Critical Role of Cysteine Residue 81 of Macrophage Migration Inhibitory Factor (MIF) in MIF-induced Inhibition of p53 Activity
2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês
10.1074/jbc.m800050200
ISSN1083-351X
AutoresHaiyoung Jung, Hyun‐A Seong, Hyunjung Ha,
Tópico(s)Nuclear Receptors and Signaling
ResumoMacrophage migration inhibitory factor (MIF) is a potent modulator of the p53 signaling pathway, but the molecular mechanisms of the effect of MIF on p53 function have so far remained unclear. Here we show that MIF physically interacts with the p53 tumor suppressor in vitro and in vivo. This association was significantly reduced by a C81S mutation but not C57S or C60S mutations, suggesting that Cys81 is essential for the in vivo association between MIF and p53. This association also depended on Cys242 (and, to some extent, on Cys238) within the central DNA binding domain of p53. Ectopic expression of MIF, but not MIF(C81S), inhibited p53-mediated transcriptional activation in a dose-dependent manner. Conversely, knockdown of endogenous MIF stimulated p53-mediated transcription. MIF inhibited p53-induced apoptosis and cell cycle arrest, whereas the MIF(C81S) mutant, which is unable to physically associate with p53, had no effect. Consistent with these findings, confocal microscopy showed that MIF prevented p53 translocation from the cytoplasm to the nucleus. We also demonstrated that MIF suppresses p53 activity by stabilizing the physical association between p53 and Mdm2. These results suggest that MIF physically associates with p53 and negatively regulates p53 function. Macrophage migration inhibitory factor (MIF) is a potent modulator of the p53 signaling pathway, but the molecular mechanisms of the effect of MIF on p53 function have so far remained unclear. Here we show that MIF physically interacts with the p53 tumor suppressor in vitro and in vivo. This association was significantly reduced by a C81S mutation but not C57S or C60S mutations, suggesting that Cys81 is essential for the in vivo association between MIF and p53. This association also depended on Cys242 (and, to some extent, on Cys238) within the central DNA binding domain of p53. Ectopic expression of MIF, but not MIF(C81S), inhibited p53-mediated transcriptional activation in a dose-dependent manner. Conversely, knockdown of endogenous MIF stimulated p53-mediated transcription. MIF inhibited p53-induced apoptosis and cell cycle arrest, whereas the MIF(C81S) mutant, which is unable to physically associate with p53, had no effect. Consistent with these findings, confocal microscopy showed that MIF prevented p53 translocation from the cytoplasm to the nucleus. We also demonstrated that MIF suppresses p53 activity by stabilizing the physical association between p53 and Mdm2. These results suggest that MIF physically associates with p53 and negatively regulates p53 function. Macrophage migration inhibitory factor (MIF) 2The abbreviations used are: MIF, macrophage migration inhibitory factor; siRNA, small interfering RNA; shRNA, short hairpin RNA; DTT, dithiothreitol; Mdm2, mouse double minute 2; FACS, fluorescence-activated cell sorting; GST, glutathione S-transferase; GFP, green fluorescent protein; DBD, DNA binding domain; 5FU, 5-fluorouracil; HA, hemagglutinin. 2The abbreviations used are: MIF, macrophage migration inhibitory factor; siRNA, small interfering RNA; shRNA, short hairpin RNA; DTT, dithiothreitol; Mdm2, mouse double minute 2; FACS, fluorescence-activated cell sorting; GST, glutathione S-transferase; GFP, green fluorescent protein; DBD, DNA binding domain; 5FU, 5-fluorouracil; HA, hemagglutinin. is known to be an important regulator of the innate and adaptive immune systems and inflammatory responses (1Calandra T. Roger T. Nat. Rev. Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1271) Google Scholar, 2Metz C.N. Bucala R. Adv. Immunol. 1997; 66: 197-223Crossref PubMed Google Scholar). Recent studies have suggested that MIF contributes to tumorigenesis by suppressing the activity of p53 (3Hudson J.D. Shoaibi M.A. Maestro R. Carnero A. Hannon G.J. Beach D.H. J. Exp. Med. 1999; 190: 1375-1382Crossref PubMed Scopus (564) Google Scholar, 4Michell R.A. Liao H. Chesney J. Fingerle-Rowson G. Baugh J. David J. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 345-350Crossref PubMed Scopus (508) Google Scholar), even though there is no evidence that MIF and p53 physically interact with each other. High levels of MIF expression have been found in human tumors, including melanomas, breast carcinomas, prostate cancer, and adenocarcinoma of the lung (5Meyer-Siegler K. J. Interferon Cytokine Res. 2000; 20: 769-778Crossref PubMed Scopus (33) Google Scholar, 6del Vecchio M.T. Tripodi S.A. Arcuri F. Pergola L. Hako L. Vatti R. Cintorino M. Prostate. 2000; 45: 51-57Crossref PubMed Scopus (68) Google Scholar). High levels of MIF also enhance tumor cell migration and the production of angiogenic factors (7Ogawa H. Nishihira J. Sato Y. Kondo M. Takahashi N. Oshima T. Todo S. Cytokine. 2000; 12: 309-314Crossref PubMed Scopus (124) Google Scholar), suggesting that MIF is an important regulator of cell growth and apoptosis. A recent gene knock-out analysis demonstrated that MIF plays a pivotal role in the regulation of cell growth and apoptosis in a p53-dependent manner (8Fingerle-Rowson G. Petrenko O. Metz C.N. Forsthuber T.G. Mitchell R. Huss R. Moll U. Muller W. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9354-9359Crossref PubMed Scopus (239) Google Scholar); however, the mechanism by which MIF regulates p53 activity remained unknown. Several studies have suggested that MIF and thiol antioxidants are bridged through interchain disulfides (9Jung H. Kim T. Chae H.-Z. Kim K.-T. Ha H. J. Biol. Chem. 2001; 276: 15504-15510Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 10Swope M.D. Sun H.W. Klockow B. Blake P. Lolis E. J. Biol. Chem. 1998; 273: 14877-14884Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). MIF exhibits a cysteine-dependent enzymatic oxidoreductase activity, and this activity is dependent on the redox-active conserved sequence motif (Cys57-Ala-Leu-Cys60) (11Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Juttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (266) Google Scholar). Consistent with this, the conserved cysteine sequence motif Cys-X-X-Cys was reported to be critical for the macrophage-activating activities and JAB1-mediated activities of MIF (12Kleemann R. Hausser A. Geiger G. Mischke R. Burger-Kentischer A. Flieger O. Johannes F.J. Roger T. Calandra T. Kapurniotu A. Grell M. Finkelmeier D. Brunner H. Bernhagen J. Nature. 2000; 408: 211-216Crossref PubMed Scopus (498) Google Scholar). Thus, MIF could regulate p53 through direct interactions mediated by cysteine residues. p53 is tightly suppressed in nonstressed cells by Mdm2, which indirectly inhibits p53-dependent gene expression by ubiquitinating and degrading p53 (13Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar, 14Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2798) Google Scholar). In addition, Mdm2 can prevent p53 transactivation through a direct interaction with the N-terminal transactivation domain of p53, blocking the interaction of p53 with the basal transcription machinery required for transactivation (15Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6673) Google Scholar, 16Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2760) Google Scholar). To grasp how p53 is activated in response to stress-induced signals, extensive efforts have been made to identify molecules that can interfere with the interaction between p53 and Mdm2. Several proteins affect the p53-Mdm2 interaction by binding to p53 or Mdm2 (17Leung K.M. Po L.S. Tsang F.C. Siu W.Y. Lau A. Ho H.T. Poon R.Y. Cancer Res. 2002; 62: 4890-4893PubMed Google Scholar, 18Hsieh J.K. Chan F.S. O'Connor D.J. Mittnacht S. Zhong S. Lu X. Mol. Cell. 1999; 3: 181-193Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). A putative tumor suppressor, ING1b, stabilizes and activates p53 function through a direct interaction with the N-terminal transactivation domain of p53, probably by disrupting the interaction between p53 and Mdm2 (17Leung K.M. Po L.S. Tsang F.C. Siu W.Y. Lau A. Ho H.T. Poon R.Y. Cancer Res. 2002; 62: 4890-4893PubMed Google Scholar). ARF, the alternative reading frame product of the INK4a/ARF locus, binds Mdm2 and prevents Mdm2-mediated p53 degradation (19Kamijo T. Weber J.D. Zambetti G. Zindy F. Roussel M.F. Sherr C.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8292-8297Crossref PubMed Scopus (781) Google Scholar, 20Bates S. Phillips A.C. Clark P.A. Scott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (809) Google Scholar). In addition, p300 and retinoblastoma augment p53 stability by binding to the site adjacent to conserved region II, a highly acidic region of Mdm2 (18Hsieh J.K. Chan F.S. O'Connor D.J. Mittnacht S. Zhong S. Lu X. Mol. Cell. 1999; 3: 181-193Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 21Grossman S.R. Perez M. Kung A.L. Joseph M. Mansur C. Xiao Z.X. Kumar S. Howley P.M. Livingston D.M. Mol. Cell. 1998; 2: 405-415Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Therefore, one way to elucidate the molecular mechanism of the effect of MIF on p53 function is to analyze MIF as an intracellular regulator of Mdm2. Here we show that MIF physically binds to p53 in vivo and that this interaction requires the participation of cysteine residues present in each of these two proteins. Moreover, MIF negatively regulates p53 activity, probably by stabilizing the association between p53 and Mdm2. Reagents and Cell Culture—Anti-GST, anti-FLAG (M2), anti-β-actin, Alexa Fluor-594 anti-mouse, Alexa Fluor-488 anti-rabbit, and anti-histone (H2B) antibodies were described previously (22Seong H.-A. Jung H. Ha H. J. Biol. Chem. 2007; 282: 12075-12096Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The anti-MIF, anti-Mdm2, anti-p53(DO-1), anti-p21, and anti-Bax, anti-histone H2B antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-HA antibody was kindly provided by Dr. S-C. Bae (Chungbuk National University, Cheongju, Korea). 293T, HEK293, MCF7, U2OS, and HCT116 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum as described previously (23Seong H.-A. Jung H. Kim K.-T. Ha H. J. Biol. Chem. 2007; 282: 12272-12289Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Generation of Inducible MIF shRNA Cell Lines—For inducible knockdown of endogenous MIF expression, double-stranded oligonucleotides (5′-TCGAGGACACCAACGTGCCCCGCGCTTCAAGAGAGCGCGGGGCACGTTGGTGTCTTTTTTA-3′, MIF sequence underlined) was cloned into the pSingle-tTS-shRNA vector (Clontech). HCT116 cells were transfected with pSingle-tTS-shRNA harboring MIF-specific shRNA or pSingle-tTS-shRNA empty vector using WelFect-Ex™ Plus (WelGENE, Daegu, Korea). Inducible MIF shRNA stable clones were screened in the presence of 450 μg/ml G418 until all control parental HCT116 cells died. Stable clones were isolated and treated with 1 μg/ml doxycycline (Sigma), a tetracycline analogue, for 72 h, and endogenous MIF knockdown was determined by immunoblot analysis using anti-MIF antibody. Plasmids and DNA Construction—The expression plasmids for wild-type p53 and its deletion derivatives were a kind gift from Dr. S-J Um (Sejong University, Seoul, Korea). To generate the p53(DBD) construct, PCR was performed using the following primers: forward primer (5′-GCGAATTCTTCTTGCATTCTGGGACA-3′) containing an EcoRI site (underlined), and reverse primer (5′-GCCTCGAGGCGGAGATTCTCTTCCTC-3′) containing an XhoI site (underlined). The amplified PCR products were cut with EcoRI plus XhoI and cloned into a pFLAG-CMV2 vector using EcoRI plus SalI sites to generate the FLAG-p53(DBD). The six Cys to Ser p53(DBD) substitution mutants (C176S, C238S, C242S, C176S/C238S, C176S/C242S, and C238S/C242S) were generated by PCR. In brief, a human p53 cDNA (GenBank™ accession number NM000546) was used as the template for amplification with either the forward primer for p53(DBD) or the reverse primer for p53(DBD), in conjunction with one of the following mutant primers containing alterations in the nucleotide sequence of p53(DBD): for p53(DBD) Cys176 to Ser (C176S), sense 5′-GTGAGGCGCTCACCCCACCAT-3′, antisense 5′-ATGGTGGGGTGAGCGCCTCAC-3′; for p53(DBD) Cys238 to Ser (C238S), sense 5′-AACTACATGTCAAACAGTTCC-3′, antisense 5′-GGAACTGTTTGACATGTAGTT-3′; for p53(DBD) Cys242 to Ser (C242S), sense 5′-AACAGTTCCTCAATGGGCGGC-3′, antisense 5′-GCCGCCCATTGAGGAACTGTT-3′. To generate the double substitution mutant C176S/C238S, FLAG-C176S was used as the template, and the sense and antisense primers of C238S and p53(DBD) were used for PCR amplification. To generate the double substitution mutants C176S/C242S and C238S/C242S, PCR was carried out using FLAG-C176S and FLAG-C238S as templates in the presence of primers for C242S and p53(DBD). The identity of all of the PCR products was confirmed by nucleotide sequencing analysis on both strands. In Vivo and in Vitro Binding Assay—Each plasmid DNA indicated under “Results” was transiently transfected into the indicated cells with WelFect-Ex™ Plus, according to the manufacturer's instructions. In vivo binding assays were performed as described previously (23Seong H.-A. Jung H. Kim K.-T. Ha H. J. Biol. Chem. 2007; 282: 12272-12289Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To determine the ability of p53 to bind to wild-type and mutant forms of MIF in vitro, p53 was translated in vitro using the TnT reticulocyte lysate system as directed by the manufacturer (Promega). In vitro-translated 35S-labeled p53 was incubated with unlabeled recombinant wild-type and mutant forms of MIF in the presence of 5 mm H2O2 at room temperature for 1 h, and then assessed by 8% nondenaturing PAGE as described previously (22Seong H.-A. Jung H. Ha H. J. Biol. Chem. 2007; 282: 12075-12096Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). RNA Interference Experiments—RNA interference experiments were performed using MIF-specific siRNAs. The sequences used were as follows: MIF-specific siRNA 1 (5′-ACACCAACGUGCCCCGCGCdTdT-3′) corresponding to a coding region (amino acids 7-13) of the human MIF (GenBank™ accession number NM002415); MIF-specific siRNA 2 (5′-CCUUCUGGUGGGGAGAAAUdTdT-3′) corresponding to a C-terminal region (nucleotides 427-445); MIF-specific siRNA 3 (5′-CAACUCCACCUUCGCCUAAdTdT-3′) corresponding toa3′-untranslated region (nucleotides 527-545); and a nonspecific control siRNA (5′-GCGCGGGGCACGUUGGUGUdTdT-3′), as described (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 25Yao K. Shida S. Selvakumaran M. Zimmerman R. Simon E. Schick J. Haas N.B. Balke M. Ross H. Johnson S.W. O'Dwyer P.J. Clin. Cancer Res. 2005; 11: 7264-7272Crossref PubMed Scopus (37) Google Scholar, 26Winner M. Koong A.C. Rendon B.E. Zundel W. Mitchell R.A. Cancer Res. 2007; 67: 186-193Crossref PubMed Scopus (82) Google Scholar, 27Rendon B.E. Roger T. Teneng I. Zhao M. Al-Abed Y. Calandra T. Mitchell R.A. J. Biol. Chem. 2007; 282: 29910-29918Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Preparation of Nuclear and Cytoplasmic Fractions—MCF7 cells (∼4 × 105 per 60-mm dish) transfected with the indicated expression vectors (wild-type and mutant forms of MIF) or a MIF-specific siRNA 1 were used for the preparation of nuclear and cytoplasmic fractions for immunoblot analyses, as described previously (22Seong H.-A. Jung H. Ha H. J. Biol. Chem. 2007; 282: 12075-12096Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Reporter Assays—Luciferase activity was monitored by using a p53-Luc reporter containing eight copies of the p53-responsive element derived from the p21 promoter (kindly provided by Dr. Y.-I. Yeom, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea). MCF7 or U2OS cells were transiently transfected according to the WelFect-Ex™ Plus method with the reporter plasmids, along with the appropriate expression vectors as indicated. Luciferase activity was assessed using a luciferase assay kit (Promega), according to the manufacturer's instructions. Ubiquitination Assay—p53-null HCT116 cells were transfected with plasmids encoding p53, wild-type, and mutant forms of MIF, MIF-specific siRNAs 1 and 2, and HA-tagged ubiquitin either alone or in combination. The cells were treated with 10 μg/ml MG132 (Calbiochem) for 4 h, harvested, and then washed twice with phosphate-buffered saline (PBS) (pH 7.4), lysed in 200 μl of Tris-buffered saline (pH 7.4) containing 2% SDS, and incubated at 95 °C for 10 min. p53 proteins were immunoprecipitated with an anti-p53 antibody and subjected to SDS-8% PAGE, followed by Western blot analysis with an anti-HA antibody. Immunodepletion of MIF—HEK293 cell extracts were incubated with 4 μg of anti-MIF antibody for 1 h at 4 °C, and 60 μl of protein A-Sepharose were subsequently added and incubated for an additional 3 h at 4 °C. After immunodepletion twice with an anti-MIF antibody, aliquots of the resulting supernatants were subjected to immunoblotting analysis with an anti-MIF antibody to confirm the immunodepletion of MIF. The MIF-depleted extracts were mixed with recombinant GST alone, GST-MIF(WT), or GST-MIF(C81S) proteins (each 4 μg). The mixtures were then purified on glutathione-Sepharose beads, followed by immunoblot analysis using anti-p53 antibody to determine the complex formation between p53 and MIF. Apoptosis Assay—The apoptosis assay was performed as described previously (24Seong H.-A. Jung H. Choi H.-S. Kim K.-T. Ha H. J. Biol. Chem. 2005; 280: 42897-42908Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). MCF7 cells undergoing apoptosis were quantified using the GFP system. The percentage of apoptotic cells was calculated as the number of GFP-positive cells with apoptotic nuclei divided by the total number of GFP-positive cells visualized under a fluorescence microscope. FACS Analysis—MCF7 cells transiently expressing wild-type and mutant forms of MIF or MIF-specific siRNAs 1-3, together with transfectants expressing p53 or empty vector alone as controls, were washed with ice-cold PBS and then treated with 5FU for 30 h or with doxorubicin for 24 h. The trypsinized cells were washed twice with ice-cold PBS and incubated at 37 °C for 30 min with a solution consisting of 50 μg/ml propidium iodide and 1 mg/ml RNase A (Sigma) in 1 mm Tris-HCl (pH 7.5). Flow cytometry was performed on a FACSCalibur-S system (BD Biosciences). Immunofluorescence Staining—The indirect immunofluorescence method was described previously in detail (22Seong H.-A. Jung H. Ha H. J. Biol. Chem. 2007; 282: 12075-12096Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Specimens were examined under a Leica Dmire2 confocal laser scanning microscope (Germany). MIF Directly Interacts with p53 in Mammalian Cells—To test whether MIF can associate with p53 in cells, we transiently expressed wild-type MIF in 293T cells as a FLAG-tagged fusion protein (FLAG-MIF) or expressed an empty vector (cytomegalovirus) as a control. The interactions of FLAG-MIF with endogenous p53 proteins were analyzed by immunoblotting with an anti-FLAG antibody. MIF was detected in the p53 immunoprecipitate (Fig. 1A). To verify the interaction of endogenous MIF with p53 in vivo, we next performed coimmunoprecipitation experiments using MCF7 and HCT116 cell extracts. Endogenous p53 was immunoprecipitated from cell lysates, and the binding of endogenous MIF was subsequently analyzed using Western blotting with an anti-MIF antibody. Again, MIF was present in the p53 immunoprecipitate (Fig. 1B, left panel). To examine whether MIF could bring down p53 in a reciprocal way, endogenous MIF was immunoprecipitated by anti-MIF antibody, followed by immunoblotting with an anti-p53 antibody. The endogenous p53 was also detected in the MIF immunoprecipitate (Fig. 1B, right panel). These data demonstrate that MIF physically interacts with p53 in vivo. Formation of the MIF-p53 Complex Requires the Cysteine Residue (Cys81) of MIF—To investigate whether the cysteine residues of MIF are necessary for the formation of the MIF-p53 complex, we transiently cotransfected 293T cells with GST-tagged wild-type MIF and its mutants, MIF(C57S), MIF(C60S), and MIF(C81S), together with FLAG-tagged wild-type p53. The expression of the MIF mutants MIF(C57S) and MIF(C60S) did not notably influence the association between MIF and p53, whereas the MIF(C81S) mutant dramatically decreased the complex formation (Fig. 2A). We also used nondenaturing PAGE to analyze the association of purified, recombinant MIF proteins with p53 that was translated in vitro. When the 35S-labeled p53 was incubated in the presence of wild-type MIF and its mutants MIF(C57S) and MIF(C60S), the mobility clearly shifted relative to incubation in the absence of MIF (Fig. 2B, 1st lane versus 2nd to 4th lanes). In contrast, the mobility shift was not observed when 35S-labeled p53 was incubated with MIF(C81S) (Fig. 2B, 5th lane). This was further confirmed by measuring the binding capacity of recombinant MIF(C81S) with endogenous p53 in MIF-depleted extracts. A weak interaction between MIF(C81S) and p53 that was observed in normal cell extracts had completely disappeared in MIF-depleted extracts (Fig. 2C), providing additional evidence of a physical association between MIF and p53 through Cys81. As a control, the efficiency of depletion was shown to be 100% by immunoblotting with an anti-MIF antibody (Fig. 2C, bottom panels). To address the question of whether MIF specifically binds to p53 through Cys81, we also employed an inducible MIF shRNA system to deplete the expression of endogenous MIF in HCT116 cells. A similar result was also observed in a stable system for tetracycline-inducible expression of MIF shRNA (Fig. 2D, right panel). As a control, HCT116 cells stably expressing pSingle-tTS-shRNA vector harboring MIF-specific shRNA (MIF shRNA) showed a doxycycline-dependent RNA interference effect on the endogenous MIF silencing (Fig. 2D, right bottom panel), whereas stable HCT116 cells containing the empty vector alone (Vector Laboratories) showed no effect on the expression of endogenous MIF in the presence or absence of doxycycline (Fig. 2D, left upper and right bottom panels). These observations led us to investigate the effect of the redox status on the formation of the endogenous MIF-p53 complex in cells. The reductants DTT and β-mercaptoethanol considerably decreased the amount of coprecipitated MIF, whereas H2O2, an oxidant, did not (Fig. 2D), suggesting that the interaction of MIF with p53 in vivo is redox-dependent. These data strongly suggest that the in vivo association of MIF and p53 requires the participation of cysteine residues. Determination of the MIF Interaction Domain of p53—To map the domain(s) within p53 required for its association with MIF, we generated a set of six p53 deletion mutants (Fig. 3A, upper panel), and we examined their ability to interact with MIF using in vivo binding assays in 293T cells. MIF interacted with wild-type p53, p53(44/393), p53(44/387), p53(319/393), and p53(DBD) but not with p53(319/360) and p53(TAD) (Fig. 3A, lower panel). These results indicate that the interaction of MIF with p53 is mediated via the DNA binding domain (DBD) within residues 113-290. The basic domain within residues 363-393 served as a secondary interaction domain. The central DBD contains all the cysteine residues of p53. Three cysteine residues in murine p53 (Cys173, Cys235, and Cys239) are essential for the suppression of transformation, transactivation, and in vitro DNA binding (28Rainwater R. Parks D. Anderson M.E. Tegtmeyer P. Mann K. Mol. Cell. Biol. 1995; 15: 3892-3903Crossref PubMed Scopus (272) Google Scholar). To examine the involvement of specific cysteines in MIF binding, we initially generated three human p53(DBD) substitution mutants, C176S, C238S, and C242S (Fig. 3B), and examined their ability to interact with MIF. MIF strongly interacted with the wild-type p53(DBD) and the C176S mutant but not with the C242S mutant (Fig. 3C, upper left panel). In addition, cells expressing the C238S mutant formed less of the complex than wild-type p53(DBD). This suggests that Cys238 of p53, like Cys242, potentially plays a role in the association of p53 with MIF, although to a somewhat lesser extent. To further examine the roles of Cys238 and Cys242 of the DBD domain of p53 in its association with MIF, we generated three p53(DBD) double substitution mutants, C176S/C238S, C176S/C242S, and C238S/C242S, and examined their binding properties in the in vivo binding assay. The expression of C176S/C242S or C238S/C242S dramatically inhibited complex formation between MIF and p53, as compared with the expression of the control p53(DBD) (Fig. 3C, upper right panel). These results suggest that both Cys238 and Cys242 of the DBD domain of p53 are important for its association with MIF; however, Cys242 had a stronger effect on formation of the complex between MIF and p53 than Cys238. We further analyzed whether the Cys81 of MIF and Cys242 (and Cys238) of p53 play a critical role in the association of MIF with p53 using an in vivo binding assay. As expected, expression of MIF(C81S) strongly inhibited the association in the presence of FLAG-C242S, whereas wild-type MIF, MIF(C57S), and MIF(C60S) did not modulate the association of the proteins (Fig. 3C, lower panel). Together, these results strongly suggest that MIF binds to p53 through Cys81 of MIF and Cys242 (and Cys238) of p53. MIF-p53 Association Is Regulated by 5FU and Doxorubicin—5FU is an anti-metabolite that inhibits thymidylate synthase and is important for the apoptotic response (29Bunz F. Hwang P.M. Torrance C. Waldman T. Zhang Y. Dillehay L. Williams J. Lengauer C. Kinzler K.W. Vogelstein B. J. Clin. Investig. 1999; 104: 263-269Crossref PubMed Scopus (915) Google Scholar); it is an important inducer of 53-mediated apoptosis. Doxorubicin induces cell cycle arrest by introducing double-stranded DNA breaks (30Kaeser M.D. Pebernard S. Iggo R.D. J. Biol. Chem. 2004; 279: 7598-7605Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). We next examined whether 5FU and doxorubicin can influence MIF-p53 complex formation in cells. Upon 5FU treatment, the association between endogenous MIF and p53 was considerably decreased compared with control cells not treated with 5FU (Fig. 4, left panel). Similarly, doxorubicin treatment resulted in a significant decrease in the association between endogenous MIF and p53 (Fig. 4, right panel). This result was also confirmed by a reciprocal immunoprecipitation experiment in which endogenous MIF was immunoprecipitated by anti-MIF antibody, followed by immunoblotting with an anti-p53 antibody (data not shown). Together, these data indicate that the interaction between MIF and p53 appears to be dependent on p53 stimulation by 5FU or doxorubicin. MIF Suppresses p53-mediated Transcription—To identify the physiological role of the MIF-p53 complex, we first analyzed the effect of MIF on p53-mediated transcription. We transiently transfected MCF7 and U2OS cells with increasing amounts of MIF, together with a p53-Luc reporter plasmid, in the presence or absence of 5FU. The addition of MIF inhibited the p53-mediated transcription in a dose-dependent manner (Fig. 5A), suggesting that MIF is a negative regulator of p53 activity. In contrast, expression of MIF alone, as a control, had no effect on the regulation of p53-mediated transcription. We also examined the effect of MIF-specific siRNAs on p53-mediated transcription in cells. The transfection of the MIF-specific siRNAs resulted in a significant increase of p53-mediated transcription that was proportional to the amount of MIF-specific siRNAs transfected (Fig. 5B, upper panel). A similar trend was also observed in p53-null HCT116 and H1299 cells transfected with the MIF-specific siRNA, supporting that the inhibitory effect of MIF on p53-mediated transcription is truly p53-dependent (Fig. 5B, lower panel). To further examine whether the MIF-induced inhibition of p53 transcriptional activity is dependent on its direct interaction with p53, we determined the effect of MIF mutants on p53-mediated transcription in MCF7 and U2OS cells. Expression of MIF(C81S) had little effect on p53-mediated transcription, whereas expression of wild-type MIF, MIF(C57S), and MIF(C60S) substantially decreased p53-mediated transcription to a similar extent in a dose-dependent manner (Fig. 5C). We extended this analysis to investigate the effect of MIF on p53 signaling. Overexpression of MIF decreased the expression of p53 target genes, including p53, p21, and BAX, whose genes are normally up-regulated by p53 signals, in cells tested (Fig. 5D); however, overexpression of MIF(C81S) did not have this effect. To further confirm the negative role of MIF in p53 signaling, we generated a stable system for tetracycline-inducible expression of MIF shRNA in HCT116 cells (MIF shRNA) and analyzed them for p53-induced gene expression. Compared with the expression in control HCT116 cells stably expressing the empty vector alone (Vector Laboratories), knockdown of endogenous MIF by doxycycline treatment significantly increased the expression of p53, p21, MDM2, and BAX (Fig. 5E, left panel). MIF-knockdown cells showed a lower induction of p53 target genes compared with parental HCT116 cells (parental cells) treated with 5FU that induces p53-mediated apoptosis (Fig. 5E, left panel, lane 4 versus lane 5). We observed similar results in HCT116 cells transiently transfected with MIF-specif
Referência(s)