Nitric oxide down-regulates polo-like kinase 1 through a proximal promoter cell cycle gene homology region

Polo-like kinase 1 (PLK1) is an evolutionarily conserved serine/threonine kinase essential for cell mitosis. As a master cell cycle regulator, p21/Waf1 plays a critical role in cell cycle progression. Nitric oxide (NO.) has been shown to down-regulate PLK1 and up-regulate p21/Waf1 independent of cGMP. Here, the respective roles of p38 MAPK and p21/Waf1 in NO.-mediated PLK1 repression were investigated using differentiated U937 cells that lack soluble guanylate cyclase. NO. was shown to down-regulate both PLK1 mRNA and protein. Nuclear run-on assays and mRNA stability studies demonstrated that the effect of NO. on PLK1 expression was associated with decreased transcription without changes in transcript stability. SB202190, a p38 MAPK inhibitor, prevented transcriptional repression of PLK1 by NO.. Transfection with dominant-negative p38 MAPK mutant eliminated the NO. effect on both p21/Waf1 and PLK1 gene expression. Knockdown of p21/Waf1 with siRNA also substantially reduced the regulatory effect of NO. on PLK1. Reporter gene experiments showed that NO. decreased activity of the PLK1 proximal promoter, an effect that was blocked by p38 MAPK inhibitor. Deletion or mutation of the CDE/CHR promoter site, an element regulated by p21/Waf1, increased base-line promoter activity and abolished NO. repression of the PLK1 promoter. Likewise, electrophoretic mobility shift assays with CDE/CHR probe revealed a NO.-mediated change in protein-probe complex formation. Competition with various unlabeled CDE/CHR mutant sequences showed that NO. increased nuclear protein binding to intact CHR. These results demonstrate that a NO.-p38 MAPK-p21/Waf1 signal transduction pathway represses PLK1 through a canonical CDE/CHR promoter element.