Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1

Abstract

Expression of p21Sdi1 downstream of p53 is essential for induction of cellular senescence, although cancer cell senescence can also occur in the p53 null condition. We report herein that senescence-associated phosphorylated extracellular signal-regulated protein kinases 1 and 2 (SA-pErk1/2) enhanced p21 Sdi1 transcription by phosphorylating Sp1 on Ser 59 downstream of protein kinase C (PKC) ␣. Reactive oxygen species (ROS), which was increased in cellular senescence, significantly activated both PKC␣ and PKC␤I. However, PKC␣, but not PKC␤I, regulated ROS generation and cell proliferation in senescent cells along with activation of cdk2, proven by siRNAs. PKC␣-siRNA also reduced SA-pErk1/2 expression in old human diploid fibroblast cells, accompanied with changes of senescence phenotypes to young cell-like. Regulation of SA-pErk1/2 was also confirmed by using catalytically active PKC␣ and its DN-mutant construct. These findings strongly suggest a new pathway to regulate senescence phenotypes by ROS via Sp1 phosphorylation between PKC␣ and SA-pErk1/2: employing GST-Sp1 mutants and MEK inhibitor analyses, we found that SA-pErk1/2 regulated Sp1 phosphorylation on the Ser 59 residue in vivo, but not threonine, in cellular senescence, which regulated transcription of p21 Sdi1 expression. In summary, PKC␣, which was activated in senescent cells by ROS strongly activated Erk1/2, and the SA-pErk1/2 in turn phosphorylated Sp1 on Ser 59. Sp1-enhanced transcription of p21 Sdi1 resulted in regulation of cellular senescence in primary human diploid fibroblast cells. PKC3 comprises a family of serine/threonine kinase that modulates a variety of signal transduction pathways, leading to gene expression, cell proliferation, and differentiation. PKC isoforms are classified into three subgroups. The conventional PKC isozymes, comprising, ␣, ␤I, ␤II, and ␥, are activated by Ca 2ϩ , phosphatidylserine, diacylglycerol, or phorbol esters; the novel PKC isozymes, consisting of PKC␦, -⑀, -, -, and -, are activated by phosphatidylserine, diacylglycerol, or phorbol esters, but insensitive to Ca 2ϩ ; and the atypical isoforms ( and /) are dependent on phosphatidylserine for activation, but not affected by Ca 2ϩ , diacylglycerol, or phorbol esters (1-3). Activated PKC translocates from cytosol to membranous organelles and/or to the nucleus (4, 5). In addition, oxidative stress has been reported to induce prolonged activation of PKC within the cells (6 -9). The growth regulatory consequences of PKC activation suggest a link between PKC signaling and control of the cell cycle machinery. Activation of PKC has been shown to result in alterations of cell cycle progression in either stimulatory or inhibitory directions in several systems (10 -15). Among the isoforms, PKC␣ has been implicated in the control of G 1 /S transition (14, 16 -18) and recently in the regulation of cancer cell senescence (19), whereas PKC␤II has been shown to play a role in progression from G 2 into M phase (15), and PKC␦ has been associated with the control of M phase (2...

Loss of p21Sdi1 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21Sdi1 gene promoter

“…Fig. 2A and B, respectively), however, it was reduced in presenescent cells after the treatment, nevertheless p53S 15 activation was observed both in young and presenescent cells. The data indicate DNA synthesis in the presenescent, but not young cells, when assayed by radioactivity; activities (cpm) of the treated vs. untreated were 22,594 ± 1197 vs. 11,903 ± 588; 2799 ± 150 vs. 5981 ± 500; and 2417 ± 194 vs. 11,617 ± 1207 in the young and the presenescent (DT5d and DT10d) cells, respectively (Fig.…”

“…Primary culture of HDF was prepared in our laboratory from foreskin of 4 year-old boy [15,[20][21][22] and maintained in DMEMhigh glucose supplemented with 10% heat inactivated fetal bovine serum under 5% CO 2 in air. To evaluate characteristics of in vivo cellular senescence, skin fibroblasts were freshly isolated from buttock of 24-75 year-old men and cultivated until use.…”

“…To evaluate characteristics of in vivo cellular senescence, skin fibroblasts were freshly isolated from buttock of 24-75 year-old men and cultivated until use. Doubling times (DT) of HDF used in this study were calculated based on the equation [15]; mean 26 h in young cells, 2-10 days in presenescent cells and over 14 days in senescent cells.…”

“…By using primary cultures of human diploid fibroblasts (HDF), we have previously reported that expression of p21 in senescent HDF is dependent of Sp1 phosphorylation on serine 59 by PKCa and its downstream kinase Erk1/2, activated by reactive oxygen species [15]. siRNA constructs against the kinases make senescent HDF to be proliferative.…”

Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

“…A number of population doublings and doubling times (DT) of HDF were calculated based on the equation, described previously [32]. DT of the young cells used in this study was about 26 h (expressed herein as 1 day), and DT of the mid-old and the old cells were 3-10 days and 14 days, respectively.…”