduction of drs into the tumor cell line also enhanced sensitivity to
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apoptosis under low serum culture conditions. These findings indicate that drs contributes to the suppression of malignant tumor formation, and that this suppression is closely correlated with drs-mediated apoptosis under stress conditions. The drs KO mice did not show the distinct character other than tumor-prone phenotype. Although these previous findings suggest that drs regulates apoptosis in tumor cells, the physiological roles of drs in normal cells under stress conditions remains to be worked out. Then, we hypothesized that drs is involved in the cellular responses for stresses via the regulation of apoptosis and/or autophagy. In the present study, we have attempted to investigate the physiological roles of drs tumor suppressor by using mouse embryo fibroblast (MEF) cells derived from drs KO mice, and have found that drs is involved in the regulation of autophagy maturation under low serum culture conditions.
2. Materials and methods 2.1. Cells MEFs deficient in drs (KO MEF) or wild-type MEFs (WT MEF) were prepared from male embryos resulting from the mating of drs heterozygous (+/ ) female and wild-type (+/Y) male mice, as the drs gene is located in chromosome X [11]. The immortalized drs KO and WT MEF cell lines, KO-LT and WT-LT, were established by expressing the large T antigen of SV40 into KO and WT MEFs, respectively. KOgLC3LT and WT-gLC3LT, the immortalized drs KO and WT MEF cell lines which express GFP-LC3, were established from male embryos resulting from the mating of drs heterozygous female and GFP-LC3 male transgenic mice (GFPLC3#53, BRC No. 00806, RIKEN BRC, Tsukuba, Japan) [12,13] followed by the immortalization with the large T antigen of SV40. EGFP-MEF and DrsEGFP-MEF are WT MEFs that expresses EGFP and EGFP-conjugated drs, respectively. 293T (a human embryonic kidney cell line expressing the E1 gene of adenovirus type 5 and the T antigen of SV40) was also used for the preparation of the recombinant retrovirus and transfection experiments. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 2.2. Plasmids and recombinant retroviruses The expression plasmids of EGFP-conjugated drs (WT, CD and TM), FLAG-tagged drs were described previously [6,10,14]. The expression plasmids of Rab24 and EGFP-conjugated Rab7, Rab3A, Rab17 and Rab21 were described previously [15]. The expression plasmid of mRFP-conjugated LC3 [16] was a kind gift from Dr. Tamotsu Yoshimori (Osaka University). Preparation and infection of pCXbsr retrovirus vectors carrying the drs cDNA (WT, CD and TM) were described previously [6,10].
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clonal antibody for Rab24 (R38620) was purchased from BD Transduction Laboratories (Lexington, KY, USA). Antia-tubulin (DM1A) and anti-FLAG M2 (F3165) monoclonal antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PDI (SPA-891) monoclonal antibody was purchased from Stressgen Biotechnologies (Victoria, BC, Canada). Anti-EGFP polyclonal (#8372-2) and monoclonal (#8371-2) antibodies (Living ColorsTM, Clontech) were purchased from Takara Bio (Shiga, Japan). Secondary fluorescence-labeled antibodies, Alexa 488- or 594-conjugated anti-rabbit or anti-mouse IgG, were purchased from Invitrogen (Carlsbad, CA, USA). 2.4. Electron microscopy MEFs, grown on gelatinized plastic coverslips (Celldesk LF1; Sumitomo Bakelite, Tokyo, Japan) were incubated with 10% or 0.1% FBS containing DMEM, fixed for 2 h with 2.5% glutaraldehyde (EM Science, Hatfield, PA, USA) in 0.1 M phosphate buffer at pH 7.4, post-fixed in 1% OsO in the same buffer and then subjected to electron microscopic analysis as described previously [17]. Representative areas were chosen for ultra-thin sectioning and viewed with a Hitachi 7600 electron microscope. The areas of autophagosomes and autolysosomes were measured using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) and the percentages of these areas in the cytoplasm area were calculated as area indexes. 2.5. Autophagy assay The early stage of autophagy development, the generation of autophagosomes, was quantified by detecting the LC3 punctuation in MEFs expressing GFP-LC3 under fluorescent microscopy [12,13]. The areas of the puncta were calculated by the top-hat detection mode of MetaMorph software as described [18]. Autophagy maturation was observed by LC3 conversion and turnover assay by immunoblotting with anti-LC3 antibody [19]. The generation of mature autophagolysosomes in living cells was quantified by detecting the development of acidic vesicular organelles stained with acridine orange [20]. Cells were stained with 0.5 lg/ml of acridine orange for 15 min, removed from the plate with trypsin–EDTA and analyzed by FACSCaliber and CellQuest software (BD, Franklin Lakes, NJ, USA). 2.6. Immunofluorescence staining Immunofluorescence staining was carried out as described previously [10]. For the visualization of lysosomes in living cells, LysoTracker Red DND-99 (50 nM, Invitrogen) was added to the cell culture and incubated for 5 min in CO2 incubator. Samples were observed with a confocal fluorescent microscope (Nikon C1si). 2.7. Immunoprecipitation and immunoblotting
2.3. Antibodies Rabbit polyclonal antibody for LC3 was a kind gift from Dr. Tamotsu Yoshimori (Osaka University). Mouse mono-
Cells were lysed in RIPA buffer containing 20 mM Tris– HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton-X100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate and
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Fig. 1. Analyses of cell death and autophagy in WT and drs KO MEFs under low serum culture conditions. (A) Cell death induced by prolonged (72 h) low serum (0.1%) treatment in WT and KO MEFs. zVAD: 2 lM of zVAD-fmk was added at 0 h. Open and closed columns show WT and drs KO MEFs, respectively. Each bar indicates mean ± s.d. *p < 0.05, **p < 0.01 by Student’s t-test. (B) Cell morphology of WT (left) and KO (right) MEFs under prolonged (48 h) low serum (0.1%) culture conditions. 5 105 cells were plated onto 60 mm dishes and cultured at 37 °C for 4 h. The medium was replaced with DMEM supplemented with 0.1% FBS (0 h, top), and then the cells were cultured for 48 h (middle and bottom). Black arrows show vacuole-like structures in cytoplasm. Scale bars represent 20 lm. (C) Quantification of aberrant MEF cells under prolonged (48 h) low serum culture conditions. Open and closed columns show WT and KO MEFs, respectively. Bars indicate mean ± s.d. of the percentage of attached cells that were aberrant. Detached dead cells were omitted from the counts. **p < 0.01 by Student’s t-test. (D) Morphological analyses of autophagy by electron microscopy. Primary WT (middle) and KO (bottom) MEFs (passage 2) were cultured in DMEM containing 0.1% FBS for 24 h. White and black arrows indicate autophagosomes (AP) and autolysosomes (AL), respectively. Representative images of autophagosomes (top left) and autolysosomes (top right) are also shown. White and black scale bars represent 1 lm and 2 lm, respectively. (E) Quantification of autophagosome and autolysosome formation under electron microscopy. Area indexes are centuple values of percentages of the organelle area in the cytoplasm area. Open and closed columns show WT and KO MEFs, respectively. Each column indicates mean ± s.d of a representative area of 50 different cells. *p < 0.05 by Student’s t-test.
1% protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan), and were centrifuged for 30 min in 10,000g at 4 °C.
Immunoprecipitation, electrophoresis and immunoblotting procedures were performed as described previously [10].
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3 Fig. 2. Analyses of the maturation process of autophagy in WT and drs KO MEFs under low serum culture conditions. (A) Time course of LC3 conversion and turnover in WT and KO MEFs. Cells were cultured in DMEM supplemented with 0.1% FBS for the represented time, and the cell lysates were analyzed by immunoblotting with anti-LC3 antibody (upper). For turnover assay, pepstatin A (10 lg/ml) and E64d (10 lg/ml) were added (+inhibitors) at the start time of low serum treatment. To evaluate the conversion of LC3 from the type I form (precursor) to the type II form (membrane-bound), the ratios of the quantity of type II form of LC3 to b-actin were calculated from the digitized image (lower). Columns indicate the relative value of the LC3-II / b-actin ratio at each point of time based on the ratio at 0 h. Open and closed columns represent WT and KO MEFs, respectively. (B) Time course of the GFP-LC3 punctuation in WT-gLC3LT and KO-gLC3LT MEFs under fluorescent microscopy. Cells were cultured in DMEM supplemented with 0.1% FBS for the represented time. Photographs represent the merged images of GFP-LC3 punctuation (green) and nuclei stained with Hoechst 33342 (blue) under low serum conditions at pretreatment (0 h), early (6 h) and late (18 h) stages of autophagy in WT-gLC3LT and KO-gLC3LT MEFs (upper). Small inserted images represent magnified views of GFP-LC3 punctuation. Yellow and white scale bars represent 20 lm and 50 lm, respectively. The percentages of the areas of GFP-LC3 dots in cytoplasm were calculated (lower). Open and closed circles represent WT-gLC3LT and KO-gLC3LT MEFs, respectively. Each value indicates a mean ± s.d. of 20 cells. p < 0.05 by Student’s t-test. (C) Time course of the GFP-LC3 punctuation in KOgLC3-LT MEFs expressing retroviral WT, CD and TM drs cDNAs under fluorescent microscope. The percentages of the area of GFP-LC3 dots in cytoplasm were calculated at pretreatment (0 h), early (6 h) and late (18 h) stage of autophagy under low serum conditions. (D) Fluorescent microscopic images of acidic organelles stained with acridine orange. The primary MEFs (passage 2) were cultured in DMEM containing 10% (upper) and 0.1% (lower) FBS for 24 h. The cytosol and nucleus fluoresce bright green and dim red, whereas acidic compartments fluoresce bright red. Small inserted images represent magnified views. Yellow and white scale bars represent 20 lm and 100 lm, respectively. Percentage of cells that were enriched with acidic organelles (A. O.) in primary WT and KO MEFs (passage 2) under high or low serum treatment (24 h) (lower). The cells with red fluorescence were quantified by FACS analyses. 3-MA: 10 mM of 3-methyladenine, an inhibitor of autophagy, was added at 0 h. Each bar indicates mean ± s.d. p < 0.05 by Student’s t-test.
2.8. Identification of drs-binding proteins by peptide mass fingerprinting analyses 293T cells were transfected with the expression plasmid of FLAG-tagged drs or vector plasmid (pCR3.1) by using Lipofectamine-PLUS reagent. After 2 days, the cells were lysed in RIPA buffer. The lysate was centrifuged at 10,000g for 30 min, and the supernatant was incubated with anti-FLAG affinity agarose (Sigma-Aldrich) at 4 °C for 2 h. Then the agarose was washed three times with RIPA buffer and the proteins bound to the agarose were eluted by FLAG-peptide (150 lM, Sigma-Aldrich). The samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins were visualized by silver staining. Each protein band was digested in the gel by modified trypsin (Promega, Madison, WI, USA). Protein identification for each band was performed by peptide mass fingerprinting analysis as described previously [21]. 3. Results 3.1. drs regulates the maturation of autophagy from autophagosomes to autolysosomes To investigate the physiological role of drs gene, we prepared the wild-type (WT) and drs KO MEFs. However, there was no significant difference between WT and KO MEFs in the cell proliferation, cellular senescence, and the cellular morphology under normal culture conditions (data not shown). Previously, we reported that the re-introduction of drs into LC-T1, a lung cancer cell line derived from a drs KO mouse, induced apoptosis after 24 h incubation under low serum culture conditions (0.1% FBS), suggesting that drs may play a role in the cell responses for stresses [11]. To investigate the role of drs gene under stress conditions, we immortalized wild-type and drs KO MEFs by introducing the large T (LT) antigen of SV40 and compared the responses of both MEF cells under low serum culture conditions. Although cell death was hardly observed in WT and KO MEFs at 24 h in low serum culture (data not shown), significant cell death (55.4%) was observed in WT MEFs in prolonged culture at 72 h (Fig. 1A). On the other hand, the percentage of cell death was significantly lower in KO MEFs (25.6%). zVAD-fmk, a pan-caspase inhibitor, did not suppress cell death in either WT or KO MEFs, indicating that this cell death was not apoptosis. During cultivation, a majority (51.9%) of morphologically aberrant cells, having a number of vacuole-like structures in the cytoplasm (Fig. 1B, black arrows), were observed in WT MEFs at 48 h, whereas the percentage of such aberrant cells was significantly lower (13.1%) in KO MEFs at 48 h (Fig. 1C). The degree of vacuolation per cell
in KO MEFs was also lower than that in WT MEFs (Fig. 1B). zVAD-fmk did not suppress the appearance of aberrant cells under low serum culture conditions in either WT or KO MEFs (Fig. 1C). These findings suggest the possibility that drs is involved in the regulation of autophagy under low serum culture conditions. We then performed a morphological analysis of autophagic vacuole formation by electron microscopy in WT and KO MEFs under low serum conditions. As shown in Fig. 1D, both the autophagosomes (AP, white arrows) and autolysosomes (AL, black arrows) were observed in WT and KO MEFs at 24 h after low serum treatment. Although the total area indexes of these autophagic vesicles were similar between WT and KO MEFs, the index of autolysosomes in KO MEFs was significantly lower than that in WT MEFs. On the other hand, the index of autophagosomes in KO MEFs was higher than that in WT MEFs (Fig. 1E). These results suggest that the maturation process from autophagosomes to autolysosomes was inhibited in drs KO cells. To confirm that drs is involved in the maturation process of autophagy, we examined the molecular behavior of LC3, a mammalian marker of autophagosomes, under low serum culture conditions in WT and KO MEFs. In the early stage of autophagy, the cytosolic form of LC3 (LC3-I) is converted into the autophagosomal membrane-bound form of LC3 (LC3-II) by conjugation with phospholipids. In a subsequent stage, LC3II is degraded in the autolysosomes by the lysosomal proteases or is recycled back to LC3-I. During low serum treatment, the relative ratio of LC3-II (LC3-II/b-actin) in WT MEFs was increased from 3 to 6 h and then gradually reduced (Fig. 2A). On the other hand, in KO MEFs, the ratio of LC3-II was increased from 3 to 6 h and remained elevated. The addition of a mixture of pepstatin A and E64d, the cell-permeable lysosomal protease inhibitors that inhibit the turnover of LC3 by delaying the degradation in autolysosomes, restored the ratio of LC3-II at the late stage in WT MEFs to the level in KO MEFs (see Fig. 2A, 18 +inhibitors). Furthermore, to visualize the molecular behavior of LC3 in cells, we established WT and drs KO MEFs expressing GFP-LC3 (WT-gLC3LT and KO-gLC3LT) by the mating of drs KO mice and GFP-LC3 transgenic mice. As shown in Fig. 2B, the punctuated GFP-LC3 area was increased from 3 to 6 h after low serum treatment, and was subsequently decreased in WT-gLC3LT. On the other hand, in KO-gLC3LT, it was similarly increased in the early stage of autophagy (6 h), but remained elevated in the late stage (up to 18 h after treatment). Re-introduction of wild-type (WT) drs by retrovirus vector into KO-gLC3LT cells decreased the punctuated GFP-LC3 area in the late stage (Fig. 2C). This effect was also observed by introduction of the CD mutant, which lacks the C-terminal region of drs, but not by introduction of the TM mutant, which lacks the transmembrane domain of drs, indicating that the transmembrane domain of drs is necessary for the maturation process of autophagy under low serum culture conditions (Fig. 2C). The formation of mature autolysosomes in the cytoplasm can be monitored by the appearance of acidic organelles, which are stained red1 with acridine orange, if newly formed acidic compartments are attributed to autolysosomes. As shown in Fig. 2D, the number of acridine orange-positive 1 For interpretation of color in Figs. 2–4, the reader is referred to the web version of this article.
Y. Tambe et al. / Cancer Letters 283 (2009) 74–83 cells in KO MEFs (12.8%) was significantly lower than that in WT MEFs (26.5%) at 24 h after low serum treatment. 3-methyladenine (3-MA), which is known to inhibit autophagy, completely suppressed acidic organelle formation in both WT and KO MEFs (Fig. 2D), suggesting that the formation of acidic organelles in these experiments reflects the induction of autophagy. These results indicate that drs is required for the maturation process of autophagic vesicles from autophagosomes to autolysosomes. 3.2. drs associates with Rab24 and related Rab family GTPases To investigate how drs regulates the autophagy maturation process, we attempted to identify the drs-binding proteins involved in the maturation of autophagic vesicles. As shown in Fig. 3A, a protein with a molecular weight of about 25 kD was co-precipitated with FLAG-tagged drs protein expressed in 293T cells. By peptide mass fingerprinting analyses, the protein was identified as Rab24, a member of the Rab GTPase protein family, has been reported to be involved in autophagy [22,23]. To confirm the association between drs and Rab24, we carried out a pulldown assay in 293T cells transfected with the plasmids expressing drs conjugated with EGFP (DrsEGFP) and/or Rab24. As shown in Fig. 3B, DrsEGFP was coprecipitated with Rab24, but EGFP alone was not. To evaluate whether endogenous Rab24 is able to bind to drs under low serum culture conditions, we also performed a pulldown assay in MEF cells expressing EGFPconjugated drs (DrsEGFP-MEF). As shown in Fig. 3C, Rab24 was coprecip-
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itated with DrsEGFP under low serum culture conditions, but hardly coprecipitated with DrsEGFP under high serum culture conditions. Endogenous Rab24 was similarly expressed in EGFP-MEF and DrsEGFPMEF cells under high and low serum conditions, although the expression of Rab24 under low serum conditions was about 2–3 fold higher than that under high serum conditions. Pulldown assays using EGFP-conjugated deletion mutants of drs (CD and TM) were also carried out in 293T cells. As shown in Fig. 3D, TM-EGFP was not coprecipitated with Rab24, whereas CD-EGFP was coprecipitated with Rab24, indicating that the transmembrane domain of drs is critical for the binding to Rab24 as well as the promotion of autophagy maturation in drs KO MEFs (Fig. 2C). To assess the association between drs and other Rab family proteins, the pulldown assay was similarly performed by using FLAG-tagged drs and the expression plasmids of EGFP-conjugated Rab3A, Rab7, Rab17 and Rab21 in 293T cells (Fig. 3E). Immunoblotting after immunoprecipitation with anti-EGFP antibody showed that drs associated with Rab7, Rab17 and Rab21, but not with Rab3A. These results indicate that drs associates with specific Rab GTPase family proteins including Rab24. 3.3. drs colocalizes with Rab24 during the autophagic process To evaluate the functional relationship between drs and Rab24 for autophagy, we investigated the molecular behavior of both proteins in DrsEGFP-MEF cells. Under high serum culture conditions, subcellular localization of drs was well merged with PDI, an ER marker protein
Fig. 3. Interaction between drs and Rab family proteins. (A) Silver-stained image of 15% SDS–PAGE gels of drs-binding proteins. 293T cells transfected by the pCR3.1 vector (vector, left) or the expression plasmid of FLAG-tagged drs (Drs-FLAG, right) were lysed, and the proteins bound to anti-FLAG affinity agarose were eluted by FLAG peptide. Eluted proteins were analyzed by 15% SDS–PAGE followed by silver staining. (B) Association of drs and Rab24 in 293T cells. The expression plasmid of EGFP-tagged drs was co-transfected with the expression plasmid of Rab24 in 293T cells. As controls, pCR3.1 vector, the expression plasmids of EGFP, EGFP-tagged drs and Rab24 were each singly transfected into 293T cells. The cell lysates were immunoprecipitated with antiRab24 antibody and immunoblotted for anti-GFP antibody. To confirm the expression of each protein in transfected cells, whole lysates were also immunoblotted for anti-EGFP or anti-Rab24 antibody. IP, immunoprecipitation; IB, immunoblotting. (C) Association of drs and endogenous Rab24 under both high and low serum conditions in DrsEGFP-MEFs. EGFP-MEF and DrsEGFP-MEF cells were cultured for 24 h under 10% or 0.1% FBS conditions. The cell lysates were immunoprecipitated with anti-GFP and immunoblotted for anti-Rab24 antibody. To confirm the expression of each protein in transfected cells, whole lysates were also immunoblotted for anti-EGFP or anti-Rab24 antibody. IP, immunoprecipitation; IB, immunoblotting. (D) Association of mutant drs and Rab24 in 293T cells. The expression plasmids of EGFP-tagged WT, CD and TM drs was co-transfected with the expression plasmid of Rab24 in 293T cells. As controls, pCR3.1 vector, the expression plasmids of EGFP, EGFP-tagged drs and Rab24 were each singly transfected into 293T cells. The cell lysates were immunoprecipitated with anti-Rab24 antibody and immunoblotted for anti-GFP antibody. To confirm the expression of each protein in transfected cells, whole lysates were also immunoblotted for anti-EGFP or anti-Rab24 antibody. IP, immunoprecipitation; IB, immunoblotting. (E) Association of drs and Rab family proteins in 293T cells. The expression plasmids of EGFP-tagged Rab7, Rab3A, Rab17 and Rab24 were co-transfected with FLAG-tagged drs in 293T cells. As controls, each expression plasmid of Rab proteins was singly transfected in 293T cells. The cell lysates were immunoprecipitated with anti-EGFP antibody and immunoblotted for anti-FLAG antibody. To confirm the expression of each protein, whole lysates were also immunoblotted for anti-EGFP or anti-FLAG antibody. IP, immunoprecipitation; IB, immunoblotting.
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Fig. 4. Subcellular localization of drs and Rab24 under confocal fluorescent microscopy. Scale bars represent 20 lm. (A) Immunofluorescence staining of PDI, an ER marker protein, in DrsEGFP-MEF cells. The cells were incubated in DMEM supplemented with 10% (upper) or 0.1% (lower) FBS for 18 h. (B) Fluorescence staining of lysosomes using LysoTracker Red in DrsEGFP-MEF cells under 10% (upper) or 0.1% (lower) serum culture conditions. (C) Immunofluorescence staining of Rab24 in DrsEGFP-MEF cells. The cells were incubated in DMEM supplemented with 10% (upper) or 0.1% (lower) FBS for 18 h. (D) Fluorescence microscopy in DrsEGFP-MEF cells transfected with mRFP-conjugated LC3. The cells were incubated in DMEM supplemented with 10% (upper) or 0.1% (lower) FBS for 18 h.
(Fig. 4A, upper), and was not detected in lysosomal vesicles that were stained with LysoTracker (Fig. 4B, upper). We then compared the behavior of DrsEGFP and endogenous Rab24. Both drs and Rab24 were localized in the ER region of the cells (Fig. 4C, upper) under high serum condition, and became aggregated and co-localized in the same vesiclelike structure (Fig. 4C, lower) when cells were cultured under low serum conditions for 18 h. PDI was not punctuated under low serum conditions in DrsEGFP-MEFs (Fig. 4A, lower), indicating that the punctuation induced by low serum treatment is not common in proteins localized in the ER. Under low serum culture conditions, most of drs was not merged with lysosomal vesicles (Fig. 4B, lower). To compare the cellular localization of drs and LC3, we transfected the plasmid expressing LC3 conjugated with monomeric red fluorescent protein (mRFP-LC3) into DrsEGFP-MEF cells. Under high serum culture conditions, mRFP-LC3 protein showed a diffuse and cytosolic distribution and was not merged with DrsEGFP (Fig. 4D, upper). After low serum treatment for 18 h, mRFP-LC3 was punctuated in the cytoplasm, and a portion of drs was also punctuated in cytoplasm and merged with
a subset of puncta with mRFP-LC3 (Fig. 4D, lower). Together, these results indicate that drs co-localized with Rab24 on the autophagic vesicles during autophagy. This, in turn, suggests that both proteins may be involved in the progression of autophagy.
4. Discussion In normal cells, autophagy occurs spontaneously at low rate and is induced in response to the cellular stresses [1,2]. These processes are considered to play physiological roles for the clearance of misfolded proteins and for the survival under the stress conditions, respectively. The present study has shown that drs plays a role in the progression of autophagy in normal cells under low serum culture conditions. There was no significant difference in the level of
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Fig. 4 (continued)
spontaneous autophagy between WT and KO MEFs and the defect of drs could not completely suppress the autophagy maturation (Figs. 1 and 2). These results suggest that drs can contribute to the autophagy maturation under the stress conditions, although it cannot govern the overall process of autophagy. We have also shown that drs is involved in ER-mediated apoptosis, which is associated with the activation of caspase -12, -9 and -3 in human and mouse cancer cells [10,11]. Both the previous and present findings suggest that drs regulates apoptosis or autophagy in response to cellular stresses such as the depletion of growth factors. Cells respond to stress through suppression of protein synthesis and cell growth, or through the induction of apoptosis and/or autophagy [1,2]. When the cells are subjected to the environmental stresses, drs positively regulates the autophagy and may help the cells to survival in the short-term. However if the stresses are prolonged and
the cells are irreversibly damaged, the cells undergo apoptosis or autophagic cell death with the regulation by drs. Thus, drs is considered to play a role in determination of cell fate in response to the environmental stresses. The determination of cell fate depends on the cellular contexts, such as the types of cells and stresses. The different responses of tumor and normal fibroblast cells to low serum treatment may be due to the difference in cell type, because normal fibroblast cells are known to be less susceptible to apoptosis than tumor cells. In normal tissues, the level of environmental stress is low and the stresses are usually not sustained. When tumors grow in vivo, their cells are subjected to prolonged environmental stresses under nutrient-, oxygen- and growth factor-limiting conditions inside the solid tumors, and many of these cells die. Our findings suggest that drs suppresses tumor growth via induction of apoptosis and/or autophagy under such stresses. Downregulation of drs has been frequently
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observed in a variety of human cancers [6–9], suggesting that tumor cells have evaded drs-mediated cell death in response to environmental stresses during malignant progression. Some tumor suppressor genes and apoptosis-regulating genes were shown to be involved in the autophagy initiation process. Beclin-1, a haploinsufficient tumor suppressor gene downregulated in breast cancer, is a mammalian homologue of a yeast autophagy-regulating gene, Atg6/ Vps30, and is necessary for the initiation of autophagy by forming a complex with class III phosphoinositide 3-kinase [24]. The anti-apoptotic and oncogenic protein Bcl-2 binds with the BH3 domain of Beclin-1 to inhibit autophagy induction [25]. The tumor suppressor UVRAG, the proapoptotic BH3-only protein Bif-1, PTEN, p53 and its target gene DRAM were also shown to be involved in the early stage of autophagy prior to autophagosome formation [25–28]. Unlike these tumor suppressor genes, the drs tumor suppressor is involved in the late maturation process, whose molecular mechanism has not been well understood. In the autophagy maturation process, autophagosomes fuse with endosomes before fusing with lysosomes [29]. Rab GTPase family proteins are key general players in membrane fusions, and it has been reported that Rab24 and Rab7 are involved in autophagy in mammals. Rab24 is predominantly localized to the ER-cis-Golgi membranes and to late endosomal structures. During autophagy, Rab24 is induced together with LC3 [23] and is punctuated on the dot structures [22]. Rab7 is also localized to late endosomes and is punctuated on the autophagic vacuoles during autophagy [30]. drs could also associate with Rab17 and Rab21 [31], which are localized to endosomes and regulate endocytosis, but not with Rab3A, which regulates exocytosis. These results suggest that drs may regulate the fusion of autophagosomes with endosomes and lysosomes through the interaction with these endosome-associated Rab proteins. The present study suggests that the drs tumor suppressor regulates the maturation process of autophagy through the interaction with Rab24. Further elucidation of the role of drs in autophagy will provide new insights into the molecular mechanisms regulating autophagy’s late maturation process and be a helpful clue to clarify the interrelationship between autophagy and carcinogenesis. Conflicts of interest The information contained there in is presented as a true representation of the data I/we have contributed to or utilized throughout this study. There are no financial conflicts of interest with any organization about this work. Acknowledgments We thank Dr. Noburu Mizushima (Tokyo Medical and Dental University) for allowing us to use the GFP-LC3 transgenic mice provided by RIKEN BRC; and Dr. Tamotsu Yoshimori (Osaka University) for providing the expression plasmids of EGFP-LC3 and mRFP-LC3 as well as the anti-
LC3 antibody. The GFP-LC3 transgenic mouse strain (BRC No. 00806) was provided by RIKEN BRC with the support of the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan. We would like to thank Ms. Hiroko Kita and Mr. Takefumi Yamamoto (Shiga University of Medical Science) for their technical assistance. This project was supported by a Grant-in-Aid for Young Scientists (B) (No. 18790270) (Y.T.); by Grants-in-Aid for Scientific Research (C) (No. 15590335 and No. 17590341); and by a Grant-in-Aid for Scientific Research on Priority Areas (No. 16021222) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.I.). This work was also supported by a grant from the NOVARTIS Foundation (Japan) for the Promotion of Science (H.I.).
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
The drs tumor suppressor is involved in the maturation process of autophagy induced by low serum Yukihiro Tambe a, Akitsugu Yamamoto b, Takahiro Isono c, Tokuhiro Chano d, Mitsunori Fukuda e, Hirokazu Inoue a,* a
Division of Microbiology and Infectious Diseases, Department of Pathology, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan Department of Cell Biology and Bioscience, The Nagahama Institute of Bioscience and Technology, Nagahama, Shiga 526-0829, Japan c Central Research Laboratory, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan d Clinical Laboratory Medicine, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan e Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Miyagi 980-8578, Japan b
a r t i c l e
i n f o
Article history: Received 2 September 2008 Received in revised form 3 February 2009 Accepted 17 March 2009
Keywords: drs Autophagy Knockout mouse Mouse embryonic fibroblasts Rab24
a b s t r a c t The drs gene is an apoptosis-inducing tumor suppressor. Previously, we showed that drs contributes to the suppression of tumor formation by generating drs-knockout mice. In this study, by using drs KO mouse embryonic fibroblasts, we found that drs is involved in the autophagy regulation under low serum culture conditions. Both electron microscopy and GFP-LC3 analyses demonstrated that drs is involved in the maturation process of autophagy from autophagosomes to autolysosomes. In addition, drs could associate with Rab24 and the association between drs and Rab24 was enhanced during autophagy. drs was also co-localized with Rab24 on punctuated structures during autophagy. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In determining whether tumor cells survive or die under stress conditions such as depletion of growth factors and nutrients, macroautophagy (hereafter referred to as autophagy) as well as apoptosis play crucial roles. Autophagy is a genetically regulated bulk degradation/recycling system conserved in eukaryotic cells [1,2]. During autophagy, parts of the cytoplasm, including macromolecules and organelles, are sequestered within autophagosomes, which are characteristic double-membraned vacuoles, and finally are degraded in the mature autolysosomes, which are formed by the fusion of autophagosomes and lysosomes. Since autophagy regenerates metabolic intermediates, it plays a critical role as a cell survival pathway in response to nutrient starvation. However, when autophagy is intensively induced and prolonged, it causes autophagic cell * Corresponding author. Tel.: +81 77 548 2177; fax: +81 77 548 2404. E-mail address: [email protected] (H. Inoue). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.03.028
death (type II programmed cell death). Recent studies suggested that autophagy is involved either in tumor development or regression together with apoptosis [1,3,4]. The drs gene was originally isolated as a transformation suppressor for the v-src oncogene [5,6], and the expression of drs mRNA has been shown to be markedly downregulated in a variety of human cancer cell lines and malignant tumor tissues [7–9]. Furthermore, ectopic expression of the drs protein induced apoptosis in human cancer cell lines via the novel pathway initiated from the endoplasmic reticulum (ER), involving the binding to ASY/Nogo-B/RTN-XS, apoptosis-inducing proteins localized in ER, and activation of caspase-12, -9, and -3 [10]. In previous study, we showed that malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of drsknockout (KO) mice [11]. Reintroduction of drs into a tumor cell line, LC-T1, derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis. Introduction of drs into the tumor cell line also enhanced sensitivity to
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apoptosis under low serum culture conditions. These findings indicate that drs contributes to the suppression of malignant tumor formation, and that this suppression is closely correlated with drs-mediated apoptosis under stress conditions. The drs KO mice did not show the distinct character other than tumor-prone phenotype. Although these previous findings suggest that drs regulates apoptosis in tumor cells, the physiological roles of drs in normal cells under stress conditions remains to be worked out. Then, we hypothesized that drs is involved in the cellular responses for stresses via the regulation of apoptosis and/or autophagy. In the present study, we have attempted to investigate the physiological roles of drs tumor suppressor by using mouse embryo fibroblast (MEF) cells derived from drs KO mice, and have found that drs is involved in the regulation of autophagy maturation under low serum culture conditions.
2. Materials and methods 2.1. Cells MEFs deficient in drs (KO MEF) or wild-type MEFs (WT MEF) were prepared from male embryos resulting from the mating of drs heterozygous (+/ ) female and wild-type (+/Y) male mice, as the drs gene is located in chromosome X [11]. The immortalized drs KO and WT MEF cell lines, KO-LT and WT-LT, were established by expressing the large T antigen of SV40 into KO and WT MEFs, respectively. KOgLC3LT and WT-gLC3LT, the immortalized drs KO and WT MEF cell lines which express GFP-LC3, were established from male embryos resulting from the mating of drs heterozygous female and GFP-LC3 male transgenic mice (GFPLC3#53, BRC No. 00806, RIKEN BRC, Tsukuba, Japan) [12,13] followed by the immortalization with the large T antigen of SV40. EGFP-MEF and DrsEGFP-MEF are WT MEFs that expresses EGFP and EGFP-conjugated drs, respectively. 293T (a human embryonic kidney cell line expressing the E1 gene of adenovirus type 5 and the T antigen of SV40) was also used for the preparation of the recombinant retrovirus and transfection experiments. All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 2.2. Plasmids and recombinant retroviruses The expression plasmids of EGFP-conjugated drs (WT, CD and TM), FLAG-tagged drs were described previously [6,10,14]. The expression plasmids of Rab24 and EGFP-conjugated Rab7, Rab3A, Rab17 and Rab21 were described previously [15]. The expression plasmid of mRFP-conjugated LC3 [16] was a kind gift from Dr. Tamotsu Yoshimori (Osaka University). Preparation and infection of pCXbsr retrovirus vectors carrying the drs cDNA (WT, CD and TM) were described previously [6,10].
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clonal antibody for Rab24 (R38620) was purchased from BD Transduction Laboratories (Lexington, KY, USA). Antia-tubulin (DM1A) and anti-FLAG M2 (F3165) monoclonal antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PDI (SPA-891) monoclonal antibody was purchased from Stressgen Biotechnologies (Victoria, BC, Canada). Anti-EGFP polyclonal (#8372-2) and monoclonal (#8371-2) antibodies (Living ColorsTM, Clontech) were purchased from Takara Bio (Shiga, Japan). Secondary fluorescence-labeled antibodies, Alexa 488- or 594-conjugated anti-rabbit or anti-mouse IgG, were purchased from Invitrogen (Carlsbad, CA, USA). 2.4. Electron microscopy MEFs, grown on gelatinized plastic coverslips (Celldesk LF1; Sumitomo Bakelite, Tokyo, Japan) were incubated with 10% or 0.1% FBS containing DMEM, fixed for 2 h with 2.5% glutaraldehyde (EM Science, Hatfield, PA, USA) in 0.1 M phosphate buffer at pH 7.4, post-fixed in 1% OsO in the same buffer and then subjected to electron microscopic analysis as described previously [17]. Representative areas were chosen for ultra-thin sectioning and viewed with a Hitachi 7600 electron microscope. The areas of autophagosomes and autolysosomes were measured using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) and the percentages of these areas in the cytoplasm area were calculated as area indexes. 2.5. Autophagy assay The early stage of autophagy development, the generation of autophagosomes, was quantified by detecting the LC3 punctuation in MEFs expressing GFP-LC3 under fluorescent microscopy [12,13]. The areas of the puncta were calculated by the top-hat detection mode of MetaMorph software as described [18]. Autophagy maturation was observed by LC3 conversion and turnover assay by immunoblotting with anti-LC3 antibody [19]. The generation of mature autophagolysosomes in living cells was quantified by detecting the development of acidic vesicular organelles stained with acridine orange [20]. Cells were stained with 0.5 lg/ml of acridine orange for 15 min, removed from the plate with trypsin–EDTA and analyzed by FACSCaliber and CellQuest software (BD, Franklin Lakes, NJ, USA). 2.6. Immunofluorescence staining Immunofluorescence staining was carried out as described previously [10]. For the visualization of lysosomes in living cells, LysoTracker Red DND-99 (50 nM, Invitrogen) was added to the cell culture and incubated for 5 min in CO2 incubator. Samples were observed with a confocal fluorescent microscope (Nikon C1si). 2.7. Immunoprecipitation and immunoblotting
2.3. Antibodies Rabbit polyclonal antibody for LC3 was a kind gift from Dr. Tamotsu Yoshimori (Osaka University). Mouse mono-
Cells were lysed in RIPA buffer containing 20 mM Tris– HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton-X100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate and
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Fig. 1. Analyses of cell death and autophagy in WT and drs KO MEFs under low serum culture conditions. (A) Cell death induced by prolonged (72 h) low serum (0.1%) treatment in WT and KO MEFs. zVAD: 2 lM of zVAD-fmk was added at 0 h. Open and closed columns show WT and drs KO MEFs, respectively. Each bar indicates mean ± s.d. *p < 0.05, **p < 0.01 by Student’s t-test. (B) Cell morphology of WT (left) and KO (right) MEFs under prolonged (48 h) low serum (0.1%) culture conditions. 5 105 cells were plated onto 60 mm dishes and cultured at 37 °C for 4 h. The medium was replaced with DMEM supplemented with 0.1% FBS (0 h, top), and then the cells were cultured for 48 h (middle and bottom). Black arrows show vacuole-like structures in cytoplasm. Scale bars represent 20 lm. (C) Quantification of aberrant MEF cells under prolonged (48 h) low serum culture conditions. Open and closed columns show WT and KO MEFs, respectively. Bars indicate mean ± s.d. of the percentage of attached cells that were aberrant. Detached dead cells were omitted from the counts. **p < 0.01 by Student’s t-test. (D) Morphological analyses of autophagy by electron microscopy. Primary WT (middle) and KO (bottom) MEFs (passage 2) were cultured in DMEM containing 0.1% FBS for 24 h. White and black arrows indicate autophagosomes (AP) and autolysosomes (AL), respectively. Representative images of autophagosomes (top left) and autolysosomes (top right) are also shown. White and black scale bars represent 1 lm and 2 lm, respectively. (E) Quantification of autophagosome and autolysosome formation under electron microscopy. Area indexes are centuple values of percentages of the organelle area in the cytoplasm area. Open and closed columns show WT and KO MEFs, respectively. Each column indicates mean ± s.d of a representative area of 50 different cells. *p < 0.05 by Student’s t-test.
1% protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan), and were centrifuged for 30 min in 10,000g at 4 °C.
Immunoprecipitation, electrophoresis and immunoblotting procedures were performed as described previously [10].
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3 Fig. 2. Analyses of the maturation process of autophagy in WT and drs KO MEFs under low serum culture conditions. (A) Time course of LC3 conversion and turnover in WT and KO MEFs. Cells were cultured in DMEM supplemented with 0.1% FBS for the represented time, and the cell lysates were analyzed by immunoblotting with anti-LC3 antibody (upper). For turnover assay, pepstatin A (10 lg/ml) and E64d (10 lg/ml) were added (+inhibitors) at the start time of low serum treatment. To evaluate the conversion of LC3 from the type I form (precursor) to the type II form (membrane-bound), the ratios of the quantity of type II form of LC3 to b-actin were calculated from the digitized image (lower). Columns indicate the relative value of the LC3-II / b-actin ratio at each point of time based on the ratio at 0 h. Open and closed columns represent WT and KO MEFs, respectively. (B) Time course of the GFP-LC3 punctuation in WT-gLC3LT and KO-gLC3LT MEFs under fluorescent microscopy. Cells were cultured in DMEM supplemented with 0.1% FBS for the represented time. Photographs represent the merged images of GFP-LC3 punctuation (green) and nuclei stained with Hoechst 33342 (blue) under low serum conditions at pretreatment (0 h), early (6 h) and late (18 h) stages of autophagy in WT-gLC3LT and KO-gLC3LT MEFs (upper). Small inserted images represent magnified views of GFP-LC3 punctuation. Yellow and white scale bars represent 20 lm and 50 lm, respectively. The percentages of the areas of GFP-LC3 dots in cytoplasm were calculated (lower). Open and closed circles represent WT-gLC3LT and KO-gLC3LT MEFs, respectively. Each value indicates a mean ± s.d. of 20 cells. p < 0.05 by Student’s t-test. (C) Time course of the GFP-LC3 punctuation in KOgLC3-LT MEFs expressing retroviral WT, CD and TM drs cDNAs under fluorescent microscope. The percentages of the area of GFP-LC3 dots in cytoplasm were calculated at pretreatment (0 h), early (6 h) and late (18 h) stage of autophagy under low serum conditions. (D) Fluorescent microscopic images of acidic organelles stained with acridine orange. The primary MEFs (passage 2) were cultured in DMEM containing 10% (upper) and 0.1% (lower) FBS for 24 h. The cytosol and nucleus fluoresce bright green and dim red, whereas acidic compartments fluoresce bright red. Small inserted images represent magnified views. Yellow and white scale bars represent 20 lm and 100 lm, respectively. Percentage of cells that were enriched with acidic organelles (A. O.) in primary WT and KO MEFs (passage 2) under high or low serum treatment (24 h) (lower). The cells with red fluorescence were quantified by FACS analyses. 3-MA: 10 mM of 3-methyladenine, an inhibitor of autophagy, was added at 0 h. Each bar indicates mean ± s.d. p < 0.05 by Student’s t-test.
2.8. Identification of drs-binding proteins by peptide mass fingerprinting analyses 293T cells were transfected with the expression plasmid of FLAG-tagged drs or vector plasmid (pCR3.1) by using Lipofectamine-PLUS reagent. After 2 days, the cells were lysed in RIPA buffer. The lysate was centrifuged at 10,000g for 30 min, and the supernatant was incubated with anti-FLAG affinity agarose (Sigma-Aldrich) at 4 °C for 2 h. Then the agarose was washed three times with RIPA buffer and the proteins bound to the agarose were eluted by FLAG-peptide (150 lM, Sigma-Aldrich). The samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins were visualized by silver staining. Each protein band was digested in the gel by modified trypsin (Promega, Madison, WI, USA). Protein identification for each band was performed by peptide mass fingerprinting analysis as described previously [21]. 3. Results 3.1. drs regulates the maturation of autophagy from autophagosomes to autolysosomes To investigate the physiological role of drs gene, we prepared the wild-type (WT) and drs KO MEFs. However, there was no significant difference between WT and KO MEFs in the cell proliferation, cellular senescence, and the cellular morphology under normal culture conditions (data not shown). Previously, we reported that the re-introduction of drs into LC-T1, a lung cancer cell line derived from a drs KO mouse, induced apoptosis after 24 h incubation under low serum culture conditions (0.1% FBS), suggesting that drs may play a role in the cell responses for stresses [11]. To investigate the role of drs gene under stress conditions, we immortalized wild-type and drs KO MEFs by introducing the large T (LT) antigen of SV40 and compared the responses of both MEF cells under low serum culture conditions. Although cell death was hardly observed in WT and KO MEFs at 24 h in low serum culture (data not shown), significant cell death (55.4%) was observed in WT MEFs in prolonged culture at 72 h (Fig. 1A). On the other hand, the percentage of cell death was significantly lower in KO MEFs (25.6%). zVAD-fmk, a pan-caspase inhibitor, did not suppress cell death in either WT or KO MEFs, indicating that this cell death was not apoptosis. During cultivation, a majority (51.9%) of morphologically aberrant cells, having a number of vacuole-like structures in the cytoplasm (Fig. 1B, black arrows), were observed in WT MEFs at 48 h, whereas the percentage of such aberrant cells was significantly lower (13.1%) in KO MEFs at 48 h (Fig. 1C). The degree of vacuolation per cell
in KO MEFs was also lower than that in WT MEFs (Fig. 1B). zVAD-fmk did not suppress the appearance of aberrant cells under low serum culture conditions in either WT or KO MEFs (Fig. 1C). These findings suggest the possibility that drs is involved in the regulation of autophagy under low serum culture conditions. We then performed a morphological analysis of autophagic vacuole formation by electron microscopy in WT and KO MEFs under low serum conditions. As shown in Fig. 1D, both the autophagosomes (AP, white arrows) and autolysosomes (AL, black arrows) were observed in WT and KO MEFs at 24 h after low serum treatment. Although the total area indexes of these autophagic vesicles were similar between WT and KO MEFs, the index of autolysosomes in KO MEFs was significantly lower than that in WT MEFs. On the other hand, the index of autophagosomes in KO MEFs was higher than that in WT MEFs (Fig. 1E). These results suggest that the maturation process from autophagosomes to autolysosomes was inhibited in drs KO cells. To confirm that drs is involved in the maturation process of autophagy, we examined the molecular behavior of LC3, a mammalian marker of autophagosomes, under low serum culture conditions in WT and KO MEFs. In the early stage of autophagy, the cytosolic form of LC3 (LC3-I) is converted into the autophagosomal membrane-bound form of LC3 (LC3-II) by conjugation with phospholipids. In a subsequent stage, LC3II is degraded in the autolysosomes by the lysosomal proteases or is recycled back to LC3-I. During low serum treatment, the relative ratio of LC3-II (LC3-II/b-actin) in WT MEFs was increased from 3 to 6 h and then gradually reduced (Fig. 2A). On the other hand, in KO MEFs, the ratio of LC3-II was increased from 3 to 6 h and remained elevated. The addition of a mixture of pepstatin A and E64d, the cell-permeable lysosomal protease inhibitors that inhibit the turnover of LC3 by delaying the degradation in autolysosomes, restored the ratio of LC3-II at the late stage in WT MEFs to the level in KO MEFs (see Fig. 2A, 18 +inhibitors). Furthermore, to visualize the molecular behavior of LC3 in cells, we established WT and drs KO MEFs expressing GFP-LC3 (WT-gLC3LT and KO-gLC3LT) by the mating of drs KO mice and GFP-LC3 transgenic mice. As shown in Fig. 2B, the punctuated GFP-LC3 area was increased from 3 to 6 h after low serum treatment, and was subsequently decreased in WT-gLC3LT. On the other hand, in KO-gLC3LT, it was similarly increased in the early stage of autophagy (6 h), but remained elevated in the late stage (up to 18 h after treatment). Re-introduction of wild-type (WT) drs by retrovirus vector into KO-gLC3LT cells decreased the punctuated GFP-LC3 area in the late stage (Fig. 2C). This effect was also observed by introduction of the CD mutant, which lacks the C-terminal region of drs, but not by introduction of the TM mutant, which lacks the transmembrane domain of drs, indicating that the transmembrane domain of drs is necessary for the maturation process of autophagy under low serum culture conditions (Fig. 2C). The formation of mature autolysosomes in the cytoplasm can be monitored by the appearance of acidic organelles, which are stained red1 with acridine orange, if newly formed acidic compartments are attributed to autolysosomes. As shown in Fig. 2D, the number of acridine orange-positive 1 For interpretation of color in Figs. 2–4, the reader is referred to the web version of this article.
Y. Tambe et al. / Cancer Letters 283 (2009) 74–83 cells in KO MEFs (12.8%) was significantly lower than that in WT MEFs (26.5%) at 24 h after low serum treatment. 3-methyladenine (3-MA), which is known to inhibit autophagy, completely suppressed acidic organelle formation in both WT and KO MEFs (Fig. 2D), suggesting that the formation of acidic organelles in these experiments reflects the induction of autophagy. These results indicate that drs is required for the maturation process of autophagic vesicles from autophagosomes to autolysosomes. 3.2. drs associates with Rab24 and related Rab family GTPases To investigate how drs regulates the autophagy maturation process, we attempted to identify the drs-binding proteins involved in the maturation of autophagic vesicles. As shown in Fig. 3A, a protein with a molecular weight of about 25 kD was co-precipitated with FLAG-tagged drs protein expressed in 293T cells. By peptide mass fingerprinting analyses, the protein was identified as Rab24, a member of the Rab GTPase protein family, has been reported to be involved in autophagy [22,23]. To confirm the association between drs and Rab24, we carried out a pulldown assay in 293T cells transfected with the plasmids expressing drs conjugated with EGFP (DrsEGFP) and/or Rab24. As shown in Fig. 3B, DrsEGFP was coprecipitated with Rab24, but EGFP alone was not. To evaluate whether endogenous Rab24 is able to bind to drs under low serum culture conditions, we also performed a pulldown assay in MEF cells expressing EGFPconjugated drs (DrsEGFP-MEF). As shown in Fig. 3C, Rab24 was coprecip-
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itated with DrsEGFP under low serum culture conditions, but hardly coprecipitated with DrsEGFP under high serum culture conditions. Endogenous Rab24 was similarly expressed in EGFP-MEF and DrsEGFPMEF cells under high and low serum conditions, although the expression of Rab24 under low serum conditions was about 2–3 fold higher than that under high serum conditions. Pulldown assays using EGFP-conjugated deletion mutants of drs (CD and TM) were also carried out in 293T cells. As shown in Fig. 3D, TM-EGFP was not coprecipitated with Rab24, whereas CD-EGFP was coprecipitated with Rab24, indicating that the transmembrane domain of drs is critical for the binding to Rab24 as well as the promotion of autophagy maturation in drs KO MEFs (Fig. 2C). To assess the association between drs and other Rab family proteins, the pulldown assay was similarly performed by using FLAG-tagged drs and the expression plasmids of EGFP-conjugated Rab3A, Rab7, Rab17 and Rab21 in 293T cells (Fig. 3E). Immunoblotting after immunoprecipitation with anti-EGFP antibody showed that drs associated with Rab7, Rab17 and Rab21, but not with Rab3A. These results indicate that drs associates with specific Rab GTPase family proteins including Rab24. 3.3. drs colocalizes with Rab24 during the autophagic process To evaluate the functional relationship between drs and Rab24 for autophagy, we investigated the molecular behavior of both proteins in DrsEGFP-MEF cells. Under high serum culture conditions, subcellular localization of drs was well merged with PDI, an ER marker protein
Fig. 3. Interaction between drs and Rab family proteins. (A) Silver-stained image of 15% SDS–PAGE gels of drs-binding proteins. 293T cells transfected by the pCR3.1 vector (vector, left) or the expression plasmid of FLAG-tagged drs (Drs-FLAG, right) were lysed, and the proteins bound to anti-FLAG affinity agarose were eluted by FLAG peptide. Eluted proteins were analyzed by 15% SDS–PAGE followed by silver staining. (B) Association of drs and Rab24 in 293T cells. The expression plasmid of EGFP-tagged drs was co-transfected with the expression plasmid of Rab24 in 293T cells. As controls, pCR3.1 vector, the expression plasmids of EGFP, EGFP-tagged drs and Rab24 were each singly transfected into 293T cells. The cell lysates were immunoprecipitated with antiRab24 antibody and immunoblotted for anti-GFP antibody. To confirm the expression of each protein in transfected cells, whole lysates were also immunoblotted for anti-EGFP or anti-Rab24 antibody. IP, immunoprecipitation; IB, immunoblotting. (C) Association of drs and endogenous Rab24 under both high and low serum conditions in DrsEGFP-MEFs. EGFP-MEF and DrsEGFP-MEF cells were cultured for 24 h under 10% or 0.1% FBS conditions. The cell lysates were immunoprecipitated with anti-GFP and immunoblotted for anti-Rab24 antibody. To confirm the expression of each protein in transfected cells, whole lysates were also immunoblotted for anti-EGFP or anti-Rab24 antibody. IP, immunoprecipitation; IB, immunoblotting. (D) Association of mutant drs and Rab24 in 293T cells. The expression plasmids of EGFP-tagged WT, CD and TM drs was co-transfected with the expression plasmid of Rab24 in 293T cells. As controls, pCR3.1 vector, the expression plasmids of EGFP, EGFP-tagged drs and Rab24 were each singly transfected into 293T cells. The cell lysates were immunoprecipitated with anti-Rab24 antibody and immunoblotted for anti-GFP antibody. To confirm the expression of each protein in transfected cells, whole lysates were also immunoblotted for anti-EGFP or anti-Rab24 antibody. IP, immunoprecipitation; IB, immunoblotting. (E) Association of drs and Rab family proteins in 293T cells. The expression plasmids of EGFP-tagged Rab7, Rab3A, Rab17 and Rab24 were co-transfected with FLAG-tagged drs in 293T cells. As controls, each expression plasmid of Rab proteins was singly transfected in 293T cells. The cell lysates were immunoprecipitated with anti-EGFP antibody and immunoblotted for anti-FLAG antibody. To confirm the expression of each protein, whole lysates were also immunoblotted for anti-EGFP or anti-FLAG antibody. IP, immunoprecipitation; IB, immunoblotting.
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Fig. 4. Subcellular localization of drs and Rab24 under confocal fluorescent microscopy. Scale bars represent 20 lm. (A) Immunofluorescence staining of PDI, an ER marker protein, in DrsEGFP-MEF cells. The cells were incubated in DMEM supplemented with 10% (upper) or 0.1% (lower) FBS for 18 h. (B) Fluorescence staining of lysosomes using LysoTracker Red in DrsEGFP-MEF cells under 10% (upper) or 0.1% (lower) serum culture conditions. (C) Immunofluorescence staining of Rab24 in DrsEGFP-MEF cells. The cells were incubated in DMEM supplemented with 10% (upper) or 0.1% (lower) FBS for 18 h. (D) Fluorescence microscopy in DrsEGFP-MEF cells transfected with mRFP-conjugated LC3. The cells were incubated in DMEM supplemented with 10% (upper) or 0.1% (lower) FBS for 18 h.
(Fig. 4A, upper), and was not detected in lysosomal vesicles that were stained with LysoTracker (Fig. 4B, upper). We then compared the behavior of DrsEGFP and endogenous Rab24. Both drs and Rab24 were localized in the ER region of the cells (Fig. 4C, upper) under high serum condition, and became aggregated and co-localized in the same vesiclelike structure (Fig. 4C, lower) when cells were cultured under low serum conditions for 18 h. PDI was not punctuated under low serum conditions in DrsEGFP-MEFs (Fig. 4A, lower), indicating that the punctuation induced by low serum treatment is not common in proteins localized in the ER. Under low serum culture conditions, most of drs was not merged with lysosomal vesicles (Fig. 4B, lower). To compare the cellular localization of drs and LC3, we transfected the plasmid expressing LC3 conjugated with monomeric red fluorescent protein (mRFP-LC3) into DrsEGFP-MEF cells. Under high serum culture conditions, mRFP-LC3 protein showed a diffuse and cytosolic distribution and was not merged with DrsEGFP (Fig. 4D, upper). After low serum treatment for 18 h, mRFP-LC3 was punctuated in the cytoplasm, and a portion of drs was also punctuated in cytoplasm and merged with
a subset of puncta with mRFP-LC3 (Fig. 4D, lower). Together, these results indicate that drs co-localized with Rab24 on the autophagic vesicles during autophagy. This, in turn, suggests that both proteins may be involved in the progression of autophagy.
4. Discussion In normal cells, autophagy occurs spontaneously at low rate and is induced in response to the cellular stresses [1,2]. These processes are considered to play physiological roles for the clearance of misfolded proteins and for the survival under the stress conditions, respectively. The present study has shown that drs plays a role in the progression of autophagy in normal cells under low serum culture conditions. There was no significant difference in the level of
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Fig. 4 (continued)
spontaneous autophagy between WT and KO MEFs and the defect of drs could not completely suppress the autophagy maturation (Figs. 1 and 2). These results suggest that drs can contribute to the autophagy maturation under the stress conditions, although it cannot govern the overall process of autophagy. We have also shown that drs is involved in ER-mediated apoptosis, which is associated with the activation of caspase -12, -9 and -3 in human and mouse cancer cells [10,11]. Both the previous and present findings suggest that drs regulates apoptosis or autophagy in response to cellular stresses such as the depletion of growth factors. Cells respond to stress through suppression of protein synthesis and cell growth, or through the induction of apoptosis and/or autophagy [1,2]. When the cells are subjected to the environmental stresses, drs positively regulates the autophagy and may help the cells to survival in the short-term. However if the stresses are prolonged and
the cells are irreversibly damaged, the cells undergo apoptosis or autophagic cell death with the regulation by drs. Thus, drs is considered to play a role in determination of cell fate in response to the environmental stresses. The determination of cell fate depends on the cellular contexts, such as the types of cells and stresses. The different responses of tumor and normal fibroblast cells to low serum treatment may be due to the difference in cell type, because normal fibroblast cells are known to be less susceptible to apoptosis than tumor cells. In normal tissues, the level of environmental stress is low and the stresses are usually not sustained. When tumors grow in vivo, their cells are subjected to prolonged environmental stresses under nutrient-, oxygen- and growth factor-limiting conditions inside the solid tumors, and many of these cells die. Our findings suggest that drs suppresses tumor growth via induction of apoptosis and/or autophagy under such stresses. Downregulation of drs has been frequently
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observed in a variety of human cancers [6–9], suggesting that tumor cells have evaded drs-mediated cell death in response to environmental stresses during malignant progression. Some tumor suppressor genes and apoptosis-regulating genes were shown to be involved in the autophagy initiation process. Beclin-1, a haploinsufficient tumor suppressor gene downregulated in breast cancer, is a mammalian homologue of a yeast autophagy-regulating gene, Atg6/ Vps30, and is necessary for the initiation of autophagy by forming a complex with class III phosphoinositide 3-kinase [24]. The anti-apoptotic and oncogenic protein Bcl-2 binds with the BH3 domain of Beclin-1 to inhibit autophagy induction [25]. The tumor suppressor UVRAG, the proapoptotic BH3-only protein Bif-1, PTEN, p53 and its target gene DRAM were also shown to be involved in the early stage of autophagy prior to autophagosome formation [25–28]. Unlike these tumor suppressor genes, the drs tumor suppressor is involved in the late maturation process, whose molecular mechanism has not been well understood. In the autophagy maturation process, autophagosomes fuse with endosomes before fusing with lysosomes [29]. Rab GTPase family proteins are key general players in membrane fusions, and it has been reported that Rab24 and Rab7 are involved in autophagy in mammals. Rab24 is predominantly localized to the ER-cis-Golgi membranes and to late endosomal structures. During autophagy, Rab24 is induced together with LC3 [23] and is punctuated on the dot structures [22]. Rab7 is also localized to late endosomes and is punctuated on the autophagic vacuoles during autophagy [30]. drs could also associate with Rab17 and Rab21 [31], which are localized to endosomes and regulate endocytosis, but not with Rab3A, which regulates exocytosis. These results suggest that drs may regulate the fusion of autophagosomes with endosomes and lysosomes through the interaction with these endosome-associated Rab proteins. The present study suggests that the drs tumor suppressor regulates the maturation process of autophagy through the interaction with Rab24. Further elucidation of the role of drs in autophagy will provide new insights into the molecular mechanisms regulating autophagy’s late maturation process and be a helpful clue to clarify the interrelationship between autophagy and carcinogenesis. Conflicts of interest The information contained there in is presented as a true representation of the data I/we have contributed to or utilized throughout this study. There are no financial conflicts of interest with any organization about this work. Acknowledgments We thank Dr. Noburu Mizushima (Tokyo Medical and Dental University) for allowing us to use the GFP-LC3 transgenic mice provided by RIKEN BRC; and Dr. Tamotsu Yoshimori (Osaka University) for providing the expression plasmids of EGFP-LC3 and mRFP-LC3 as well as the anti-
LC3 antibody. The GFP-LC3 transgenic mouse strain (BRC No. 00806) was provided by RIKEN BRC with the support of the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan. We would like to thank Ms. Hiroko Kita and Mr. Takefumi Yamamoto (Shiga University of Medical Science) for their technical assistance. This project was supported by a Grant-in-Aid for Young Scientists (B) (No. 18790270) (Y.T.); by Grants-in-Aid for Scientific Research (C) (No. 15590335 and No. 17590341); and by a Grant-in-Aid for Scientific Research on Priority Areas (No. 16021222) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.I.). This work was also supported by a grant from the NOVARTIS Foundation (Japan) for the Promotion of Science (H.I.).
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