p53 regulates the transcription of the anti-inflammatory molecule developmental endothelial locus-1 (Del-1)

Abstract

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.

Figure 1. Schematic representation of the region upstream of the Del-1 gene

Putative p53 response elements (p53REs) were predicted using the Genometrix program. CATG-containing p53REs in the upstream regions are indicated. The first nucleotide of CATG is numbered relative to the translation start site (TSS) of the Del-1 gene.

Figure 2. Relative transcriptional activity is dependent on the presence of proximal p53 response elements in the upstream region of the Del-1 gene

(A) Schematic diagram of the Del-1 promoter constructs. Nucleotides are indicated relative to the TSS. Upstream fragments of the Del-1 gene were cloned into the pGL3 vector to generate four Del-1 promoter deletion constructs containing multiple putative p53 response elements. (B) Relative luciferase activity of these constructs. The Del-1 promoter constructs shown in (A) were individually transfected into HEK293T cells. Luciferase activity was determined 24 h after transfection and is expressed as the fold activity over that of the empty pGL3 vector (EV). A Renilla luciferase vector was co-transfected for normalization of transfection efficiency. Values are the means ± standard deviations (SD) from triplicate transfections. Data represent four independent experiments. **, p < 0.01; ***, p < 0.001; n.s., non-significant vs. the Del-1_luc 2k construct (i.e., the construct exhibiting the highest activity).

Figure 3. Functional p53 response elements are required to enhance Del-1 transcription

(A) Schematic diagram of wild-type (WT) and mutant Del-1 promoter constructs. The Del-1_luc 2k construct was mutated at the consensus sequence (CATG→TATA) of either or both p53 response elements. (B) The WT and mutant Del-1_luc 2k constructs were independently transfected into HEK293T cells. Luciferase activity was determined 24 h after transfection and is expressed as the fold activity over that of the empty pGL3 vector (EV). Values are means ± SD from triplicate transfections. Data represent four independent experiments. *, p < 0.05; **, p < 0.01; n.s., non-significant vs. the WT Del-1_luc 2k construct.

Figure 4. p53 positively regulates Del-1 transcription

(A) Mouse WT p53 or p53 with mutated DNA binding sites (G239A, R242A, and R243A) was transfected along with the 2k Del-1 promoter construct into HEK293T cells. Luciferase activity was determined 24 h after transfection and is expressed as the fold activity over that of the empty pGL3 vector. Values are means ± standard deviations from triplicate transfections. *, p < 0.05; ***, p < 0.001, vs. the 2k Del-1 promoter construct. (B) Mouse primary endothelial cells were treated with increasing concentrations of tenovin-1 and incubated for 24 h. The Del-1 mRNA level was measured and is expressed as the fold increase over dimethyl sulfoxide (DMSO)-treated cells. Values are means ± SD from triplicate treatments. Data represent three independent experiments. *, p < 0.05; **, p < 0.01 vs. the DMSO-treated cells.

Figure 5. p53 directly binds to p53 response elements to enhance Del-1 transcription

(A) Representative ChIP analysis. HEK293T cells were transfected with a mouse p53-expressing plasmid, together with either the WT or mutant (mutated at both p53REs) 2k Del-1 promoter construct. Nuclear lysates were analyzed 48 h after transfection using a p53 antibody or control IgG and promoter regions containing the p53REs were amplified by PCR. (B) HEK293T cells were transfected with the plasmids in (A). Luciferase activity was determined 24 h after transfection and is expressed as the fold activity over that of the empty pGL3 vector. Values are means ± SD from triplicate transfections. Data represent three independent experiments. *, p < 0.05; ***, p < 0.001 vs. the cells co-transfected with the WT p53 and p53REwt Del-1 constructs.

Figure 6. p53 levels determine Del-1 expression in mouse primary endothelial cells

Mouse p53 (A) and Del-1(B) mRNA levels were analyzed by real-time RT-PCR in primary endothelial cells from p53^+/+, p53^+/−, and p53^−/− mice. The relative Del-1 mRNA levels in cells heterozygous or homozygous null for p53 were compared to those of p53^+/+ mice. Values are means ± SD (n = 4–5 mice/group). Data represent three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 7. Del-1 reciprocally regulates p53 expression in mouse primary endothelial cells

(A) Levels of p53 protein was analyzed by western blotting in primary endothelial cells from Del-1^+/+ and Del-1^−/− mice. Values are means ± SD (n = 3 mice/group). (B) Transcript levels of p53 were analyzed by real-time RT-PCR. WT primary endothelial cells were incubated in the absence or presence of recombinant Del-1 protein (10 nM) for 24 h. Values are means ± SD (n = 4−5 mice/group). *, p < 0.05.