Putative P53 Response Elements Research Articles

P53 alters intracellular Ca2+ signaling through regulation of TRPM4

Altered expression of transient receptor potential channel melastatin 4 (TRPM4) contributes to several diseases, including cardiac conduction disorders, immune diseases, and cancer. Yet the underlying mechanisms of TRPM4 expression changes remain elusive. In this study, we report that loss of tumor suppressor protein p53 or p63γ function or mutation of a putative p53 response element in the TRPM4 promoter region increase TRPM4 promoter activity in the colorectal cancer cell line HCT 116. In cells that lack p53 expression, we observed increased TRPM4 mRNA and protein levels and TRPM4-mediated Na+ currents. This phenotype can be reversed by transient overexpression of p53. In the prostate cancer cell line LNCaP, which expresses p53 endogenously, p53 overexpression decreases TRPM4-mediated currents. As in other cancer cells, CRISPR-Cas9 mediated knockout of TRPM4 in p53 deficient HCT 116 cells results in increased store-operated Ca2+entry. The effect of the TRPM4 knockout is mimicked by p53 mediated suppression of TRPM4 in the parental cell line expressing TRPM4. In addition, a TRPM4 knockout-mediated shift in cell cycle is abolished upon loss of p53. Taken together, these findings indicate that p53 represses TRPM4 expression, thereby altering cellular Ca2+ signaling and that TRPM4 adds to cell cycle shift dependent on p53 signaling. One sentence summary: TRPM4 is repressed in the p53 pathway leading to reduced currents and increased calcium signaling.

The Alzheimer's disease-associated TREM2 gene is regulated by p53 tumor suppressor protein

TREM2 mutations evoke neurodegenerative disorders, and recently genetic variants of this gene were correlated to increased risk of Alzheimer's disease. The signaling cascade originating from the TREM2 membrane receptor includes its binding partner TYROBP, BLNK adapter protein, and SYK kinase, which can be activated by p53. Moreover, in silico identification of a putative p53 response element (RE) at the TREM2 promoter led us to hypothesize that TREM2 and other pathway elements may be regulated in p53-dependent manner. To stimulate p53 in synergistic fashion, we exposed A549 lung cancer cells to actinomycin D and nutlin-3a (A + N). In these cells, exposure to A + N triggered expression of TREM2, TYROBP, SYK and BLNK in p53-dependent manner. TREM2 was also activated by A + N in U-2 OS osteosarcoma and A375 melanoma cell lines. Interestingly, nutlin-3a, a specific activator of p53, acting alone stimulated TREM2 in U-2 OS cells. Using in vitro mutagenesis, chromatin immunoprecipitation, and luciferase reporter assays, we confirmed the presence of the p53 RE in TREM2 promoter. Furthermore, activation of TREM2 and TYROBP by p53 was strongly inhibited by CHIR-98014, a potent and specific inhibitor of glycogen synthase kinase-3 (GSK-3). We conclude that TREM2 is a direct p53-target gene, and that activation of TREM2 by A + N or nutlin-3a may be critically dependent on GSK-3 function.

Tumor suppressor protein p53-mediated repression of human mitotic centromere-associated kinesin gene expression is exerted via down-regulation of Sp1 level.

The repressive role of p53 on the human mitotic centromere-associated kinesin (MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53−/−) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53V143A, p53R248W, and p53R273H) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.

Tumor suppressor p53 links ceramide metabolism to DNA damage response through alkaline ceramidase 2.

p53 mediates the DNA damage response (DDR) by regulating the expression of genes implicated in cell cycle arrest, senescence, programmed cell death (PCD), and metabolism. Herein we demonstrate that human alkaline ceramidase 2 (ACER2) is a novel transcriptional target of p53 and that its transactivation by p53 mediates the DDR. We found that p53 overexpression or its activation by ionizing radiation (IR) upregulated ACER2 in cells. Two putative p53 responsive elements (p53REs) were found in its first intron of the ACER2 gene, and Chromatin Immunoprecipitation (ChIP) assays in combination with promoter activity assays demonstrated that these p53REs are the bona fide p53 binding sites that mediate ACER2 transactivation by p53. As ACER2 catalyzes the hydrolysis of ceramides into sphingosine, which in turn is phosphorylated to form sphingosine-1-phosphate (S1P), ACER2 upregulation increased the levels of both sphingosine and S1P while decreasing the levels of ceramides in cells. A moderate upregulation of ACER2 inhibited cell cycle arrest and cellular senescence in response to low-level expression of p53 or low-dose IR by elevating S1P, a pro-proliferative and pro-survival bioactive lipid, and/or decreasing ceramides whereas its robust upregulation mediated PCD in response to high-level expression of p53 or high-dose IR likely by accumulating cellular sphingosine, a pro-death bioactive lipid. ACER2 is frequently inactivated in various cancers due to its deletion or mutations, and restoring its expression inhibited the growth of tumor xenografts in mice. These results suggest that p53 mediates DDR and exerts its tumor suppressive role in part by regulating the expression of ACER2, which in turn regulates the bioactive sphingolipid lipids.

Reciprocal repression between TUSC7 and miR-23b in gastric cancer.

Recently, long noncoding RNAs (lncRNAs) were demonstrated to play important regulatory roles in biological processes and cancer biology. However, the overall pathophysiological contribution of lncRNAs to gastric cancer (GC) remains largely unknown. In this study, differentially expressed lncRNAs in GC and paired adjacent normal tissue samples were identified by microarray and were validated using quantitative real-time polymerase chain reaction (qRT-PCR). One particular lncRNA, tumour suppressor candidate 7 (TUSC7), was analyzed in sequential large cohorts, and the Kaplan-Meier method with the log-rank test for comparisons was used to analyse the survival data. The results indicated that TUSC7 was downregulated in GC samples and was an independent prognostic indicator of disease-free survival (DFS) and disease-specific survival (DSS) in GC patients. Applying loss-of-function and gain-of-function approaches, we determined that TUSC7 suppressed tumour cell growth in vitro and in vivo. Furthermore, we showed that TUSC7 was a direct transcriptional target of p53 via interaction of p53 with the putative p53-response element in the upstream region of TUSC7. Finally, we demonstrated reciprocal repression between TUSC7 and miR-23b; in contrast to TUSC7, miR-23b promoted cell growth. The results indicated that TUSC7 is a p53-regulated tumour suppressor that acts in part by repressing miR-23b and that TUSC7 may be a key regulatory hub in GC.

Apoptotic Effect of STAMP2 Transfection to PC3 Cells and Caspase Induction with Hydrogen Peroxide Incubation at Lncap Cells- Role of P53

STAMP genes STAMP1/STEAP2 and STAMP2/STEAP4 are expressed in androgen receptor positive-prostate cancer cell line LNCaP, and this regulation was reported, previously. The androgen induction was done at LNCaP cells. STAMP2/STEAP4 was silenced for 1, 2 and 5 days. Using real-time RT-PCR, an apoptosis panel including proapoptotic and/or apoptotic genes was investigated for gene expression alterations in STAMP expressing LNCaP cells in comparison to P53-null PC3 cells in which STAMP2 gene was transfected. In addition, promoter region of the STAMP2 gene was analyzed and putative p53 and androgen receptor (AR) response elements were identified. Thus, p53 gene was silenced using siRNA approach and was induced by hydrogen peroxide. Taken together, prostate-specific STAMP2 gene and its regulation with the p53- and caspase-related pathway was characterized. These results will shed light on understanding the control of proliferation in neoplastic transformation in prostate cancer.

P53 regulates the transcription of the anti-inflammatory molecule developmental endothelial locus-1 (Del-1)

Developmental endothelial locus-1 (Del-1) is an endothelium-derived anti-inflammatory molecule that is downregulated by inflammatory stimuli. Little is known about the molecular mechanisms by which Del-1 transcription is regulated. In the present study, a DNA sequence upstream of the Del-1 gene was analyzed and putative p53 response elements (p53REs) were identified. An approximately 2 kb fragment upstream of the translation start site displayed the highest Del-1 transcriptional activity, and the transcriptional activity of this fragment was enhanced by overexpression of p53. Chemical activation of endogenous p53 elevated the levels of Del-1 mRNA. Site-directed mutagenesis of CATG in the consensus sequences of the 2 kb fragment to TATA significantly reduced the transcription of Del-1. Chromatin immunoprecipitation revealed recruitment of p53 to the p53REs of the Del-1 promoter, resulting in increased Del-1 transcription. Finally, primary endothelial cells isolated from mice with reduced levels of p53 showed a decrease in Del-1 mRNA compared to wild-type endothelial cells. Moreover, Del-1 reciprocally enhanced p53 expression in primary endothelial cells. Thus, these findings suggest that Del-1 is a novel transcriptional target gene of p53.

P38α and p38δ MAPK are key regulators of p21Cip1 gene expression in human keratinocytes

PKCδ increases keratinocyte differentiation via a mechanism that involves activation of p38δ MAPK. We recently showed that PKCδ also suppresses cell proliferation by increasing expression of the p21Cip1 cyclin‐dependent kinase inhibitor. However, the downstream effectors that mediate this PKCδ‐dependent regulation are not known. Our present studies suggest that PKCδ activates p38δ which regulates p21Cip1 promoter activity, as exogenously expressed p38δ increases p21Cip1 mRNA and protein level in human keratinocytes. Treatment with SB202190, a p38α/β inhibitor, or dominant negative p38α, leads to a decrease in PKCδ‐dependent p21Cip1 promoter activity, implicating p38α as a second mediator of this regulation. p21Cip1 promoter truncation experiments indicate that p38δ increases the activity via a putative p53 response element. This suggests that p38α and δ may act in conjunction with or through p53 to regulate p21Cip1 gene expression. In addition, we show that p38α and p38δ overexpression increases p53 promoter activity, suggesting that p38α and δ regulate p53 gene expression. We propose that PKCδ activates p38α and p38δ which in turn activates p53 to increase p21Cip1 expression leading to cessation of keratinocyte proliferation.

Poly(ADP-ribosyl)ation of p53 induces gene-specific transcriptional repression of MTA1

The metastasis-associated protein 1 (MTA1) is overexpressed in various human cancers and is closely connected with aggressive phenotypes; however, little is known about the transcriptional regulation of the MTA1 gene. This study identified the MTA1 gene as a target of p53-mediated transrepression. The MTA1 promoter contains two putative p53 response elements (p53REs), which were repressed by the p53-inducing drug 5-fluorouracil (5-FU). Notably, 5-FU treatment decreased MTA1 expression only in p53 wild-type cells. p53 and histone deacetylases 1/2 were recruited, and acetylation of H3K9 was decreased on the promoter region including the p53REs after 5-FU treatment. Proteomics analysis of the p53 repressor complex, which was pulled down by the MTA1 promoter, revealed that the poly(ADP-ribose) polymerase 1 (PARP-1) was part of the complex. Interestingly, p53 was poly(ADP-ribose)ylated by PARP-1, and the p53-mediated transrepression of the MTA1 gene required poly(ADP-ribose)ylation of p53. In summary, we report a novel function for poly(ADP-ribose)ylation of p53 in the gene-specific regulation of the transcriptional mode of p53 on the promoter of MTA1.

P53 suppresses lung resistance-related protein expression through Y-box binding protein 1 in the MCF-7 breast tumor cell line

Lung resistance-related protein (LRP) has roles in multi-drug resistance of tumor cells. Understanding the mechanisms that regulate LRP expression in tumor cells is an important research area. A putative p53 response element in the LRP promoter has been found. Thus, p53-related regulation of LRP expression was explored in this study. We first demonstrated that p53 overexpression inhibited LRP expression both at the protein and mRNA levels. Then, using a dual-luciferase reporter assay, we located the p53 response element to the Y-box (-263~-407) of the LRP promoter, the YB-1 binding site, but not the putative p53 response element. Furthermore, coimmunoprecipitation and chromatin immunoprecipitation showed p53 could bind to the Y-box of the LRP promoter through interaction of p53 with YB-1. YB-1 coexpression with p53 facilitated p53-induced suppression of endogenous LRP expression in MCF-7 cells. HDAC2, a corepressor of p53, was found to also interact with YB-1, and this interaction was mediated by p53. These results showed that the p53-HDAC2 transcriptional repressor complex can bind to the Y-box of the LRP promoter and repress LRP expression through interaction with YB-1. p53-related suppression of LRP expression was completely reversed by doxorubicin treatment and Adr, whereas CP and VP-16 treatment induced LRP expression increased significantly. Inhibition of LRP expression by siRNA facilitated Adr induced apoptosis of MCF-7 cells. All these findings indicated that loss of p53-related suppression of LRP may be the reason for LRP expression increase, and, therefore, chemotherapy resistance in tumor cells.

P53 Negatively Regulates RGS13 Protein Expression in Immune Cells

RGS13, a member of the regulator of G protein signaling (RGS) family, inhibits G protein-coupled receptor signaling in B cells and mast cells (MCs) and suppresses IgE-antigen-induced MC degranulation and anaphylaxis. Although RGS13 expression is induced by immune receptor and chemokine receptor stimulation, the molecular regulation of RGS13 transcription is unknown. Here, we investigated the role of two p53 response elements (REs) in the regulation of RGS13 promoter activity and expression. We found that a 1000-bp DNA fragment upstream of the ATG translation start site (TSS) had promoter activity in reporter gene assays, and deletion or mutation of a p53-binding motif nearest the TSS abolished promoter activity. Notably, p53 bound to both REs in the RGS13 promoter in vivo as assessed by chromatin immunoprecipitation, suggesting that the p53 RE most distal to the TSS is physiologically inactive. We detected reduced RGS13 expression in MCs exogenously expressing p53 or treated with doxorubicin, which induces genotoxic stress and leads to p53 accumulation. RNA silencing of p53 up-regulated RGS13 expression in B lymphocytes, and bone marrow-derived MCs from p53(-/-) mice had increased RGS13 expression. Finally, p53-depleted B cells with increased RGS13 expression had reduced Ca(2+) mobilization in response to sphingosine 1-phosphate. These studies indicate that p53 may modulate immune responses through suppression of RGS13 transcription in MCs and B cells.

Abstract 3804: The pathobiology of perturbed SEPT9 expression in ovarian neoplasia

Abstract We have made observations of the deregulated expression of certain SEPT9 transcripts and we wish to place these in a pathobiological context. Septins are an evolutionarily conserved family of GTP binding proteins which have been implicated in a wide range of cellular processes. SEPT9 was initially identified through mapping of a region of chromosome 17q which demonstrated allelic imbalance in sporadic ovarian tumours. The large SEPT9 locus is complex since up to 18 splice variants are possible through alternate splicing of six 5’ ends and three 3’ ends. Of note are two transcripts SEPT9_v4 and v4* differing only in their 5’UTRs but encoding the same polypeptide SEPT9 isoform 4 (SEPT9_i4). Ovarian neoplasia is accompanied by down regulation of SEPT9_v4 and up regulation of SEPT9_v4*. We have also shown that translation of the former is by cap-dependant mechanisms while translation of the later is cap-independant and stress responsive (Hum Mol Gen 2007;6:742-52). We therefore sought to understand the mechanism of control of transcription of these two transcripts and how this is perturbed in neoplasia. Using bioinformatic analysis we have identified multiple putative p53 responsive elements upstream of the transcription start site of SEPT9_v4*. Such sites are rare upstream of all other SEPT9 transcripts. In wild type p53 cells but not an isogenic p53 null cell line, the SEPT9_v4* transcript and isoform 4 are induced under conditions in which p53 is induced. No other SEPT9 transcript responded in this way. We used chromatin immunoprecipitation assays to demonstrate the validity of at least some of these putative p53 sites. Using a luciferase assay we show that these same sites bind and respond to wild type but not mutant p53. Previous studies from our lab have shown that over expression of isoform 4 perturbs microtubule dynamics in cells such that depolymerisation of microtubules (in response to cold) and their subsequent repolymerisation is delayed. We therefore sought to determine if the p53 dependant upregulation of SEPT9_v4* might also be associated with altered microtubule dynamics and resistance to microtubule-interacting drugs. In a tissue culture model we observation that induction of p53 and SEPT9_v4* using a DNA damaging agent reduces the cellular response to paclitaxel. Further, the transfection of SEPT9_v4* specific siRNA (with SEPT9_i4 knock down) leads to dramatically enhanced cell kill after taxol treatment compared with scrambled siRNAs. The clinical relevance of this is emphasised by the observation that SEPT9_v4* expression is associated with disease stage in ovarian cancer. Our data provide novel insight into septin function and the role of stress pathways including p53 in its regulation and has clinical significance as a determinant of drug sensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3804. doi:10.1158/1538-7445.AM2011-3804

P53 downregulates the gene expression of mitochondrial aconitase in human prostate carcinoma cells

Mitochondrial aconitase (mACON) is regarded as the key enzyme in citrate oxidation in human prostate epithelial cells, and its abnormal expression has been implicated in tumorigenesis of the prostate. Evidence also supports a broad role for the p53 gene in suppressing prostatic tumorigenesis. We investigated whether p53 regulates mACON expression and explore the potential mechanisms responsible for its effect on prostate cancer cells. Camptothecin (CPT) treatments and transient overexpression of p53 were used to investigate p53 regulation of mACON and may effects were assessed using immunoblotting and transient gene expression assays. In vitro enzymatic activity assays and immunoblot assays showed that CPT treatment induced p53 expression while reducing mACON protein biosynthesis in wild-type p53 expressing LNCaP cells. Immunoblot assays and reporter assays revealed that transient transfection of a p53 expression vector into p53-null PC-3 cells decreased mACON expression. Cyclic pifithrin-α, an inhibitor of p53 transcriptional activity, blocked the decrease in mACON gene expression resulting from CPT treatment in LNCaP cells. Two putative p53 response elements were identified within the mACON promoter; however, mutation of these putative p53 response elements did not abolish the effect of CPT whereby it decreased mACON expression. A similar result was obtained for the effect of these mutants on the promoter activity of the mACON gene after transient overexpression of p53. Together these results suggest that p53 downregulation of mACON gene expression in human prostate carcinoma cells may not occur through the putative consensus p53 response elements found within the mACON promoter.

Respiratory syncytial virus hijacks the tumor-suppressor p53 to enhance its replication in lung epithelial cells (137.3)

Abstract Respiratory syncytial virus (RSV) infection is the major cause of childhood lower respiratory illness and has been associated with the induction and exacerbation of asthma. Previously, we reported that RSV infection induced TGF-β expression that caused cell cycle arrest and subsequently enhanced RSV replication. In the current study, we demonstrate that a downstream target of TGF-β signaling; P53, is a pivotal transcription factor for RSV replication. Over-expression of p53 protein by an inducible system (Tet-off) enhanced RSV replication by 9 folds, whereas, knockdown of p53 using siRNA significantly reduced RSV replication in H1299 and A549 lung epithelial cell lines. Stabilization of p53 with nutlin-3 epithelial (PHBE) cells. We hypothesized that a plausible mechanism of enhanced RSV replication by p53 was through direct binding of p53 to RSV RNA. A search of RSV genome revealed several putative p53-responsive elements. To confirm interaction of p53, EMSA analysis was performed which showed a specific shift with one of the p53 response elements. Taken together, we have identified a novel role for p53 in positive regulation of RSV replication. These findings suggest that enhanced p53 expression through lung injury, smoking or air pollution could facilitate a significant increase in RSV replication leading to more severe respiratory pathologies.

Gene expression patterns in heterozygous Plk4 murine embryonic fibroblasts

BackgroundThe polo-like kinases (Plks) are a group of serine/threonine kinases which have roles in many aspects of cellular function including the regulation of mitotic activity and cellular stress responses. This study focuses on Plk4, the most divergent member of the Plk family, which is necessary for proper cellular proliferation. More specifically, alterations in Plk4 levels cause significantly adverse mitotic defects including abnormal centrosome duplication and aberrant mitotic spindle formation. We sought to clarify the effect of reduced Plk4 levels on the cell by examining transcript profiles of Plk4 wild-type and heterozygous mouse embryonic fibroblasts (MEFs). Subsequently, the levels of several key proteins involved in the DNA damage response were examined.Results143 genes were found to be significantly up-regulated in the heterozygous MEFs compared to their wild-type counterparts, while conversely, 9 genes were down-regulated. Numerous genes with increased transcript levels in heterozygous MEFs were identified to be involved in p53-dependent pathways. Furthermore, examination of the promoter regions of all up- and down-regulated genes revealed that the majority contained putative p53 responsive elements.An analysis of transcript levels in MEFs after exposure to either ionizing or ultraviolet radiation revealed a significant change between wild type and heterozygous MEFS for Plk4 transcript levels upon only UV exposure. Furthermore, changes in protein levels of several important cell check-point and apoptosis regulators were examined, including p53, Chk1, Chk2, Cdc25C and p21. In heterozygous MEFs, p53, p21 and Chk2 protein levels were at significantly higher levels. Furthermore, p53 activity was increased 5 fold in the Plk4 heterozygous MEFs.ConclusionGlobal transcript profiles and levels of key proteins involved in cellular proliferation and DNA damage pathways were examined in wild-type and Plk4 heterozygous MEFs. It was determined that Plk4 haploinsufficiency leads to changes in the levels of RNA accumulation for a number of key cellular genes as well as changes in protein levels for several important cell cycle/DNA damage proteins. We propose a model in which reduced Plk4 levels invoke an increase in p53 levels that leads to the aforementioned changes in global transcription profiles.

The p53HMM algorithm: using profile hidden markov models to detect p53-responsive genes

BackgroundA computational method (called p53HMM) is presented that utilizes Profile Hidden Markov Models (PHMMs) to estimate the relative binding affinities of putative p53 response elements (REs), both p53 single-sites and cluster-sites. These models incorporate a novel "Corresponded Baum-Welch" training algorithm that provides increased predictive power by exploiting the redundancy of information found in the repeated, palindromic p53-binding motif. The predictive accuracy of these new models are compared against other predictive models, including position specific score matrices (PSSMs, or weight matrices). We also present a new dynamic acceptance threshold, dependent upon a putative binding site's distance from the Transcription Start Site (TSS) and its estimated binding affinity. This new criteria for classifying putative p53-binding sites increases predictive accuracy by reducing the false positive rate.ResultsTraining a Profile Hidden Markov Model with corresponding positions matching a combined-palindromic p53-binding motif creates the best p53-RE predictive model. The p53HMM algorithm is available on-line: ConclusionUsing Profile Hidden Markov Models with training methods that exploit the redundant information of the homotetramer p53 binding site provides better predictive models than weight matrices (PSSMs). These methods may also boost performance when applied to other transcription factor binding sites.

A newly identified dependence receptor UNC5H4 is induced during DNA damage-mediated apoptosis and transcriptional target of tumor suppressor p53

UNC5H4 is a netrin-1 receptor UNC5H family member. In this study, we found that UNC5H4 is a direct transcriptional target of p53. During adriamycin (ADR)-mediated apoptosis, UNC5H4 was significantly induced in p53-proficient U2OS cells but not in p53-deficient H1299 cells. Enforced expression of p53 induced UNC5H4. Consistent with these results, siRNA-mediated knockdown of p53 in U2OS cells attenuated ADR-dependent induction of UNC5H4. Indeed, we found four putative p53-responsive elements within intron 1 of UNC5H4 gene. Luciferase reporter assay and ChIP analysis demonstrated that, among them, two tandem elements respond to exogenous p53 which is efficiently recruited onto them. Furthermore, enforced expression of UNC5H4 remarkably reduced number of drug-resistant colonies in p53-proficient cells but not in p53-deficient cells, suggesting that UNC5H4-induced apoptosis is dependent on p53 status. siRNA-mediated knockdown of UNC5H4 rendered U2OS cells resistant to ADR. Collectively, our present results suggest that UNC5H4 amplifies p53-dependent apoptotic response.

Functional analysis of p53 response elements in the human Polycystic Kidney Disease Gene 1 (PKD‐1) promoter

We have previously shown that p53 represses human PKD1 expression in vivo and in vitro, and that this effect is dependent on histone deacetylase activity (JBC, 281, p.31234, 2006). The 5′‐regulatory region of PKD1 contains 4 putative p53 response elements (p53RE4‐1) at positions: −2.7kb, −1.2kb, −0.8kb and −0.2kb respectively. In this study, we delineate the differential roles of the p53RE's in transcriptional repression of the PKD1 gene. We performed site‐directed mutagenesis of distal p53RE4, proximal p53RE1. The wild‐type and mutant PKD1 promoter‐reporter constructs were co‐transfected with a p53 expression plasmid in IMCD3, HCT116(p53+/+) and HCT116(p53−/−) cells. p53RE4 deletion decreased PKD1 baseline promoter activity (2.4, 1.8 and 1.4‐fold) in IMCD3, HCT116(p53+/+) and HCT116(p53−/−) cells, respectively, and significantly enhanced p53‐mediated repression in all cell lines, indicating that p53RE4 is a positive cis‐response element. In contrast, deletion of the p53RE1 induced a 1.6‐fold increase in baseline PKD1 promoter activity; this was observed in p53+/+ but not p53−/ − cells. p53RE1 failed to mediate repression of a known p53‐target gene ‐the bradykinin B2 receptor‐ indicating that p53RE1 functions in a context‐specific manner. Chromatin Immunoprecipitation assays in IMCD3 cells revealed the DNA regions surrounding p53RE1/2 are occupied by HDAC 1 and 2. We conclude that the balance between p53RE2 and p53RE1 occupancy by p53 and HDACs determines the final response of PKD1 to p53 within a given cell.Supported by NIH grant DK‐062250

P53 Regulates Cellular Resistance to Complement Lysis through Enhanced Expression of CD59

It has been recently hypothesized that the CD59 gene has two putative p53-responsive elements that may be involved in defense of host cells from damage by the complement system in inflammation. Here we have examined the roles of these putative p53-binding sequences within the CD59 gene in regulation of CD59 expression. We have shown that both of these potential responsive elements bind p53 in vitro. Knocking down expression of p53 using small interfering RNA led to a 6-fold decrease in CD59 protein expression in HeLa cells. We have previously observed a decrease of CD59 in camptothecin-induced apoptotic IMR32 cells, whereas expression was increased in the surviving fraction compared with untreated cells. Here, we have shown that these changes are associated with altered expression levels and acetylation status of p53. We have also shown that acetylation status of p53 regulates CD59 expression on cells exposed to inflammatory cytokines to model inflammation. Our data suggest that p53 and in vivo positive/negative regulators of p53 could be used to modulate susceptibility of tumor cells to complement lysis in chemotherapy.

A SNP in the flt-1 promoter integrates the VEGF system into the p53 transcriptional network

The VEGF system is essential for angiogenesis. VEGF overexpression frequently correlates with increased microvascularity and metastasis and decreased spontaneous apoptosis. Although a precise mechanism has not been established, studies suggest that VEGF expression is negatively regulated by p53, a master regulator and tumor suppressor. There are no reports of additional components of the VEGF signal transduction pathway being part of the p53 transcriptional network. A target of VEGF, the VEGF receptor 1/flt-1, can regulate growth and migration of endothelial cells and modulate angiogenesis. VEGF appears to be up-regulated in various cancers in which flt-1 may have a role in tumor progression and metastasis. We identified a C-to-T SNP upstream of the transcriptional start site in approximately 6% of the people examined. The SNP is located within a putative p53 response element. Only the promoter with the T SNP (FLT1-T) was responsive to p53 when examined with reporter assays or by endogenous gene expression analysis in cell lines with different SNP status. In response to doxorubicin-induced DNA damage, there was clear allele discrimination based on p53 binding at the FLT1-T but not FLT1-C promoters as well as p53-dependent induction of flt-1 mRNA, which required the presence of FLT1-T. Our results establish that p53 can differentially stimulate transcription at a polymorphic variant of the flt-1 promoter and directly places the VEGF system in the p53 stress-response network via flt-1 in a significant fraction of the human population. We suggest that the p53-VEGF-flt-1 interaction is relevant to risks in angiogenesis-associated diseases, including cancer.