PDGF-BB Regulates p27 Expression through ERK-dependent RNA Turn-over inVascular Smooth MuscleCells

Abstract

Cyclin-dependent kinase inhibitor p27, a critical determinant for cell cycle progression, is an important regulation target of mitogenic signals during arterial injury. In this study, we show in rat aortic smooth muscle cells that PDGF-BB down-regulated p27 protein and mRNA in an ERK-dependent mechanism. Inhibition of ERK, but not other subtypes of the mitogen-activated protein kinase family, prevented the reduction of p27 protein and mRNA. Conversely, direct activation of ERK via adenovirus-mediated expression of a constitutively active form of MEK led to a reduction of p27 protein and mRNA, further supporting the central role of ERK in regulation of p27 expression. Rapamycin, which potently inhibited PDGF-induced activation of p70 S6 kinase as well as proliferation of smooth muscle cells, did not alter the expression of p27. To delineate the molecular mechanism underlying the p27 down-regulation, we examined the effect of PDGF-BB on p27 promoter activity as well as mRNA stability. Stimulation with PDGF-BB significantly shortened the half-life of p27 mRNA without affecting its promoter activity. To further understand the PDGF-stimulated p27 mRNA turnover, we inserted the 5-and/or 3-untranslated regions of p27 cDNA into a non-PDGF-responsive luciferase gene. Only those chimeric genes that contained the 3-untranslated region responded to PDGF-BB with reduced expression. Moreover, inhibition of ERK completely prevented the effect of PDGF on the chimera expression. In summary, our data suggest that p27 is down-regulated by PDGF-BB in vascular smooth muscle cells through an ERK-dependent posttranscriptional mechanism.

The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition

“…The levels of p27 are high in quiescent cells and decline in response to mitogenic factor stimulation. Several studies have suggested that the Ras-ERK1/2 signaling pathway is involved in the mitogen-induced downregulation of p27 (Kawada et al, 1997;Kerkhoff and Rapp, 1997;Woods et al, 1997;Greulich and Erikson, 1998;Rivard et al, 1999;Treinies et al, 1999;Lenferink et al, 2000;Yang et al, 2000;Delmas et al, 2001;Mirza et al, 2004;Gysin et al, 2005;Sakakibara et al, 2005). In contrast, other studies failed to document any significant change in p27 levels following inhibition of ERK1/2 signaling by synthetic MEK1/2 inhibitors or dominant-negative ERK2, or after conditional activation of the pathway by activated Raf-1 or MEK1 (Sewing et al, 1997;Takuwa and Takuwa, 1997;Weber et al, 1997a;Cheng et al, 1998;Ladha et al, 1998;Chen et al, 1999;Tetsu and McCormick, 2003).…”

Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression

“…It is well established that p27 expression is mainly posttranslationally regulated by the ubiquitin-proteasome pathway (Pagano et al, 1995;Montagnoli et al, 1999;Malek et al, 2001;Kamura et al, 2004). However, some studies demonstrate that p27 might also be transcriptionally regulated (Servant et al, 2000;Sakakibara et al, 2005). Nevertheless, at present, the mechanisms involved in such regulation remain unidentified.…”

Inhibition of p27Kip1gene transcription by mitogens

“…However, as shown above, PDGF reduced p27 Kip1 promoter activity and mRNA abundance in Balb/c-3T3 cells, NIH 3T3 cells and MEFs examples, p27 Kip1 promoter activity declined 90% in PDGF-treated aortic smooth muscle cells, 60% in eicosanoid-treated endothelial cells, and 23% in muscle satellite cells exposed to insulin-like growth factor 1. 25,26,28 Changes in p27 Kip1 stability contributed to changes in p27 Kip1 mRNA abundance in PDGF-treated smooth muscle cells and histidine-deprived hepatoma cells 26,75,76 although not in other systems. 41,42,74 Deletion analysis of the p27 Kip1 promoter localized the PDGFresponsive element to a region approximately 600 bp upstream (or slightly downstream) of the most 3' transcription start site.…”