Zongyu Zhang

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.

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition.

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.

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.

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.

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.

CSIG Inhibits PTEN Translation in Replicative Senescence

ABSTRACT Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) that was abundant in young human diploid fibroblast cells but declined upon replicative senescence. Overexpression or knockdown of CSIG did not influence p21Cip1 and p16INK4a expressions. Instead, CSIG negatively regulated PTEN and p27Kip1 expressions, in turn promoting cell proliferation. In PTEN-silenced HEK 293 cells and PTEN-deficient human glioblastoma U87MG cells, the effect of CSIG on p27Kip1 expression and cell division was abolished, suggesting that PTEN was required for the role of CSIG on p27Kip1 regulation and cell cycle progression. Investigation into the underlying mechanism revealed that the regulation of PTEN by CSIG was achieved through a translational suppression mechanism. Further study showed that CSIG interacted with PTEN mRNA in the 5′ untranslated region (UTR) and that knockdown of CSIG led to increased luciferase activity of a PTEN 5′ UTR-luciferase reporter. Moreover, overexpression of CSIG significantly delayed the progression of replicative senescence, while knockdown of CSIG expression accelerated replicative senescence. Knockdown of PTEN diminished the effect of CSIG on cellular senescence. Our findings indicate that CSIG acts as a novel regulatory component of replicative senescence, which requires PTEN as a mediator and involves in a translational regulatory mechanism.

Sp1 is involved in the transcriptional activation of p16INK4 by p21Waf1 in HeLa cells

Both p16INK4 and p21Waf1 are very important negative regulators of the cell cycle. In this study we examined the effects of p21Waf1 on the transcription of p16INK4. We determined that p21Waf1 can activate the transcription of p16INK4, and that this effect is GC‐box dependent. We also found that the transcription factor Sp1 plays a key role in this event. Upregulation of Sp1 contributes to the transcriptional activation and protein level of p16INK4 mediated by p21Waf1, and is a potential point of cooperation between the p16/pRb and p14 (ARF)/p53 tumor suppressor pathways.

B-MYB delays cell aging by repressing p16 (INK4α) transcription.

Abstractp16INK4α, an inhibitor of cyclin-dependent kinase 4 and 6, has been proposed to play an important role in cellular aging and in premature senescence. The expression of the p16INK4α is primarily under transcriptional control. Our previous data showed that a negative regulation element lies in its promoter. In that element, a MYB-binding site (MBS) was uncovered by transcription analysis. Here, we report that MBS is a negative regulation element and B-MYB binds to this site in vivo. In human embryonic lung fibroblast cells, B-MYB downregulated p16INK4α expression, whereas knocking down of B-MYB upregulated it. Evidence also showed that overexpression of B-MYB in cells could increase the number of utmost passage and decrease G1 block, whereas knocking down of B-MYB could impair their replicative ability. This study provides evidence of the capacity of B-MYB not only to regulate p16INK4α expression but also the phenotypic consequence on cellular senescence.