PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF-beta signaling pathway - PubMed (original) (raw)

PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF-beta signaling pathway

M Tan et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Identification and characterization of p53 target genes would lead to a better understanding of p53 functions and p53-mediated signaling pathways. Two putative p53 binding sites were identified in the promoter of a gene encoding PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member. Gel shift assay showed that p53 bound to both sites. Luciferase-coupled transactivation assay revealed that the gene promoter was activated in a p53 dose- as well as p53 binding site-dependent manner by wild-type p53 but not by several p53 mutants. The p53 binding and transactivation of the PTGF-beta promoter was enhanced by etoposide, a p53 activator, and was largely blocked by a dominant negative p53 mutant. Furthermore, expression of endogenous PTGF-beta was remarkably induced by etoposide in p53-positive, but not in p53-negative, cell lines. Finally, the conditioned medium collected from PTGF-beta-overexpressing cells, but not from the control cells, suppressed tumor cell growth. Growth suppression was not, however, seen in cells that lack functional TGF-beta receptors or Smad4, suggesting that PTGF-beta acts through the TGF-beta signaling pathway. Thus, PTGF-beta, a secretory protein, is a p53 target that could mediate p53-induced growth suppression in autocrinal as well as paracrinal fashions. The finding made a vertical connection between p53 and TGF-beta signaling pathways in controlling cell growth and implied a potential important role of p53 in inflammation regulation via PTGF-beta.

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Figures

Figure 1

Figure 1

p53 binds to putative p53 binding sites in the promoter of the PTGF-β gene. Synthetic oligonucleotides (PTGF-FBS01 and PTGF-SBS01) were labeled with [γ-32P]ATP and were used as probes in gel shift assays. (A) The oligonucleotide PTGF-FBS01. (B) The oligonucleotide PTGF-SBS01. Lanes: 1–4, partially purified p53 protein (3 μg) with or without pAb421 antibody; 5–12, nuclear extracts (8.5 μg) prepared from U2-OS cells treated with etoposide (25 μM) for 0 hr (lanes 5 and 6), 6 hr (lanes 7 and 8), and 24 hr (lanes 9–12). Competition was performed with a 100-fold excess of unlabeled specific oligonucleotides, PTGF-FBS01 or PTGF-SBS01, respectively, or nonspecific oligonucleotide mT3SF (5′-GGGGTTGCTTGAAGAGCGTC-3′) (29).

Figure 2

Figure 2

p53 binding site-dependent activation of the PTGF-β promoter by wild-type p53, but not by most p53 mutants. (A) Diagrammatic presentation of four luciferase reporter constructs driven by the PTGF-β promoter, containing, respectively, two (W/p53BS), the first one (W/Fp53BS), the second one (W/Fp53BS), and none of p53 binding sites (W/Op53BS). (B) Activation of luciferase activity in p53-binding site dependent manner. (C) Lack of transactivation of PTGF-β promoter by most p53 mutants. Four constructs were transiently co-transfected with or without p53 expressing plasmids, respectively, along with a β-galactosidase-expressing vector into human Saos-2 cells, followed by luciferase assay. The results are presented as fold activation ± standard error derived from three independent transfections, each run in duplicate, after normalization with β-galactosidase activity for transfection efficiency.

Figure 3

Figure 3

Induction of p53-dependent transactivation of the PTGF-β promoter by etoposide. Subconfluent U2-OS cells were co-transfected with β-galactosidase-expressing construct and PTGF-β W/p53BS luciferase reporters, individually, or in combination with a construct expressing a dominant negative p53 mutant (p53-280T) or the vector control by the Lipofectamine method. Cells were treated with etoposide (25 μM) 24 hr after transfection for 0, 2, 6, 12, 24, and 48 hr, respectively, followed by luciferase activity measurements. Three independent transfections, each run in duplicate, were performed, and results are presented as fold activation ± standard error after normalization with β-galactosidase activity for transfection efficiency. To calculate the fold activation, the luciferase activity from PTGF-β W/p53BS construct after 0 hr of etoposide treatment was arbitrarily set as 1.

Figure 4

Figure 4

Induction of endogenous PTGF-β expression by etoposide in p53 positive cells. (A) Subconfluent U2-OS, Saos-2, H460, and H1299 cells were subjected to etoposide (25 μM) treatment for various times up to 48 hr, followed by total RNA isolation and Northern blot analysis, using PTGF-β cDNA as a probe. (B) The time course of PTGF-β mRNA induction by etoposide in U2OS cells. (C) PTGF-β protein induction by etoposide. Subconfluent H460 cells were subjected to DMSO or etoposide (25 μM) treatment for 24 hr under serum-free condition. The media were collected, and proteins were TCA-precipitated and subjected to Western blot analysis using rabbit anti-PTGFβ antibody.

Figure 5

Figure 5

Expression of PTGF-β in conditioned medium and requirement of intact TGFβ signaling pathway for growth inhibition. (A) Expression of PTGF-β in the conditioned medium collected from PTGF-β-expressing DLD-Cl7 cells, but not from Dn1-3 vector control. Conditioned medium was collected and subjected (85 μg) to Western blot analysis using Flag-tag antibody. (B) PTGF-β-induced growth inhibition of TGF-β-sensitive but not TGF-β-resistant cells. The target cells used are TGF-β-sensitive Mv1Lu or Du145 cells as well as TGF-β-resistant cells, including RKO (TβRII mutation), R1B/L17 (TβRI mutation), and MDA-MB468 (Smad4-null). Conditioned medium was collected from PTGF-β expressing clone, DLD-Cl7 or the vector control, and Dn1-3 cells and was concentrated and added into the target cells at various protein concentrations, followed by BrdUrd incorporation assay. All results were presented as percent of growth rate ± standard error of the mean from three independent experiments, each run in quadruplet. The growth rate was calculated with the control group (without any addition of conditioned medium) setting as 100%.

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