PARP-1 modulates Ets-1-mediated transactivation of the stromelysin-1 promoter by interaction and by PARylation.

<p>(<b>A</b>) Schematic representation of the luciferase gene under the control of the human stromelysin-1 promoter. Palindromic Ets-binding sites (EBS) are shown bound with two Ets-1 molecules. (<b>B</b>) Effect of PARP-1 knock out (KO) on Ets-1-mediated stromelysin-1 promoter activity. pGL3 luciferase reporter constructs (100 ng) were transfected into 3T3 mouse cells wild type (WT) (lanes 1 and 3) or KO for the PARP-1 gene (lanes 2, 4 and 5) at 50–80% confluence in the absence (lanes 1 and 2) or in the presence (lanes 3, 4 and 5) of the Ets-1 expression vector (25 ng) and in the rescue experiments with PARP-1 expression vector (100 ng; lane 5). (<b>C</b>) Effect of PARP-1 catalytic inhibition on the Ets-1-mediated stromelysin-1 promoter activity in the presence or absence of PARP-1. pGL3 luciferase reporter constructs (100 ng) were transfected into 3T3 mouse cells WT (lanes 1- 4) or KO for the PARP-1 gene (lane 5–8) at a 50–80% confluence in the absence (lanes 1, 2, 5 and 6) or in the presence (lanes 3, 4, 7 and 8) of Ets-1 expression vector (25 ng) and in the absence (lanes 1, 3, 5 and 7) or in the presence (lanes 2, 4, 6 and 8) of PJ-34, a catalytic inhibitor of PARP-1 (10 µM). (<b>D</b>) Effect of PARP-1 catalytic inhibition on Ets-1-mediated stromelysin-1 promoter activity in a cancer cell model. pGL3 luciferase reporter constructs (100 ng) were transfected into HeLa cells at a 50–80% confluence in the absence (lanes 1–4) or in the presence (lanes 5–8) of Ets-1 expression vector (100 ng) and with increasing doses of PJ-34 (0–20 µM; lanes 1–4 and 5–8). In (B), (C) and (D), luciferase activity, measured 48 h after transfection and 20 h after incubation with PJ-34, is expressed as a percentage with the activity induced by Ets-1 in a PARP-1 WT context indicated as 100%. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055883#s2" target="_blank">Results</a> are the average of three experiments performed in triplicate. (<b>E</b>) Effect of PARP-1 catalytic inhibition on the level of Ets-1 protein. HeLa cells were grown in 6-well plates until 70% confluence, transfected with pcDNA3 (1 µg; left panel) or pcDNA3-Ets1 (1 µg; right panel) vectors for 24 h and treated with PJ-34 (1 µM) (lanes 2 and 6), 5-AIQ (1 µM) (lanes 3 and 7) or ABT-888 (1 µM) (lanes 4 and 8) for 20 h. In all experiments, cell lysates (30 µg total proteins) were analysed by Western blot using different antibodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055883#s4" target="_blank">Materials and Methods</a>).