p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop
2003; Springer Nature; Volume: 22; Issue: 6 Linguagem: Inglês
10.1093/emboj/cdg129
ISSN1460-2075
Autores Tópico(s)Cancer-related Molecular Pathways
ResumoArticle17 March 2003free access p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop Pat P. Ongusaha Pat P. Ongusaha Cancer Biology Program, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Jong-il Kim Jong-il Kim Present address: Department of Biochemistry, College of Medicine, Hallym University, Chunchon, 200-702 Korea Search for more papers by this author Li Fang Li Fang Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, 10029 USA Search for more papers by this author Tai W. Wong Tai W. Wong Oncology Drug Discovery Group, Bristol-Meyer Squibb Pharmaceutical Research Institutes, Princeton, NJ, 08543 USA Search for more papers by this author George D. Yancopoulos George D. Yancopoulos Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591 USA Search for more papers by this author Stuart A. Aaronson Stuart A. Aaronson Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, 10029 USA Search for more papers by this author Sam W. Lee Corresponding Author Sam W. Lee Cancer Biology Program, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Pat P. Ongusaha Pat P. Ongusaha Cancer Biology Program, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Jong-il Kim Jong-il Kim Present address: Department of Biochemistry, College of Medicine, Hallym University, Chunchon, 200-702 Korea Search for more papers by this author Li Fang Li Fang Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, 10029 USA Search for more papers by this author Tai W. Wong Tai W. Wong Oncology Drug Discovery Group, Bristol-Meyer Squibb Pharmaceutical Research Institutes, Princeton, NJ, 08543 USA Search for more papers by this author George D. Yancopoulos George D. Yancopoulos Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591 USA Search for more papers by this author Stuart A. Aaronson Stuart A. Aaronson Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, 10029 USA Search for more papers by this author Sam W. Lee Corresponding Author Sam W. Lee Cancer Biology Program, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Author Information Pat P. Ongusaha1, Jong-il Kim2, Li Fang3, Tai W. Wong4, George D. Yancopoulos5, Stuart A. Aaronson3 and Sam W. Lee 1 1Cancer Biology Program, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine and Harvard Medical School, Boston, MA, 02115 USA 2Present address: Department of Biochemistry, College of Medicine, Hallym University, Chunchon, 200-702 Korea 3Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY, 10029 USA 4Oncology Drug Discovery Group, Bristol-Meyer Squibb Pharmaceutical Research Institutes, Princeton, NJ, 08543 USA 5Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:1289-1301https://doi.org/10.1093/emboj/cdg129 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DDR1, discoidin domain receptor 1, belongs to a subfamily of tyrosine kinase receptors with an extracellular domain homologous to Dictyostellium discoideum protein discoidin 1. We showed that DDR1 is a direct p53 transcriptional target, and that DNA damage induced a p53-dependent DDR1 response associated with activation of its tyrosine kinase. We further demonstrated that DDR1 activated the MAPK cascade in a Ras-dependent manner. Whereas levels of p53, phosphoserine-15 p53, p21, ARF and Bcl-XL were increased in response to exogenous overexpression of activated DDR1, dominant-negative DDR1 inhibited irradiation-induced MAPK activation and p53, phosphoserine-15 p53, as well as induced p21 and DDR1 levels, suggesting that DDR1 functions in a feedforward loop to increase p53 levels and at least some of its effectors. Nonetheless, inhibition of DDR1 function resulted in strikingly increased apoptosis of wild-type p53-containing cells in response to genotoxic stress through a caspase-dependent pathway. These results strongly imply that this p53 response gene must predominately act to alleviate the adverse effects of stress induced by p53 on its target cell. Introduction The p53 tumor suppressor gene plays a crucial role in cancer progression, and it is inactivated in the majority of human tumors. Depending on cell context, wild-type (wt) p53 limits cellular proliferation in response to DNA damage and other cellular stresses by inducing cell cycle arrest, apoptosis or senescence (Ko and Prives, 1996; Levine, 1997; Sugrue et al., 1997; Lin et al., 2000; Oda et al., 2000; Vogelstein et al., 2000; Vousden, 2000). It has been shown that inhibition of cell proliferation by p53 is largely due to its ability to transcriptionally activate genes that directly control cell fate (Ko and Prives, 1996; Levine, 1997; Vogelstein et al., 2000). We have shown previously that the MAPK cascade can be activated in a sustained manner by p53 (Lee et al., 2000) and that one upstream activator of this pathway is HB-EGF, a growth factor of the EGF family (Fang et al., 2001). These findings, as well as evidence that inhibition of HB-EGF signaling following p53 induction by cellular stresses is pro-apoptotic, support the concept that the p53 program induces pro-survival as well as pro-death cell fate decisions (Fang et al., 2001). In an effort to identify other downstream target genes of p53 and, in particular, those that might be involved in p53-mediated MAPK activation, we performed expression array analysis using tetracycline (tet) regulatable p53-expressing EJ tumor cells that have lost p53 function. We identified the discoidin domain receptor 1 (DDR1) as a p53 response gene, whose expression levels increase markedly in wt-p53-containing cells in response to DNA damage. The DDR1 was isolated as a novel subfamily of receptor tyrosine kinases (RTK) (Di Marco et al., 1993; Johnson et al., 1993; Alves et al., 1995). DDR1 is characterized by a 160 amino acid segment within the extracellular domain that exhibits strong sequence similarity to the Dictyostelium discoideum protein discoidin 1 (Springer et al., 1984), coagulation factors V and VIII, and a Xenopus laevis recognition protein, A5 (Takagi et al., 1991), which has an unusually long cytoplasmic juxtamembrane region and a kinase domain that has high homology to that of the NGF receptor, TrkA (Di Marco et al., 1993; Vogel, 1999). DDR1 has been reported to be overexpressed in mammary, ovarian and lung carcinomas, suggesting that it may play a role in the progression of certain carcinomas (Johnson et al., 1993; Zerlin et al., 1993; Laval et al., 1994; Alves et al., 1995; Barker et al., 1995; Perez et al., 1996). Various types of collagen have been identified as ligands capable of activating both DDR receptor kinases (Shrivastava et al., 1997; Vogel et al., 1997). However, the kinetics of DDR receptor activation are very different from those observed in most RTK (Schlessinger, 1997; Shrivastava et al., 1997; Vogel et al., 1997). Whereas activation of DDR1 by collagen is direct, tyrosine phosphorylation of the receptors occurs with delayed but sustained tyrosine kinase activation (Schlessinger, 1997). In addition, DDR activation by collagen can occur in the absence of a functional integrin collagen receptor (Vogel et al., 2000). There is, as yet, relatively little information concerning DDR downstream signaling pathways or functions. Here, we show that DDR1 is a direct p53 transcriptional target. In response to DNA damage, DDR1 was induced and activated/phosphorylated in a p53-dependent manner. We also present evidence that DDR1 plays a central role in p53 regulation through a positive feedback loop of the DDR1-Ras/MAPK-p53-p21 module. Nonetheless, blocking endogenous DDR1 function in wt-p53-containing cells following genotoxic stimuli resulted in significantly increased apoptosis. These results provide strong evidence that, unlike other known p53 direct target genes, the DDR1 receptor can function as a survival effector directly in the wt-p53-containing cell exposed to genotoxic stress. Since many human cancers maintain wt-p53 status, inhibition of DDR1 function may provide a novel approach to selectively enhancing the targeting of such tumors. Results DDR1 is a direct transcriptional target of p53 We showed previously that the Ras/Raf/MAPK signaling pathway is activated in response to p53 and that the ability of p53 to activate this pathway is dependent on its transcriptional activity (Lee et al., 2000). To identify potential p53 transcriptional target genes that might be involved in this signaling response, we used a DNA chip expression array to compare genes expressed in the presence or absence of p53. Affymetrix GeneChips were used for hybridization. Among upregulated genes detected, the transcript for DDR1 was found to increase in response to p53 induction. A previous study indicated that transient transfection of p53 induced DDR1 mRNA in an osteosarcoma line (Sakuma et al., 1996). However, no evidence was presented as to whether DDR1 was a direct p53 transcriptional target or whether DDR1 could be induced in response to genotoxic stress. To quantitate the level of DDR1 induction, we performed northern and western blot analysis using several different p53 expression systems. As shown in Figure 1A, as early as 12 h after tet removal in Saos2-p53 cells, the transcripts of DDR1 were easily induced and detectable, as well as p53 and p21CIP1/WAF1. In contrast, DDR1 was not induced in Saos2 control cells after tet removal. In addition, western blotting showed that DDR1 was induced in EJ-p53 cells after tet removal with kinetics like those of p53 as well as p21. Figure 1.Wt-p53-dependent induction of DDR1. (A) Induction of DDR1 mRNA and protein after tet removal in Saos2-p53 and EJ-p53 cells. Total RNA or protein extracts were prepared from EJ-p53 or Saos2-p53 cells grown in the presence or absence of tet for 0.5, 1, 2 or 3 days. Northern blots were performed sequentially using a 32P-labeled probe against DDR1, p21, Mdm2 and 36B4 (loading control). Western blotting was performed using antibodies against DDR1, p53, p21 and β-actin. β-actin was used as a loading control. (B) DDR1 induction in response to DNA damage. HCT116 cells with or without p53 were exposed to 1, 2, 2.5 or 5 Gy of γ-irradiation, and total proteins were collected at the indicated times following irradiation. DDR1 protein was significantly increased in HCT116 cells with p53 but not in HCT116-p53−/− cells. Normal diploid fibroblasts (IMR90) with wt-p53 and two human cancer cell lines (PC3, prostate cancer cell line; Saos2, osteosarcoma cell line) that had lost wt-p53 were exposed to irradiation (5 Gy). Then total RNA and proteins were collected at the indicated times. In p53+/+ or p53−/− MEF cells, cells were treated with DNA-damaging agents, mitomycin C (MMC, 2.5 μg/ml) and adriamycin (Adr, 0.25 μg/ml), at the indicated times. Download figure Download PowerPoint We next analyzed the effects of various genotoxic stresses such as γ-irradiation, actinomycin D, adriamycin or mitomycin C on DDR1 upregulation in wt-p53-containing cells. First, human normal diploid fibroblasts (IMR90) and a human colon cancer cell line (HCT116), both containing wt-p53, were treated with three different doses, 1, 2.5 and 5 Gy, in a γ-irradiator with a mark I135 Cs source. The result at all doses was a consistent stepwise increase in DDR1 expression levels (Figure 1B), whereas the expression of β-actin protein remained unchanged. The results from northern and western blot experiments indicate that DDR1 was upregulated as early as 4 h after irradiation in wt-p53-containing cells. The functionality of p53 was monitored by detecting transactivation of the p53 target gene, p21. In contrast, irradiated PC3 and Saos2 cell lines, which contained mutant forms of p53, show no detectable upregulation of DDR1 mRNA and protein (Figure 1B). To further confirm DDR1 increases following DNA damage and to verify that the response was p53 dependent, wt mouse embryonic fibroblasts (MEF) were treated with DNA-damaging agents, actinomycin D, adriamycin and mitomycin C. Exposures to these agents also resulted in a marked induction of DDR1 expression at the mRNA and protein levels (Figure 1B). In contrast, DDR1 expression levels remained unchanged when p53-null MEF was exposed to the agents. These data support the finding that DDR1 is induced by wt-p53 in response to genotoxic stress. Since the p53 dependence and kinetics of DDR1 expression in response to p53 and DNA damage resemble those of other well-defined p53 target genes, we investigated whether the DDR1 gene contains p53 response elements. The human DDR1 gene is composed of 17 exons (Figure 2A) (Playford et al., 1996; Sakuma et al., 1996). An earlier report indicated that the promoter region of DDR1 contains a consensus sequence of the p53-binding site (Sakuma et al., 1996) but the site lacks a critical nucleotide and does not contain a good consensus half-site. Moreover, a reporter assay revealed that the promoter region containing a potential p53-binding site did not confer p53 responsiveness upon the luciferase reporter (data not shown). These results led us to seek other p53-responsive site(s). We found a potential p53-responsive site of 20 nucleotides in intron 3 of the DDR1 gene, which matched the consensus p53 binding sequence by 90% (Figure 2A). To determine whether this sequence exhibited p53-dependent transcription-enhancing activity, a 0.73 kb DNA fragment (MluI and BglII) corresponding to intron 3 was fused to the luciferase reporter construct (subcloned upstream of the SV40 minimal promoter, pGL3prom-DDR1-intron 3) (Figure 2A and C). The same 0.73 kb intron 3 segment containing a mutated p53-BS sequence (G→T) (DDR1-intron-mt) was generated and also fused upstream of the SV40 minimal promoter. To examine the p53 responsiveness of the potential p53-responsive site in intron 3, the pGL3-DDR1 intron 3 reporter vector was co-transfected into Saos2 cells (p53-null osteosarcoma cell line) with plasmids expressing wt or mutant p53 (p53V143A). The luciferase activities shown by these constructs were then measured and compared. As a negative control, we tested the pGL3-promoter vector, which served as the backbone for the DDR1-p53RE reporter construct. As a positive control for p53 responsiveness, we used the pGL3-p21CIP1/WAF1 promoter construct containing p53-responsive sites. Figure 2.The DDR1 is directly p53 responsive. (A) Schematic of the genomic structure of the DDR1 gene. Open boxes indicate locations and sizes of the exons. The location of a potential p53-binding site (p53-BS) indicated in intron 3 and the consensus p53-binding sequence are also shown. Required bases for the p53-binding site are in bold (C and G). (B) EMSA of a potential p53-BS in intron 3 of DDR1. An EMSA was performed using oligos corresponding to a potential p53-BS in DDR1 intron 3 (20 bp) and a mutant BS-p53 (GAATCGTCCAGGGCTCGTTC, bold mutated from CAAG) as radiolabeled probes. Saos2-p53 (10 μg) nuclear extract was incubated with 3 ng of probe alone, 30- or 100-fold molar excess of either the unlabeled DDR1 wt-p53-BS (competitor, self) or mutant p53-BS (mutant competitor). Anti-p53 antibody, p53 Ab1 (Pab421), was present in the designated lanes. The arrows indicate the position of the p53–DNA complex (arrow 1) and supershifted complex containing antibody, p53 and DNA (arrow 2). Right panel: competition was also performed using the unlabeled p53 consensus oligo from the human p21 promoter, 5′-AGCTTG AACATGTCCCAACATGTTGA-3′. (C) Luciferase assay of each construct. The DDR1 intron sequence containing potential p53-BS is activated by wt-p53. pGL3-DDR1-p53-BS-luciferase reporter construct is also shown. The pGL3-promoter-DDR1 p53-BS plasmid (DDR1-intron-wt) or pGL3-DDR1-mutant-BS plasmid (DDR1-intron-mt), and pCMV-β-gal plasmid (for normalization) were co-transfected with either wt-p53 or mutant p53 expression plasmid, as well as the vector (pcDNA3 alone) into Saos2 cells that were grown to 60% confluence in 60 mm diameter dishes 24 h prior to lipofectin transfection. Intron 3 mutant vector (DDR1-intron-mt) contains a point mutation that changes the seventh nucleotide ‘G’ of p53 BS to ‘T’. After transfection, cells were incubated for 48 h. Samples were then collected, and luciferase and β-galactosidase activities were measured. Download figure Download PowerPoint As shown in Figure 2C, introduction of the pGL3-DDR1-intron 3 construct increased luciferase activity in response to wt-p53 expression but not to mutant p53 or vector alone. Moreover, a point mutation (G to T in p53-BS consensus) in the intron 3 mutant reporter abolished luciferase activity (Figure 2C) in response to wt-p53. Introduction of the pGL3 control vector in Saos2 cells resulted in only a marginal change in activity with/without wt-p53. To determine whether p53 could bind to this putative p53 response element in intron 3, we per formed an electrophoretic mobility shift assay (EMSA) and observed the shift in mobility of the 20 bp sequence by analyzing nuclear extracts including wt-p53. Supershift of this band by addition of anti-p53 monoclonal antibody p53 Ab-1 (Oncogene) confirmed that the 20 bp sequence was a p53-binding site (Figure 2B). Moreover, the addition of increasing amounts of the unlabeled wt-DDR1 p53-BS oligo, as well as an oligo containing the p53 binding site from the the p21 promoter (El-Deiry et al., 1993), competed with the formation of the electrophoretically shifted complex (Figure 2B), confirming the specificity of p53 binding to the DNA sequence from the DDR1 intron region. These results support the conclusion that DDR1 is a direct p53 transcriptional target. DDR1 is activated/tyrosine phosphorylated in response to genotoxic stress We next determined whether DDR1 was activated, as measured by tyrosine phosphorylation, in response to DNA damage such as γ-irradiation via p53. HCT116 cells containing wt-p53 were irradiated (5 Gy) and 12 h later analyzed for levels of tyrosine phosphorylation of endogenous DDR1 receptor by immunoprecipitation with DDR1 antibodies, followed by an immunoblot analysis using PY20 phosphotyrosine antibodies. As shown in Figure 3A, DDR1 receptor expression was dramatically increased after γ-irradiation above an undetectable level in untreated cells. Following irradiation, tyrosine phosphorylation of the induced receptor was readily detectable as well. To further investigate the kinetics of DDR1 activation/phosphorylation by γ-irradiation, we transfected HCT116 cells with DDR1b-myc to exogenously express the myc-tagged receptor. The transfected cells were exposed to γ-irradiation (5 Gy), and cell lysates were analyzed at the indicated times (1, 3, 6 and 12 h) by immunoprecipitation with anti-myc followed by immunoblot analysis with anti-myc or anti-pTyr. Of note, activation of the exogenously expressed receptor was not detectable in the absence of irradiation but was readily observed 3–6 h following exposure (Figure 3B). As a control, similar levels of myc-DDR1 were observed in each cell lysate. Taken together, these results demonstrate that the DDR1 receptor is both induced and activated/phosphorylated in response to DNA damage such as γ-irradiation in wt-p53-containing cells. Figure 3.DDR1 is activated/phosphorylated in response to genotoxic stress. (A) Tyrosine phosphorylation of endogenous DDR1 proteins following γ-irradiation. p53+/+ HCT116 cells were exposed to γ-irradiation (5 Gy) and incubated for 12 h. Cells were then lysed and DDR1 proteins were immunoprecipitated. The amount of endogenous DDR1 proteins in the immunoprecipitate was determined by western blot analysis using DDR1 antibodies (lower blot). The levels of tyrosine phosphorylation of endogenous DDR1 receptor in the immunoprecipitate were measured by western blotting using PY20 phosphotyrosine antibodies. (B) Tyrosine phosphorylation of exogenous DDR1 proteins by γ-irradiation treatment. HCT116 cells were transiently transfected with 2 μg of pcDNA3-DDR1-myc. Sixteen hours after transfection, cells were left untreated (control; no irradiation, -IR) or treated with γ-irradiation (5 Gy). At the indicated times after treatment, cells were lysed and myc-tagged proteins were immunoprecipitated, and the levels of tyrosine phosphorylation were determined by western blot analysis using PY20 phosphotyrosine antibodies. The quantity of DDR1-myc proteins in the myc immunoprecipitate was determined by western blot analysis using myc-tagged antibodies. Download figure Download PowerPoint Effects of p53-induced and activated DDR1 on downstream signaling pathways To investigate whether the DDR1 receptor might be involved in the p53-mediated MAPK signaling, human 293T cells were transfected with DDR1 (pcDNA3-DDR1b-myc) or vector (pcDNA3). As shown in Figure 4A, exogenously expressed DDR1 was tryosine phosphorylated with slow but sustained kinetics in response to collagen IV stimulation, and MAPK (p-MAPK) was activated with similar kinetics. As reported previously (Vogel et al., 2000), MAPK was not detectably activated upon DDR1 stimulation by collagen I (data not shown). It is well established that truncated forms of RTK that lack intracellular catalytic domains can function as dominant-negative (DN) mutant proteins by oligomerizing with endogenous wt RTKs (Hunter, 2000; Schlessinger, 2000; Simon, 2000). A truncated DDR1 mutant that lacks the catalytic domain has been shown to act as a DN, inhibiting DDR1 tyrosine phosphorylation in response to collagen in a dose-dependent manner (Bhatt et al., 2000; Vogel et al., 2000). Thus, we generated such a DN-DDR1 and transfected it into 293T cells together with an expression construct containing myc-tagged full-length wt-DDR1. Transfected cells were then stimulated with collagen IV for up to 4 h. As shown in Figure 4A, tyrosine phos phorylation of DDR1 was significantly inhibited by the DN-DDR1 construct, as were the levels of phosphorylated MAPK compared with levels induced by DDR1 alone. These results confirm that MAPK was a downstream effector in the DDR1 pathway. Figure 4.Sustained activation of the Ras/raf/MAPK cascade by DDR1 receptor. (A) 293T cells were transiently transfected with vector (pcDNA3), DDR1 (pcDNA3-DDR1-myc) or DDR1 + DN-DDR1 (pcDNA3-DDR1-myc + pcDNA3-DN-DDR1-myc). Twenty-four hours after transfection, cells were serum starved for an additional 24 h. Collagen IV (final concentration 10 μg/ml) was added and total lysates were prepared at the times (0, 0.5, 1, 2, 3 or 4 h) indicated at the top of each lane. Immunoblots were performed using antibodies against myc tag, phospho-MAPK/ERK and β-actin, respectively. The levels of tyrosine phosphorylation of DDR1 receptor protein were analyzed by immunoprecipitation with DDR1 antibodies, followed by western blot analysis using PY20 phosphotyrosine antibodies. Expression of DDR1-myc was detected by using myc-tagged antibodies. Lysates from transfected cells stimulated with collagen IV were immunoprecipitated by DDR1 antibodies. (B) Inhibition of DDR1-induced MAPK activation by DN-Ras and DN-Raf. 293T cells were transfected with vector, DDR1-myc, DDR1b plus DN-Raf, or DDR1b plus DN-Ras as indicated at the top of each lane. Lysates were prepared at 48 h post-transfection. Immunoblots were performed using antibodies against phospho-MAPK, total MAPK, myc tag (DDR1b-myc) or flag tag (DN-Raf). Download figure Download PowerPoint Through the use of DN mutants of Ras and Raf, whose products block the functions of the MAPKs within this cascade, we next sought to identify upstream effectors involved in activating the MAPK signaling by DDR1 activation. When DDR1 and either a DN mutant form of Ras (N17Ras) or Raf1 (DN-Raf-Flag), a direct downstream effector of Ras, were transiently transfected into 293T cells stimulated with collagen IV, activation levels of ERK were substantially decreased (Figure 4B). These findings demonstrated that DDR1 activates MAPK through Ras and Raf, as has been demonstrated for p53-induced MAPK activation (Lee et al., 2000). It is well established that Ras activation of the MAPK cascade can induce p53 through the p19/ARF pathway (Serrano et al., 1997; Lin et al., 1998; Michael and Oren, 2002). Therefore, it was possible that exogenous DDR1 expression and activation might cause accumulation of p53, p21 and ARF through activation of the Ras/MAPK cascade. Infection of adenovirus-expressing DDR1 (Ad-DDR1) into wt-p53-containing cells, IMR90, MCF7 and HCT116, in the presence of collagen IV led to high levels of exogenous DDR1 expression and tyrosine phosphorylation along with increased levels of both phosphorylated MAPK and AKT. Under these conditions, Mdm2 and ARF expression levels also increased associated with increased levels of p53, phosphoserine-15 p53 (pSer15-p53) and p21 (Figure 5A). Moreover, in all three lines tested, the expression and activation of DDR1 increased expression levels of Bcl-XL, an anti-apoptotic protein, while pro-apoptotic proteins including Noxa (Figure 5A) and Puma (data not shown) were expressed at similar levels with or without DDR1. Figure 5.Effects of p53-induced and -activated DDR1 on downstream signaling pathways. (A) Activated DDR1 induces accumulation of p53, p21 and ARF in wt-p53-containing cells. IMR90, MCF7 and HCT116 cells were used for this study. Immunoblot analyses of cellular lysates corresponding to cells infected with control GFP-expressing (C) or DDR1-expressing (D) adenoviruses were performed using antibodies against p53, pSer15-p53, DDR1, phospho-MAPK, phospho-AKT, p21, p19/ARF, Mdm2, Bcl-XL, Noxa and β-actin. (B) The effects of DDR1 on p53-downstream signaling after genotoxic stress. IMR90 and HCT116 cells were infected with Ad-GFP (GFP) or Ad-DN-DDR1 (DN), followed by exposure to γ-irradiation (5 Gy). Wt-MEFs were also infected with Ad-GFP (GFP) or Ad-DN-DDR1 (DN), followed by exposure to adriamycin (0.3 μg/ml). Twelve hours after irradiation or adriamycin treatment, cell lysates from treated or untreated cells were collected for western blot analysis using DDR1, p53, pSer15-p53, phospho-MAPK, p21, Mdm2 and β-actin (loading control) antibodies. Download figure Download PowerPoint To assess whether DDR1 induction by genotoxic stress had similar effects on induced p53 levels, DDR1 function was inhibited in wt-p53 cells including IMR90, HCT116 and MEF by expressing DN-DDR1. Normal diploid fibroblasts (IMR90) and HCT116 cells were infected with Ad-GFP or Ad-DN-DDR1, followed by exposure to γ-radiation (5 Gy). MEF cells were exposed to adriamycin (0.3 μg/ml) after infection with either Ad-GFP or Ad-DN-DDR1. Twelve hours after irradiation or adriamycin treatment, cells were collected for immunoblotting analysis using DDR1, phospho-MAPK, p53, pSer15-p53, p21 and Mdm2 antibodies. As shown in Figure 5B, these genotoxic treatments led to increased p53, pSer15-p53, p21 and Mdm2 levels as well as increased DDR1 expression associated with MAPK activation. Inhibition of DDR1 function by DN-DDR1 inhibited irradiation-induced MAPK phosphorylation associated with a detectable decrease in DDR1, p53, pSer15-p53, p21 and Mdm2 levels. Taken together, these findings indicate that DDR1 is an upstream regulator of the Ras/Raf/MEK/MAPK cascade as well as of AKT activation, and that DDR1 induction/activation by genotoxic insult results in a positive feedback loop to increase p53. Induction of DDR1 increases cell survival in response to p53-mediated apoptosis and genotoxic stress To investigate the biological functions of p53-mediated DDR1 induction, we determined the effects of DDR1 inhibition on p53-induced cellular responses such as apoptosis by expression of the DN form of DDR1 (DN-DDR1) or antisense (AS)-DDR1. An AS-DDR1 cDNA construct containing the 0.9 kb XhoI fragment of DDR1 was made in the same adenovirus expression system and amplified. Co-expression of p53 and AS-DDR1 repressed p53-induced DDR1 expression by ∼60–70%, as compared with the level of DDR1 expression in response to p53 alone. Expression levels from each adenovirus co-infection were examined by western blot analysis and are shown in Figure 6A and B (left panels). Following infection of Saos2 cells that lack functional p53 with Ad-DDR1-myc (Ad-DDR1-myc) or p53 (Ad-p53), high levels of DDR1 expression and an increased level of phospho-MAPK were observed (Figure 6A). Co-infection of Ad-p53 and Ad-DDR1-myc further enhanced the level of an active form of MAPK, while co-expression of p53 and DN-DDR1 abrogated p53-induced MAPK activation (Figure 6A). Of note, DN-DDR1 expression in p53-expressing cells reduced p21 and Mdm2 expression, as was observed when DN-DDR1 was expressed in irradiated IMR90 or HCT116 cells (Figure 5B). DN-DDR1 expression in p53-expressing cells also reduced Bcl-XL expression. Co-expression of p53 and DN-DDR1 or AS-DDR1 resulted in a dramatic increase in p53-induced apoptosis (DN-DDR1, ∼13.4–32%; AS-DDR1, ∼15–33%) in p53-null Saos2 cells compared with that induced by co-expression of p53 with L
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