Identification of a functional link for the p53 tumor suppressor protein in dexamethasone-induced growth suppression

Serine/threonine phosphatase 5 (PP5) can act as a suppresser of p53-dependent growth suppression and has been reported to associate with several proteins, including the glucocorticoid receptor/heat-shock protein-90 complex. Still, the physiological/pathological roles of PP5 are unclear. To characterize the relationship of PP5, glucocorticoid receptor activation and p53, here we describe the development of chimeric antisense oligonucleotides that potently inhibit human p53 expression. This allowed us to regulate the expression of either p53 (e.g. with ISIS 110332) or PP5 (e.g. with ISIS 15534) in genetically identical cells. Studies with ISIS 110332 revealed that the suppression of p53 expression is associated with a decrease in the basal expression of the cyclin-dependent kinase inhibitor protein, p21(WAF1/Cip1), and a concomitant increase in the rate of cell proliferation. Suppression of p53 also blocks dexamethasone-induced p21(WAF1/Cip1) expression and G(1)-growth arrest. Furthermore, treatment with ISIS 110332, but not the mismatched controls, ablates the suppression of growth produced by prior treatment with dexamethasone. Additional studies revealed that dexamethasone-dependent p21(WAF1/Cip1) expression occurs without an apparent change in p53 protein levels or the phosphorylation status of p53 at Ser-6, -37, or -392. However, dexamethasone treatment is associated with an increase in p53 phosphorylation at Ser-15. Suppression of PP5 expression with ISIS 15534 also results in the hyperphosphorylation of p53 at Ser-15. Together, these findings indicate that the basal expression of p53 plays a functional role in a glucocorticoid receptor-mediated response regulating the expression of p21(Waf1/Cip1) via a mechanism that is suppressed by PP5 and associated with the phosphorylation of p53 at Ser-15.     

Protein phosphatase 2Cγ regulates the level of p21 by Akt signaling

PP2Cγ is a splicing factor that dephosphorylates specific substrates required for the formation of the spliceosome. In a previous study, we reported that the degradation of p21^Cip1/WAF1was affected by PP2Cγ, causing an accumulation of cells in S phase. Here, we demonstrate that the PP2Cγ-induced degradation of p21^Cip1/WAF1 is mediated by Akt signaling. In cells expressing PP2Cγ, Akt1 protein was phosphorylated. When PP2Cγ expression was knocked down, the phosphorylation of Akt1 was reduced and the level of p21^Cip1/WAF1 protein was increased. Interestingly, the stability of p21^Cip1/WAF1 was highly maintained in Akt1-depleted cells despite the ectopic expression of PP2Cγ. Taken together, these results suggest that PP2Cγ is a novel regulator of p21^Cip1/WAF1 protein stability via the Akt signaling pathway.     

BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21Cip1/Waf1

Bone-morphogenetic proteins (BMP)-2 and -7, multifunctional members of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, cause cell cycle arrest in a variety of transformed cell lines by activating signaling cascades that involve several cyclin-dependent kinase inhibitors (CDKIs). CDKIs in the cip/kip family, p21(Cip1/Waf1) and p27(Kip1), have been shown to negatively regulate the G1 cyclins and their partner cyclin-dependent kinase proteins, resulting in BMP-mediated growth arrest. Bone morphogens have also been associated with antiproliferative effects in vascular tissue by unknown mechanisms. We now show that BMP-2-mediated inhibition of platelet-derived growth factor (PDGF)-stimulated human aortic smooth muscle cell (HASMC) proliferation is accompanied by increased levels of p21 protein. Antisense oligodeoxynucleotides specific for p21 attenuate BMP-2-induced inhibition of proliferation when transfected into HASMCs, demonstrating that BMP-2 inhibits PDGF-stimulated proliferation of HASMCs through induction of p21. Whether p21-mediated induction of cell cycle arrest by BMP-2 sets the stage for osteogenic differentiation of vascular smooth muscle cells, ultimately leading to vascular mineralization, remains to be investigated.     

Proinflammatory cytokine-induced cellular senescence of biliary epithelial cells is mediated via oxidative stress and activation of ATM pathway: a culture study

Cellular senescence is reportedly involved in cholangiopathy in primary biliary cirrhosis and oxidative stress is proposed as a pathogenetic factor in biliary epithelial cells (BECs). This study investigated the involvement of proinflammatory cytokines (IFN-beta, IFN-gamma and TNF-alpha) and ataxia telangiectasia-mutated (ATM)/p53/ p21(WAF1/Cip1) pathway with respect to oxidative stress in cellular senescence of BECs. H(2)O(2) treatment (oxidative stress) induced phosphorylation (activation) of ATM and p53 and also p21(WAF1/Cip1) expression in BECs. Treatment with inflammatory cytokines generated reactive oxygen species (ROS) in cultured BECs followed by activation of the ATM/p53/p21(WAF1/Cip1) pathway and the induction of cellular senescence. Pre-treatment with ATM inhibitor (2-aminopurine) and antioxidant (N-acetylcysteine) significantly blocked the cellular senescence of BECs induced by oxidative stress or inflammatory cytokines. In conclusion, proinflammatory cytokines induce ROS generation and activate the ATM/p53/p21(WAF1/Cip1) pathway, followed by biliary epithelial senescence. This senescent process may be involved in the development of destructive cholangiopathy in humans.