Nitric Oxide Down-regulates Polo-like Kinase 1 through a Proximal Promoter Cell Cycle Gene Homology Region

Abstract

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 ⅐ ) 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 ⅐ -mediated PLK1 repression were investigated using differentiated U937 cells that lack soluble guanylate cyclase. NO ⅐ was shown to down-regulate both PLK1 mRNA and protein. Nuclear run-on assays and mRNA stability studies demonstrated that the effect of NO ⅐ 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 ⅐ . Transfection with dominant-negative p38 MAPK mutant eliminated the NO ⅐ effect on both p21/Waf1 and PLK1 gene expression. Knockdown of p21/Waf1 with siRNA also substantially reduced the regulatory effect of NO ⅐ on PLK1. Reporter gene experiments showed that NO ⅐ 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 ⅐ repression of the PLK1 promoter. Likewise, electrophoretic mobility shift assays with CDE/CHR probe revealed a NO ⅐ -mediated change in protein-probe complex formation. Competition with various unlabeled CDE/CHR mutant sequences showed that NO ⅐ increased nuclear protein binding to intact CHR. These results demonstrate that a NO ⅐ -p38 MAPK-p21/Waf1 signal transduction pathway represses PLK1 through a canonical CDE/CHR promoter element.

Nitric oxide-p38 MAPK signaling stabilizes mRNA through AU-rich element-dependent and -independent mechanisms

“…Consistent with this microarray result, p38 MAPK signaling has been implicated in regulating the mRNA half-life of more than 40 AUREcontaining genes [18], including TNF-␣ [27], p21/Waf1 [26,28], and IL-8 [27,29]. Importantly, NO • was shown in LPSstimulated THP-1 cells [4,30] and PMA-differentiated U937 cells [26,28] to stabilize mRNA through p38 MAPK activation. However, the specific 3ЈUTR sequences and configuration of elements that transduce NO • signaling to individual transcripts as well as the identity of the binding proteins involved remain unknown.…”

“…We have previously shown that NO • stabilizes IL-8 and p21/ Waf1 mRNA through p38 MAPK activation [26,28,30]. Here, the 3ЈUTRs of IL-8, p21/Waf1, and TNF-␣ were shown to destabilize the mRNA of an artificial construct, an effect closely associated with the presence of AUUUA sequences.…”

“…NO • -p38 MAPK signaling was found to stabilize a different set of genes that had a preponderance of AURE sites in their 3ЈUTRs [4,26]. Consistent with this microarray result, p38 MAPK signaling has been implicated in regulating the mRNA half-life of more than 40 AUREcontaining genes [18], including TNF-␣ [27], p21/Waf1 [26,28], and IL-8 [27,29]. Importantly, NO • was shown in LPSstimulated THP-1 cells [4,30] and PMA-differentiated U937 cells [26,28] to stabilize mRNA through p38 MAPK activation.…”

“…p21/Waf1 is a master regulator of the cell cycle that inhibits cell proliferation [26,28]. IL-8 and TNF-␣ are potent cytokines that play major roles in propagating the innate immune response [30].…”

Cx43 Hemichannels and Gap Junction Channels in Astrocytes Are Regulated Oppositely by Proinflammatory Cytokines Released from Activated Microglia

“…The reducing agent dithiothreitol (DTT; 10 mM; Sigma-Aldrich) was applied similar to the hemichannels blockers. SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1 H-imidazole] (10 M; Sigma-Aldrich), a specific p38 mitogen-activated protein kinase (MAPK) inhibitor (Fukunaga and Hunter, 1997;Zhang et al, 2007), or L-name (1 mM; Sigma-Aldrich), a nitric oxide synthase (NOS) inhibitor, were coapplied with CM* or the mix of cytokines, depending of the experiment.…”