Báo cáo khoa học: The regulation of the endosomal compartment by p53 the tumor suppressor gene docx

The regulation of the endosomal compartment by p53 the tumor suppressor gene Xin Yu 1 , Todd Riley 2,3 and Arnold J. Levine 1,3 1 The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ, USA 2 The BioMaPS Institute at Rutgers University, Piscataway, NJ, USA 3 School of Natural Sciences, The Institute for Advanced Study, Princeton, NJ, USA The p53 protein is a transcription factor that is acti- vated by a wide variety of stress signals [1]. This results in a transcriptional program that responds to the stress and returns the cell to homeostasis, permit- ting it to function normally without errors. The most commonly studied stress is DNA damage, and the p53 program responds with cell-cycle arrest and the synthe- sis of repair functions, cellular senescence or apoptosis. However, the p53 pathway also responds to stress sig- nals, resulting in a transcriptional program impacting upon a large number of other cellular processes. For example, the p53 pathway shuts down the insulin-like growth factor-1 (IGF-1) ⁄ AKT-1 ⁄ mammalian target of rapamycin (mTor) pathways in response to nutrient starvation [2–4] and activates autophagy, monitors ribosomal biogenesis and regulates the metabolic path- ways [5]. The p53 pathway also regulates the synthesis of cytokines that can attract cells to a senescent signal- ing cell [6–11]. Recently, it has become clear that p53- activating stress signals can have an impact upon the endosomal compartment in a cell and alter membrane and vesicle trafficking [3,12], leading to autophagy and exosome production. Exosomes are 50–150 nm vesicles generated from the late endosome ⁄ multivesicular bodies (MVBs) in a cell by invagination into the MVB, trapping cytoplasmic components and mem- brane proteins. Exosomes exit into the extracellular space after MVBs fuse with the plasma membrane. Keywords EGF receptor; endosomal compartment; exosome production; internalization; p53 regulation Correspondence A. J. Levine, School of Natural Sciences, The Institute for Advanced Study, Princeton, NJ 08540, USA Fax: +1 609 951 4438 Tel: +1 609 734 8118 E-mail: alevine@ias.edu (Received 23 September 2008, revised 29 January 2009, accepted 4 February 2009) doi:10.1111/j.1742-4658.2009.06949.x The endosomal compartment of the cell is involved in a number of func- tions including: (a) internalizing membrane proteins to multivesicular bodies and lysosomes; (b) producing vesicles that are secreted from the cell (exosomes); and (c) generating autophagic vesicles that, especially in times of nutrient deprivation, supply cytoplasmic components to the lysosome for degradation and recycling of nutrients. The p53 protein responds to various stress signals by initiating a transcriptional program that restores cellular homeostasis and prevents the accumulation of errors in a cell. As part of this process, p53 regulates the transcription of a set of genes encod- ing proteins that populate the endosomal compartment and impact upon each of these endosomal functions. Here, we demonstrate that p53 regulates transcription of the genes TSAP6 and CHMP4C, which enhance exosome production, and CAV1 and CHMP4C, which produce a more rapid endosomal clearance of the epidermal growth factor receptor from the plasma membrane. Each of these p53-regulated endosomal functions results in the slowing of cell growth and division, the utilization of cata- bolic resources and cell-to-cell communication by exosomes after a stress signal is detected by the p53 protein. These processes avoid errors during stress and restore homeostasis once the stress is resolved. Abbreviations ChIP, chromatin immunoprecipitation; Chmp, charged multivesicular body protein; EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor; mTOR, mammalian target of rapamycin; MVB, multivesicular body. FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS 2201 These vesicles can communicate with the immune sys- tem (dendritic cells) immunizing the host, fuse with adjacent cells, presumably communicating physiologi- cal signals, or contribute to the extracellular matrix [13,14]. The increased rate of exosome production in cells with an activated p53 response is caused, in part, by the p53-regulated gene TSAP6, a member of the Steap family of proteins (Steap3), which functions in an unknown way to enhance exosome production [12,15–19]. Endosomes have a number of functions in a cell. Endosomal vesicles sample the environment and bring components to the lysosome for degradation. They transport membrane proteins, including receptors for growth or cell maintenance, to the intracellular com- partments (MVBs and lysosomes). They internalize receptors that have engaged their ligands and are sig- naling, either reducing the signals or setting up new locations for signaling. Some of these receptors are degraded in lysosomes, whereas others are trafficked back onto the cell surface in a regulated process. Reg- ulation of these processes permits cells to be responsive to outside signaling or to ignore such messages. The MVB contains a set of  30 different sorting proteins that are quite conserved from yeast to humans [20–23]. The mammalian MVB is composed of several sets of protein complexes termed Stam ⁄ Hrs, ESCRT-I, ESCRT-II, ESCRT-III and Vps4 [20,21]. These com- plexes are sequentially recruited to the site of MVB formation and result in the progressive trafficking of vesicles (cargo) through this organelle. It is during this process that decisions are made to traffic cargo outside the cell (exosomes), into the plasma membrane for degradation (lysosomes) or into an autophagic vesicle. The ESCRT-III protein complex on the MVB is com- posed of a series of charged MVB proteins (Chmp) 1A, 1B, 2A, 2B, 3, 4A, 4B, 4C, 5 and 6 [20,21]. The experiments presented here demonstrate that CHMP4C is a p53-regulated gene whose transcription and protein increase after a p53 stress response. This is correlated with higher rates of exosome production and faster rates of clearing the epidermal growth factor receptor (EGFR) from the plasma membrane. There are at least two routes via which to clear pro- teins such as EGFR from the plasma membrane into the cell: clathrin-coated pits or a caveolae-mediated pathway [24,25]. In this study, the caveolin-1 (CAV1) gene, encoding one of three caveolin proteins, is shown to be a p53-regulated gene. The EGFR and caveolin-1 proteins colocalize in the plasma membrane and the EGFR is then internalized at a faster rate after a p53 stress response, demonstrating for the first time that the p53 response down-modulates the availability of growth receptors at the cell surface, making the cell less sensitive to growth and division signals. Interest- ingly, the CAV1 gene has been called by some a tumor suppressor gene that is absent in some breast cancer cells [26]. In some animal models that deleted the CAV1 gene, animals were more susceptible to oncogene- or carcinogen-induced tumorigenesis [27]. However, caveolin-1 protein levels were found to be very high in some multidrug resistant cells [28], aggres- sive prostate cancers [29] and malignant breast lesions [30,31]. Clearly, this is not a consistent pattern of observations from which to draw any firm conclusions. Thus, the functions of the endosome compartment, exosome production, endosome production, and the regulation and recycling of cell-surface receptors all increase after a p53 response to stress. The net result is to shut down growth and division, utilize the cell’s reserves and communicate stress signals to other cells. In this fashion, the p53 protein helps to down- modulate cell growth and division after stress, and utilizes cellular reserves to maintain cells during periods of stress. Results CHMP4C is a p53-regulated gene Previous experimental results [2,3,12] have identified the endosome compartment of the cell as a place where several types of cellular stress are responded to by a p53-mediated transcription of genes that enhances endosomal functions such as autophagy and exosome production. For this reason, the DNA sequences in the promoter–enhancer regions of all known genes for endosome compartment components in the human cell were screened for potential p53 DNA-binding sites. To carry out this screening, we developed an algorithm designed to detect p53 regulatory DNA sequences (p53MHH). The position of )512 to )450 nucleotides 5¢ to the transcriptional start site of the first exon of the CHMP4C gene was a perfect match to a p53 DNA-binding site, with the two sites separated by an 18 bp spacer (Fig. 1A). CHMP4C is part of the ESC- RT-III protein complex that is essential for endosome function in a cell. Regulation of CHMP4C expression by p53 was tested in the human cell lines H460 (wild- type p53) and H1299 (null-p53). For this we used quantitative real-time PCR to follow the steady-state levels of CHMP4C mRNA in the cell at 24 h after irradiation. Activation of p53 by irradiation increased the levels of CHMP4C RNA by 4.5-fold in H460 cells, although H1299 cells did not show increased CHMP4C RNA (Fig. 1B). A different stress agent, p53-regulated endosomal compartment X. Yu et al. 2202 FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS 0 1 2 3 4 5 H460 0 1 2 3 4 5 Relative expression level 0 0.5 1 1.5 2 2.5 3 3.5 4 No treatment IR H1299 0 0.5 1 1.5 2 2.5 3 3.5 4 No treatment IR M 300 bp 200 bp Input -Ab +IgG +DO-1 +DO-1 H1299 - + -1 H460 -1 0 1 2 3 4 5 6 7 8 9 Relative activity of Luciferase pGL3- Vector pGL3- promoter pGL3-CHMP4Cseq wt- p53 mt-p53 (22/23) mt-p53 (273) - No treatment Etop No treatment Etop 0 50 100 150 200 250 siRNA-NS siRNA-p53 p21 in H460 0 0.5 1 1.5 2 2.5 3 siRNA-NS siRNA-p53 p53 in H460 - 0 1 2 3 4 5 6 7 siRNA-NS siRNA-p53 CHMP4C in H460 Relative expression level A B CD Fig. 1. Expression of CHMP4C was regulated by p53. (A) A p53 recognition sequence (p53RE) was identified by the algorithm p53MHH. Uppercase letters represent the two repeats of PuPuPuC(A ⁄ T)(T ⁄ A)GPyPyPy in the p53RE. The lowercase letters within the repeats repre- sent the spacer. The lowercase letters on the flank of the repeats represent the flanking sequences around the p53RE. (B) Regulation of gene expression was measured by real-time PCR after the cells (H460 and H1299) had been treated with c-radiation and the cells (H460) had undergone transfection of siRNA against nonspecific sequence (siRNA-NS) or siRNA against p53 (siRNA-p53), followed by etoposide treatment. (C) Putative p53RE was able to be bound by the p53 protein shown in the ChIP assay. The samples were the input, the reco- vered DNA from incubation with no antibody, with IgG or with antibody against p53 (DO-1). Both H460 and H1299 cells were treated with irradiation. (D) DNA sequence, including the p53RE, induced luciferase activity with co-transfection of wild-type p53, but not with p53 mutants (22 ⁄ 23) and (273), in the luciferase activity assay. The pGL3–vector and pGL3–promotor plasmids were tested as parallel controls to the constructs of pGL3–p53RE in the CHMP4C sequence. X. Yu et al. p53-regulated endosomal compartment FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS 2203 etoposide, also increased CHMP4C mRNA levels five- fold in H460 cells, and this induction was decreased nine-fold by an siRNA directed against the p53 mRNA (Fig. 1B). When V138 cells, a cell line with a tempera- ture-sensitive p53 protein, were shifted from a nonper- missive temperature to a permissive one, the levels of CHMP4C RNA increased by 2.5-fold (data not shown). These data make it clear that the p53 protein can regu- late the levels of this endosomal protein after activation of p53 by a series of diverse stress exposures in several different cell lines. Cell lines with a mutant p53 gene (e.g. H1299 ⁄ V138 with a ts mutation in Val138 of p53 protein, or H1299 with a deletion in p53 gene) failed to regulate CHMP4C. We used chromatin immunoprecipi- tation (ChIP) to show that, after a stress response in H460 cells, the p53 protein could be shown to bind to chromatin in the )512 to )450 nucleotide region, the predicted DNA site that regulates the CHMP4C gene (Fig. 1C). In order to test the p53-dependent transcrip- tional activity through this putative p53 responsive element, a construct containing this sequence cloned in front of a luciferase expression vector was introduced into a p53-null H1299 cell, with co-transfection of a wild-type p53 expression vector. There was a seven-fold increase in luciferase activity. By contrast, there was no increase in luciferase activity when it was co-transfected with a vector of no p53 expression, or two different p53-mutant vectors (codon 22 ⁄ 23 mutant or codon 273 mutant) of the p53 protein (Fig. 1D). Clearly, CHMP4C is a p53-regulated gene. Chmp4C plays an important role in exosome production Previously, it has been shown that exosome production is regulated by p53 [12]. In p53-null cells, no exosomes were detected in the cell media, even after c-radiation [12]. Exosome production is conveniently isolated by differential centrifugation to pellet the exosomes from the culture medium. The many cellular proteins in an exosome preparation can then be visualized either by staining the proteins (Fig. 2A) or by using an antibody to Hsp90b or PGK1 (Fig. 2B) as a marker to show exosome production after separating by SDS ⁄ PAGE. H1299 cells with no p53 fail to produce exosomes with or without c-radiation, whereas the introduction of a wild-type p53 expression vector into these same cells produces high levels of exosomes, as shown in a SDS ⁄ PAGE by staining [12]. The addition of the CHMP4C cDNA (added as a YFP–CHMP4C so as to visualize Chmp4C protein) to H1299 cells restored exo- some production in the medium, as measured by either stained proteins in exosomes (Fig. 2A) or western blots for Hsp90b and PGK1 (Fig. 2B). In addition, the Chmp4C protein was detected in both the cell extract and in exosomes (Fig. 2B). In H460 cells, in which exosomes were produced only after activation of p53 (by etoposide) (Fig. 2C), pretreatment of the cells with siRNA directly against CHMP4C, followed by exposure with etoposide, led to a failure to produce exosomes (Fig. 2C). Thus, it is clear that the p53-mediated increase in the transcrip- tion rate of CHMP4C is required to increase the levels of exosome production. Two different p53-regulated genes, TSAP6 and CHMP4C, can each increase the rate of exosome production when introduced sepa- rately into cells with no p53 [12,15,16]. Although there was an occasional overlap in the localizations of the two proteins, Tsap6 and Chmp4C, in some cells by fluorescent staining, no consistent evidence was found that these two proteins act together in a complex in a cell (data not shown). This is interpreted most simply as either CHMP4C and TSAP6 are on different, but parallel, pathways for exosome production, or they are in the same pathway, but each raises the rate of pro- duction of exosomes (two distinct rate-limiting steps). p53 regulates the internalization of EGFR from the plasma membrane into the endosome Because the p53 response regulates the activity of the endosome compartment, and endosomal processes reg- ulate the levels and activity of growth factor receptors at the cell surface, we next explored whether a p53 response could accelerate removal of the EGFR from the plasma membrane. In H460 cells, the localization of the EGFR was determined by fluorescent immuno- staining. In the absence of etoposide, EGFR was all at the plasma membrane (Fig. 3A). After treatment with etoposide, EGFR molecules progressively moved into the internal compartments of the cells so that by 6–8 h most of the EGFR was internalized (Fig. 3A). In order to determine if EGFR molecules were moving into the endosome, the molecules were stained with red fluores- cence and several endosomal proteins were counter- stained with green fluorescence. Figure 3B shows the staining of the TfR protein, an early endosome compo- nent, whereas Fig. 3C shows staining of the LAMP1 protein, a late endosome–lysosome protein, and Fig. 3D shows staining of Chmp4C in the MVB late endosome. Clearly, EGFR proteins become progres- sively associated with the different endosomal com- partments, with a rather clear colocalization with Chmp4C in the MVB. To confirm that p53 activity was responsible for the rapid clearance of EGFR from the plasma membrane, p53-regulated endosomal compartment X. Yu et al. 2204 FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS we tested for EGFR internalization in H1299 cells, which do not express p53 protein. Even though the H1299 cells were treated with etoposide, the location of the EGFR molecules did not change from predomi- nantly the plasma membrane to the cytoplasm (Fig. 4A). We also tested EGFR internalization in H24 cells in which p53 expression is controlled by the pres- ence of tetracycline. With tetracycline withdrawal, the p53 expression is increased (Fig. 4B), resulting in EGFR internalization in these cells (Fig. 4C). Colocal- ization of EGFR with the endosomal compartment proteins TfR and LAMP1 was also observed in these experiments (Fig. 4C). Clearly, EGFR internalization from the plasma membrane into the endosomal com- partment can be regulated by a p53 response. Removal of the EGFR from the cell surface by a p53-responsive mechanism was shown to occur in several very differ- ent cell lines: H460 treated with a DNA damaging agent, H24 with a Tet-off controlled p53 expression, and V138 with a temperature-sensitive p53 protein (data not shown); this failed to occur in cells without a p53 gene (H1299 cells). The EGFR protein level decreased upon p53 activation, as determined by western blot (Fig. S1), and this is consistent with the observation of EGFR clearance from the plasma membrane to the endosome compartment and then to the lysosomes for degradation. Caveolin-1 expression is regulated by p53 Receptors such as EGFR may utilize a caveolae-medi- ated pathway for internalization [32]. Caveolin-1 is the major component of caveolae [33]. Based solely upon increased levels of CAV1 mRNA [34–36], or, separately reported, p53-binding assays (EMSA and luciferase assay) [34–36], it had previously been reported that CAV1 is a p53-regulated gene. However, the CAV1 gene has a rather poor consensus p53 DNA-binding site in the 5¢ location (Fig. 5A), which is nonetheless predicted to be a p53-responsive element by the p53MH algorithm [37]. When H460 cells were treated with etoposide or radiation the steady-state levels of CAV1 mRNA increased, by seven- and two-fold respectively, at 24 h after treatment (Fig. 5B). Simi- larly, when V138 cells were shifted to the permissive temperature for activation of p53 protein, there was a five-fold increase in the levels of CAV1 mRNA at 24 h after the temperature shift (Fig. 5B). Irradiation of H1299 cells with no p53 protein failed to increase the levels of CAV1 mRNA in those cells (Fig. 5B). YFP- Chmp4C Mock 210 125 80 49 35 29 21 7.1 kDa Exosomes YFP- Chmp4C Mock GAPDH Cell lysates YFP- Chmp4C Mock Hsp90β PGK1 Exosomes YFP- Chmp4C YFP- Chmp4C 210 125 80 49 35 29 21 7.1 kDa Exo s om es Etop – + + M H460 siRNA- Chmp4C A B C Fig. 2. Exosome production was regulated by expression of Chmp4C. (A,B) Overexpression of Chmp4C restored exosome production. (A) Silver staining of a typical SDS ⁄ PAGE of the exosome production isolated by ultracentrifugation from H1299 cells (p53 null), with or without overexpression of Chmp4C. (B) Western blot of the isolated exosomes and the cell lysates. Hsp90b and PGK1 were used as markers for exosome production. (C) Suppression of exosome production by knockdown of Chmp4C expression. The silver staining of an SDS ⁄ PAGE is shown. ), no etoposide (Etop) addition; +, the addition of etoposide. M, molecular markers. X. Yu et al. p53-regulated endosomal compartment FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS 2205 Irradiation of HCT116 cells with a wild-type p53 gene increased gene expression by four-fold, but the isogenic cell line without a p53 gene failed to produce more CAV1 mRNA after this treatment (Fig. 5B). Caveolin- 1 protein levels in H460 and V138 cells were induced after p53 activation (Fig. S1). In H460 cells, ChIP with a p53-specific antibody (DO-1) immunoselected the same region of DNA predicted in Fig. 5A to regulate this gene, whereas a no-antibody control failed to detect this DNA, and H1299 cells with no p53 protein also failed to bind to this DNA (Fig. 5C). Clearly, this DNA sequence can bind the p53 protein after a stress signal. When this sequence ()202 to )185 bp in Fig. 5A) was cloned and placed into a luciferase expres- sion vector it stimulated luciferase activity more than 80-fold compared with wild-type p53 protein, but not compared with the two different p53 mutant proteins that fail to stimulate p53-regulated transcription (Fig. 5C). Clearly, these additional criteria demonstrate that the p53 protein regulates the CAV1 gene at the promoter region ()202 to )185 bp), rather than at the reported sequence ()297 to )259 bp) [34–36], increas- ing its rate of transcription. It has previously been reported that caveolin-1 interacted with EGFR under various conditions [38– 40]. Using coimmunoprecipitation, we provided 0 h 6 h 4 h 2 h EGFR LAMP1 EGFR/LAMP1 C 0 h 6 h 4 h 2 h EGFR TfR EGFR/TfR B EGFR Chmp4C EGFR/Chmp4C D 0 h 2 h 4 h 6 h 8 h A Fig. 3. p53 activation promoted EGFR internalization through the endosome compartment. (A) H460 cells were treated with etoposide and at 0, 2, 4, 6 and 8 h cells were washed and stained for EGFR (red). (B) H460 cells treated with etoposide were stained for EGFR (red) and TfR (green). (C) H460 cells treated with etoposide were stained for EGFR (red) and LAMP1 (green). (D) H460 cells transfected with YFP– CHMP4C, followed by etoposide treatment for 8 h, were stained for EGFR (red). The location of Chmp4C protein was visualized in green. Bars, 10 lm. p53-regulated endosomal compartment X. Yu et al. 2206 FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS evidence that, in the H460 cell line, caveolin-1 inter- acted with EGFR (Fig. S1). To test the role caveolin- 1 may have in EGFR clearance from the plasma membrane after p53 activation, we treated H460 cells with etoposide and costained the cells with antibodies against EGFR and caveolin-1. The green fluorescent signals represent EGFR and the red signals represent caveolin-1 (Fig. 5D). Without etoposide treatment, EGFR and caveolin-1 are localized mainly on the plasma membrane, and both molecules show signifi- cant overlap in their locations at the membrane (Fig. 5D, 0 h, no etoposide treatment). With time after etoposide treatment and p53 activation (4, 6 and 24 h), caveolin-1 has a stronger signal, forms patched structures and moves into the cell (Fig. 5D, b, e, h, k). At the same time, EGFR also changes to a more granulated appearance and moves into the cell (Fig. 5D, a, d, g, j). Merger of these two mole- cules shows a progressive colocalization inside the cell in the endosomal compartment with increasing time (Fig. 5D, c, f, i, l). A similar experiment was carried out with V138 cells (ts p53) employing a temperature shift from a nonpermissive to a permissive tempera- ture for p53 activity. The results confirmed the con- clusions presented in Fig. 5D, activation of p53 increased the removal of both EGFR and caveolin-1 proteins from the surface and they colocalized within the cell. These data provide clear evidence, in several independent cell lines with several diverse ways to activate p53, that the internalization of EGFR and caveolin-1 from the cell surface can be mediated by gene products produced after the activation of p53. EGFR TfR Merge Day 0 EGFR LAMP1 Merge Day 2 Day 1 EGFR Merge dag ebh fci Relative expression level 0 2 4 6 8 10 12 Day 0 Day 1 Day 2 LAMP1 24 h4 h0 h A B C Fig. 4. EGFR internalization was mediated by p53 activation. (A) H1299 cells (without p53 expression) were treated with etopo- side at 0, 4 and 24 h, followed by staining for EGFR (red). (B) In H24 cells in which p53 expression was under the control of tetracycline, the expression of p53 was determined by quantitative PCR after tetra- cycline withdrawal. (C) H24 cells were incu- bated with and without tetracycline and stained for EGFR (red) and TfR (green) or LAMP1 (green). Bars, 10 lm. X. Yu et al. p53-regulated endosomal compartment FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS 2207 M 300 bp 200 bp Input CAV1 +DO-1 H460 +DO-1 H1299 IR+++ No Ab 100 bp M 300 bp 200 bp Input CAV1 +DO-1 H460 +DO-1 H1299 IR–+ + + No Ab 100 bp Relativeluciferase activity 0 20 40 60 80 100 120 –202 to –185 bp (A) –297 to –259 bp (B) No p53 wt-p53 mt-p53 (22/23) mt-p53 (273) No p53 wt-p53 mt-p53 (22/23) mt-p53 (273) V138 0 1 2 3 4 5 6 39 °C 32°C H1299 0 1 2 3 No treatment IR H460 0 1 2 3 No treatment IR Relative expression level 0 1 2 3 4 5 6 7 8 9 10 No treatment Etoposide 0 1 2 3 4 5 HCT116wt HCT116-p53null HCT116wt HCT116-p53null No treatment IR No treatment IR Relative expression level Intron 1 +1 297 to –259bp (B) Consensus p53 responsive element (p53RE): PuPuPuC(A/T)(T/A)GPyPyPy N(spacer) PuPuPuC(A/T)(T/A)GPyPyP y Intron 1 +1 Exon Exon Intron 1 +1 –297 to –259bp (B) ––202 to -185bp (A) CAV1EGFR Merge 0 h 4 h 24 h 6 h abc def ghi jkl A B C D Fig. 5. Caveolin-1 expression was regulated by p53. (A) A putative p53RE was identified. The uppercase letters at )202 to )185 bp repre- sent the p53RE predicted by p53MH. The uppercase letters at )297 to )259 bp represent the p53RE as reported previously [34,35]. The lowercase letters represent the genomic sequence. (B) Caveolin-1 expression was measured by quantitative PCR in H460, H1299, HCT116 and V138 cells upon treatment with etoposide, irradiation or temperature shift. (C) The putative p53RE was able to be bound by the p53 protein shown in the ChIP assay, and the DNA sequence including the p53RE induced luciferase activity with co-transfection of wild-type p53, but not with p53 mutants (22 ⁄ 23) and (273) in the luciferase activity assay. (D) H460 cells were treated with etoposide followed by staining for EGFR (green) and caveolin-1 (red). Bar, 10 lm. p53-regulated endosomal compartment X. Yu et al. 2208 FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS Discussion The p53 protein responds to stress signals in a cell by initiating cell-cycle arrest, senescence or apoptosis. At the same time, the p53 transcriptional program shuts down the IGF-1 ⁄ AKT-1 and mTOR pathways [2,3] and activates several of the endosomal compartment activi- ties including autophagy and exosome production [12]. As part of this p53 program to down-modulate cellular growth and division, the levels of several proteins in the endosomal compartment are increased (caveolin-1, Chmp4C), resulting in lower amounts of cell surface growth receptors (EGFR) which are internalized and sent to the MVB. At the same time, there is an increased rate of exosome production, which results from the stimulation of the transcription of TSAP6 and CHMP4C by p53. These events communicate a cellular stress event to the immune system, adjacent cells and the extracellular matrix. Thus, the endosomal compartment participates in a coordinated response to both shut down cellular processes after stress and to alert adjacent cells and the immune system of these events (Fig. 6). The p53 network also alters several other physiologi- cal processes in the cell that are undoubtedly related to the functions of the endosome compartment. p53 regulates genes encoding proteins that alter glycolytic activities and oxidative phosphorylation [5]. Thus, a p53-responsive stress signal can result in: (a) the cell shutting down its commitment to cell growth and divi- sion; (b) the removal of growth receptors from the cell surface; (c) an increase in the rate of exosome produc- tion signaling to surrounding cells and the immune system (along with secreting cytokines); (d) alteration of its metabolic and energy production sources; and (e) depending upon the cell type and whether it is a nor- mal cell or cancer cell, the cell undergoing cell-cycle arrest, senescence or apoptosis and autophagy. Throughout this process, there is an elaborate set of negative and positive feedback loops to regulate and increase or decrease p53 levels and its activities [41]. We are beginning to appreciate the coordinated nature of these networks and how each p53-regulated gene fits into this picture. Clearly, an integral part of this coordi- nated system is the p53-regulated control of the endo- somal compartment of the cell (Fig. 6). The results presented here begin to outline the way in which the p53 protein regulates the transcription of selected genes to accomplish this integrated response. There is an interest- ing level of redundancy in these endosomal-regulated processes: (a) four p53-regulated genes turn off the IGF–mTOR pathways; (b) autophagy is activated by the negative control of mTOR and the positive control of an autophagy gene MAP1LC3A (unpublished data); (c) exosome production is stimulated by both Chmp4C and Tsap6; and (d) both Chmp4C and caveolin-1 enhance removal of the EGFR from the cell surface. These activi- ties all function to slow cell growth and division, con- serve and reutilize cellular resources, and notify other cells and organ systems (the immune response) about the stresses. These functions are also an important part of cell and tissue repair after cell damage (DNA or chemical damage), virus infection or hypoxia. This coordinated effort by the p53 pathway integrates the molecular, cellu- lar and systemic levels of activities and demonstrates how a stress response is independent of scale. The endo- somal compartment of a cell, regulated by its protein constituents, can coordinate interactions at each of these scales and respond to stress in a p53-regulated fashion. Experimental procedures Cell culture, DNA damage treatment and transfection H460 and H1299 cells were cultured as described previously [12]. H1299 ⁄ V138 cells (from J. Chen, H Lee Moffitt Cancer Stress signals (such as DNA damage) Upstream mediators (such as ATM/ATR, Chk1/Chk2) p53Mdm2 Core regulation Cell cycle arrest Apoptosis Senescence Chmp4C Caveolin-1 TSAP6 Autophagy Lysosome function Exosome secretion Communication between cells Receptor endocytosis, Protein trafficking Endosome functions Downstream effectors and pathways Fig. 6. p53 regulation of cellular pathways upon stress responses. p53 activation by stress signals regulates not only cell-cycle arrest and apoptosis, but also endosome functions and autophagy which are involved in protein trafficking and signal transferring inside the cell and between the cells. See text for details. X. Yu et al. p53-regulated endosomal compartment FEBS Journal 276 (2009) 2201–2212 ª 2009 The Authors Journal compilation ª 2009 FEBS 2209 Center, FL, USA), with a stably transfected temperature- sensitive mutant form of p53 (Ala138 to valine) into H1299 cells [42], were cultured in Dulbecco’s modified Eagle’s med- ium, supplemented with 10% fetal bovine serum and 500 gÆmL )1 G418. The H24 cell line was from C. Prives (Columbia University, NY, USA), established to express tet- racycline-regulated p53 [43]. HCT116 p53 + ⁄ + and HCT116 p53 ) ⁄ ) cells (from B. Vogelstein at John Hopkins University, USA) were cultured in McCoy’s 5A with 10% fetal bovine serum. All cells were grown at 37 °C with 5% CO 2 . The cells were treated with DNA damage reagent, 20 lm etoposide or irradiated with 5 Gy as described previously [12]. pRC ⁄ CMV-wt p53 and mutant p53 (mt22 ⁄ 23 and mt273H) expression plasmids were generated as described previously [44]. The plasmids of pcDNA-3.1–HA–TSAP6 and the vector were from A. Telerman (Molecular Engines Laboratories, France). The plasmid of YFP– CHMP4C was from P. Bieniasz (Rockefeller University, USA). The pGL3 luciferase reporter vectors (pGL3-Basic and pGL3-promoter vectors) were purchased from Pro- mega (Madison, WI, USA). siRNA against CHMP4C and p53 was purchased from Dharmacon (Chicago, IL, USA). The siRNA for a nonspecific siRNA (NS) was purchased from Qiagen (Valencia, CA, USA). Plasmids were transfected with Lipofectamin 2000 (Invitrogen, Carlsbad, CA, USA) or jetPEI (ISC Bioexpress, Kays- ville, UT, USA), and the siRNA was transfected with oligofectamin (Invitrogen). Exosome isolation Exosome isolation was carried out as described previously [12]. Western blot Cell lysates were made as described previously [12]. The cell lysates or isolated exosomes were run on SDS ⁄ PAGE (4–20%) (Invitrogen) and transferred to Immobilon-P mem- branes (Millipore). Membranes were blotted with the following antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA): Hsp90b (D-19, sc-1057); PGK1 (Y-12, sc-17943); GFP (FL, sc-8334); and GAPDH (FL-335, sc-25778). Real-time PCR Total RNA was isolated at different time points using a RNeasy Kit (Qiagen) followed by one-step reverse tran- scription (TaqMan Reverse Transcription Reagents; Applied Biosystems, Foster City, CA, USA) and real-time PCR (ABI Prism 7000 sequence detection system; Applied Biosystems). b-Actin was used as an internal control. Probes and primer sets were purchased as predeveloped assays from Applied Biosystems. Triplicate samples were taken and each experiment was repeated. The relative induction ⁄ repression level was calculated by the ratio of the value of the gene to that of b-actin and then to the controls. ChIP assay ChIP assays were performed using a Upstate ChIP Assay Kit (Lake Placid, NY, USA) according to the manufac- turer’s instructions. The antibody against p53 (DO-1, sc-126) was from Santa Cruz Biotechnology. The primer sets were designed to encompass the potential p53-binding elements in the human CHMP4C and CAV1 genes. The sequences for the promoter region of CHMP4C gene are as follows: 5¢-CCTGACATTAGGAAAAGAGATGGCC-3¢ and 5¢-ATGAGTGTGTGGACACAAAGGCTTCC-3¢. The sequences for the CAV1 gene are as follows: 5¢-CGGGG TACCGGGAAAATTGTTGCCTCAGG-3¢,5¢-CCGCTCG AGGGTTTGTTCTGCTCGCGG-3¢ (A) and 5¢-CCGCTC GAGCCCCAAGGTTCTGGCAGCAG-3¢ (B). Luciferase activity reporter assay H1299 cells (p53 null) were plated in a 12-well plate, one day before transfection. Cells were transiently co-trans- fected with the constructed luciferase reporter plasmids pGL3-putative p53RE sequences and either wild-type or a mutant p53 plasmid, and pRL-TK plasmid (Promega) was used as an internal control. Forty-eight hours after transfection, whole-cell extracts were prepared and a luciferase assay was performed according to the manufac- turer’s instructions (Promega). Each transfection was performed with repeats and standard deviations were calculated. Immunofluorescent staining and confocal microscopy Cells were cultured on glass coverslips, treated with etoposide (20 lm), temperature shifting for various periods or cultured in the media without tetracycline (see above for details), and rinsed with phosphate buffered saline (NaCl/ P i ). Cells were fixed in 4% paraformaldehyde in NaCl ⁄ P i for 10 min, followed by permeabilization with 0.5% Triton X-100 in NaCl ⁄ P i for 10 min. Cells were then incubated with primary antibody for 1 h followed by washing with NaCl ⁄ P i and incubation with Alexa Fluor )555 or )488 conjugated secondary antibody. The cells were visualized with a Zeiss Axiovert 200M fluorescence microscope under confocal settings. 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Fig S1 Upon p53 activation, he EGFR protein level was decreased and the caveolin-1 protein level was increased This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding...X Yu et al p53- regulated endosomal compartment Acknowledgements We thank Agelika Teresky for her expert technical assistance; and Drs S Ganesan and M Yao for help in fluorescent microscopy 12 13 References 1 Levine AJ (1997) p53, the cellular gatekeeper for growth and division Cell 88, 323–331 2 Feng Z, Hu W, de Stanchina E, Teresky AK, Jin S, Lowe S & Levine AJ (2007) The regulation of AMPK beta1,... 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G, Bouvard V, Tuynder M, Susini L et al (2003) The p53- inducible TSAP6 gene product regulates apoptosis and the cell cycle and interacts with Nix and the Myt1 kinase Proc Natl Acad Sci USA 100, 2284–2289 Lespagnol A, Duflaut D, Beekman C, Blanc L, Fiucci G, Marine JC, Vidal M, Amson R & Telerman A (2008) Exosome secretion, including the DNA damageinduced p53- dependent secretory pathway, is severely compromised . The regulation of the endosomal compartment by p53 the tumor suppressor gene Xin Yu 1 , Todd Riley 2,3 and Arnold J. Levine 1,3 1 The Cancer. is the p53- regulated control of the endo- somal compartment of the cell (Fig. 6). The results presented here begin to outline the way in which the p53 protein