Tanjun Tong

SIRT1 Overexpression Antagonizes Cellular Senescence with Activated ERK/S6k1 Signaling in Human Diploid Fibroblasts

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.

SIRT1 is regulated by a PPARγ–SIRT1 negative feedback loop associated with senescence

Human Silent Information Regulator Type 1 (SIRT1) is an NAD+-dependent deacetylase protein which is an intermediary of cellular metabolism in gene silencing and aging. SIRT1 has been extensively investigated and shown to delay senescence; however, less is known about the regulation of SIRT1 during aging. In this study, we show that the peroxisome proliferator-activated receptor-γ (PPARγ), which is a ligand-regulated modular nuclear receptor that governs adipocyte differentiation and inhibits cellular proliferation, inhibits SIRT1 expression at the transcriptional level. Moreover, both PPARγ and SIRT1 can bind the SIRT1 promoter. PPARγ directly interacts with SIRT1 and inhibits SIRT1 activity, forming a negative feedback and self-regulation loop. In addition, our data show that acetylation of PPARγ increased with increasing cell passage number. We propose that PPARγ is subject to regulation by acetylation and deacetylation via p300 and SIRT1 in cellular senescence. These results demonstrate a mutual regulation between PPARγ and SIRT1 and identify a new posttranslational modification that affects cellular senescence.

HuR Uses AUF1 as a Cofactor To Promote p16INK4 mRNA Decay

ABSTRACT In this study, we show that HuR destabilizes p16INK4 mRNA. Although the knockdown of HuR or AUF1 increased p16 expression, concomitant AUF1 and HuR knockdown had a much weaker effect. The knockdown of Ago2, a component of the RNA-induced silencing complex (RISC), stabilized p16 mRNA. The knockdown of HuR diminished the association of the p16 3′ untranslated region (3′UTR) with AUF1 and vice versa. While the knockdown of HuR or AUF1 reduced the association of Ago2 with the p16 3′UTR, Ago2 knockdown had no influence on HuR or AUF1 binding to the p16 3′UTR. The use of EGFP-p16 chimeric reporter transcripts revealed that p16 mRNA decay depended on a stem-loop structure present in the p16 3′UTR, as HuR and AUF1 destabilized EGFP-derived chimeric transcripts bearing wild-type sequences but not transcripts with mutations in the stem-loop structure. In senescent and HuR-silenced IDH4 human diploid fibroblasts, the EGFP-p16 3′UTR transcript was more stable. Our results suggest that HuR destabilizes p16 mRNA by recruiting the RISC, an effect that depends on the secondary structure of the p16 3′UTR and requires AUF1 as a cofactor.

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organisms aging. However, less is known about the methylation changes of age‐related specific genes in aging. p21Waf1/Cip1 and p16INK4a are cyclin‐dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21Waf1/Cip1 and p16INK4a have different methylation patterns during the aging process of normal human 2BS and WI‐38 fibroblasts. p21Waf1/Cip1 promoter is gradually methylated up into middle‐aged fibroblasts but not with senescent fibroblasts, whereas p16INK4a is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle‐aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21Waf1/Cip1 promoter methylation directly represses its expression and blocks the radiation‐induced DNA damage‐signaling pathway by p53 in middle‐aged fibroblasts. More importantly, demethylation by 5‐aza‐CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21Waf1/Cip1 level and premature senescence of middle‐aged fibroblasts demonstrated by cell growth arrest and high β‐Galactosidase expression. Our results suggest that p21Waf1/Cip1 but not p16INK4a is involved in the DNA methylation mediated aging process. p21Waf1/Cip1 promoter methylation may be a critical biological barrier to postpone the aging process. J. Cell. Biochem. 98: 1230–1248, 2006.

Senescence Delay of Human Diploid Fibroblast Induced by Anti-sense p16INK4a Expression

p16INK4a, a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and cyclin-dependent kinase 6, is also implicated in the mechanisms underlying replicative senescence, because its RNA and protein accumulate as cells approach their finite number of population doublings in tissue culture. To further explore the involvement of p16INK4a in replicative senescence, we constructed a retroviral vector containing antisense p16INK4a, pDOR-ASp16, and introduced it into early passages of human diploid fibroblasts. The introduction of this construct significantly suppressed the expression of wild-type p16INK4a. It also imposed a finite increase in proliferative life span and significant delay of several other cell senescent features, such as cell flattening, cell cycle arrest, and senescence-associated β-galactosidase positivity. Moreover, telomere shortening and decline in DNA repair capacity, which normally accompany cell senescence, are also postponed by the ASp16 transfection. The life span of fibroblasts was significantly extended, but the onset of replicative senescence could not be totally prevented. Telomerase could not be activated even though telomere shortening was slowed. These observations suggest that the telomere pathway of senescence cannot be bypassed by ASp16 expression. These data not only strongly support a role for p16INK4a in replicative senescence but also raise the possibility of using the antisense p16INK4atherapeutically.

Reduced nuclear export of HuR mRNA by HuR is linked to the loss of HuR in replicative senescence

The RNA-binding protein, HuR, associates with the HuR mRNA, but the consequences of this interaction are unknown. Here, we use human diploid fibroblasts (HDFs) and cervical carcinoma cells to study this regulatory paradigm. Ectopic overexpression of HuR potently enhanced the translation and cytoplasmic levels of endogenous HuR, but did not affect HuR mRNA levels. Inhibition of CRM1 function by Lemptomycin B or by knockdown of CRM1 greatly diminished the cytoplasmic levels of endogenous HuR mRNA and hence blocked the induction of endogenous HuR by exogenous HuR. Further studies showed that HuR interacted with the 3′-untranslated region (UTR) of HuR and that overexpression of HuR increased the cytoplasmic levels of a chimeric luciferase-HuR 3′-UTR reporter transcript, as well as luciferase activity; conversely, HuR knockdown reduced both parameters. Moreover, the loss of HuR in senescent, late-passage HDFs was accompanied by a reduced cytoplasmic presence of endogenous HuR mRNA, ectopic Luc-HuR-3′UTR reporter transcript, and luciferase activity relative to what was observed in young, early-passage cells. Our results reveal a positive feedback mechanism for the regulation of HuR, which may play an important role in the regulation of HuR during replicative senescence.

Cell aging of human diploid fibroblasts is associated with changes in responsiveness to epidermal growth factor and changes in HER-2 expression

The limited replicative life span of diploid human cells in vitro (cellular senescence) serves as a cellular model of aging. We examined the proliferative response of 2BS cells of different population doubling levels to epidermal growth factor (EGF). DNA synthesis was measured by thymidine incorporation. As the cells aged, there was a significant decrease both in the baseline level of DNA synthesis and in the stimulation of DNA synthesis by EGF addition. The effective concentration of EGF and the latent period prior to DNA synthesis did not change. EGF receptor mRNA expression also remained unchanged as the cells aged, in the absence or presence of EGF, suggesting that the defect in old cells lies downstream in the EGF signaling pathway. As the cells reached 100% of their life span, however, there was a 70% decrease in EGF receptor mRNA. Expression of the EGF receptor homologue HER-2 was also examined. The HER-2 mRNA level was significantly reduced in old cells. Moreover, HER-2 expression was stimulated by EGF addition in young cells but not in old cells. The results suggest that cell aging is associated with a progressive loss in the ability of cells to respond to growth factors.

PPARγ accelerates cellular senescence by inducing p16INK4α expression in human diploid fibroblasts

Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in the inhibition of cell growth by promoting cell-cycle arrest, and PPARγ activation induces the expression of p16INK4α (CDKN2A), an important cell-cycle inhibitor that can induce senescence. However, the role of PPARγ in cellular senescence is unknown. Here, we show that PPARγ promotes cellular senescence by inducing p16INK4α expression. We found several indications that PPARγ accelerates cellular senescence, including enhanced senescence-associated (SA)-β-galactosidase staining, increased G1 arrest and delayed cell growth in human fibroblasts. Western blotting studies demonstrated that PPARγ activation can upregulate the expression of p16INK4α. PPARγ can bind to the p16 promoter and induce its transcription, and, after treatment with a selective PPARγ agonist, we observed more-robust expression of p16INK4α in senescent cells than in young cells. In addition, our data indicate that phosphorylation of PPARγ decreased with increased cell passage. Our results provide a possible molecular mechanism underlying the regulation of cellular senescence.

Loss of Repression of HuR Translation by miR-16 May Be Responsible for the Elevation of HuR in Human Breast Carcinoma

Elevated levels of RNA binding protein HuR were found in various human cancers. However, the mechanisms underlying HuR over‐expression in cancers have not been fully elucidated. Here, we show that miR‐16 acts as a novel post‐transcriptional regulator for HuR. Knockdown of miR‐16 increased HuR protein levels in MDA‐MB‐231 cells, while over‐expression of pre‐miR16 reduced HuR expression. Neither knockdown nor over‐expression of miR‐16 could alter the mRNA levels of HuR. Instead, knockdown of miR‐16 increased the level of de novo synthesized HuR protein. Importantly, mechanistic studies showed that miR‐16 associated with the 3′UTR of HuR, and knockdown of miR‐16 markedly increased the luciferase activity of a HuR 3′UTR‐containing reporter. We further demonstrate that the level of miR‐16 was inversely correlated with HuR protein level in human breast carcinoma. Together, our results suggest an important role of miR‐16 in regulating HuR translation and link this regulatory pathway to human breast cancer. J. Cell. Biochem. 111: 727–734, 2010.

Sp1 Is Essential for p16INK4a Expression in Human Diploid Fibroblasts during Senescence

Background p16 INK4a tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16 INK4a gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from −466 to −451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts. Methodology/Principal Findings Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16 INK4a gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16 INK4a. Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16 INK4a gene expression. In addition, the enhanced binding of Sp1 to p16 INK4a promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level. Conclusions/Significance All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16 INK4a expression during cell aging.