Limin Han

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.

FOXA1 mediates p16 INK4a activation during cellular senescence

Mechanisms governing the transcription of p16INK4a, one of the master regulators of cellular senescence, have been extensively studied. However, little is known about chromatin dynamics taking place at its promoter and distal enhancer. Here, we report that Forkhead box A1 protein (FOXA1) is significantly upregulated in both replicative and oncogene‐induced senescence, and in turn activates transcription of p16INK4a through multiple mechanisms. In addition to acting as a classic sequence‐specific transcriptional activator, FOXA1 binding leads to a decrease in nucleosome density at the p16INK4a promoter in senescent fibroblasts. Moreover, FOXA1, itself a direct target of Polycomb‐mediated repression, antagonizes Polycomb function at the p16INK4a locus. Finally, a systematic survey of putative FOXA1 binding sites in the p16INK4a genomic region revealed an ∼150 kb distal element that could loop back to the promoter and potentiate p16INK4a expression. Overall, our findings establish several mechanisms by which FOXA1 controls p16INK4a expression during cellular senescence.

Senescence delay and repression of p16INK4a by Lsh via recruitment of histone deacetylases in human diploid fibroblasts.

Lymphoid specific helicase (Lsh) belongs to the family of SNF2/helicases. Disruption of Lsh leads to developmental growth retardation and premature aging in mice. However, the specific effect of Lsh on human cellular senescence remains unknown. Herein, we report that Lsh overexpression delays cell senescence by silencing p16INK4a in human fibroblasts. The patterns of p16INK4a and Lsh expression during cell senescence present the inverse correlation. We also find that Lsh requires histone deacetylase (HDAC) activity to repress p16INK4a and treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Lsh. Moreover, overexpression of Lsh is correlated with deacetylation of histone H3 at the p16 promoter, and TSA treatment in Lsh-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate an interaction between Lsh, histone deacetylase 1 (HDAC1) and HDAC2 in vivo. Furthermore, we demonstrate that Lsh interacts in vivo with the p16 promoter and recruits HDAC1. Our data suggest that Lsh represses endogenous p16INK4a expression by recruiting HDAC to establish a repressive chromatin structure at the p16INK4a promoter, which in turn delays cell senescence.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) delays cellular senescence by promoting p27(Kip1) degradation in human diploid fibroblasts.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27Kip1 and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27Kip1 expression through ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyubiquitination of p27Kip1 in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27Kip1 by promoting p27Kip1 degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.

Protein kinase D1 is essential for Ras-induced senescence and tumor suppression by regulating senescence-associated inflammation

Significance Oncogene-induced senescence (OIS) is an initial barrier for cancer development. Reactive oxygen species (ROS) play critical roles in oncogenic Ras OIS. Senescent cells develop a senescence-associated secretory phenotype (SASP), which has important role in tumor suppression and tissue repair. However, the mechanisms underlying the SASP regulation are not clear. In this paper, we show that ROS-protein kinase Cδ (PKCδ)-protein kinase D1 (PKD1) axis is essential for SASP induction and maintenance via modulation of NF-κB activity. Considering the pivotal role of both SASP and ROS in systemic aging and age-related diseases, the link between ROS-PKCδ-PKD1 pathway and SASP regulation elucidated here may provide a new target to intervene in age-related inflammation and diseases. Oncogene-induced senescence (OIS) is an initial barrier to tumor development. Reactive oxygen species (ROS) is critical for oncogenic Ras OIS, but the downstream effectors to mediate ROS signaling are still relatively elusive. Senescent cells develop a senescence-associated secretory phenotype (SASP). However, the mechanisms underlying the regulation of the SASP are largely unknown. Here, we identify protein kinase D1 (PKD1) as a downstream effector of ROS signaling to mediate Ras OIS and SASP. PKD1 is activated by oncogenic Ras expression and PKD1 promotes Ras OIS by mediating inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) via modulation of NF-κB activity. We demonstrate that ROS-protein kinase Cδ (PKCδ)-PKD1 axis is essential for the establishment and maintenance of IL-6/IL8 induction. In addition, ablation of PKD1 causes the bypass of Ras OIS, and promotes cell transformation and tumorigenesis. Together, these findings uncover a previously unidentified role of ROS-PKCδ-PKD1 pathway in Ras OIS and SASP regulation.

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.

Upregulation of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation of PPAR-γ mediated by NEDD4-1

Abstract17β-estradiol (E2) treatment of cells results in an upregulation of SIRT1 and a down-regulation of PPARγ. The decrease in PPARγ expression is mediated by increased degradation of PPARγ. Here we report that PPARγ is ubiquitinated by HECT E3 ubiquitin ligase NEDD4-1 and degraded, along with PPARγ, in response to E2 stimulation. The PPARγ interacts with ubiquitin ligase NEDD4-1 through a conserved PPXY-WW binding motif. The WW3 domain in NEDD4-1 is critical for binding to PPARΓ. NEDD4-1 overexpression leads to PPARγ ubiquitination and reduced expression of PPARγ. Conversely, knockdown of NEDD4-1 by specific siRNAs abolishes PPARΓ ubiquitination. These data indicate that NEDD4-1 is the E3 ubiquitin ligase responsible for PPARγ ubiquitination. Here, we show that NEDD4-1 delays cellular senescence by degrading PPARΓ expression. Taken together, our data show that E2 could upregulate SIRT1 expression via promoting the PPARΓ ubiquitination-proteasome degradation pathway to delay the process of cell senescence.

HDAC4 stabilizes SIRT1 via sumoylation SIRT1 to delay cellular senescence.

The nicotinamide adenine dinucleotide‐dependent protein deacetylase silent information regulator 2 (Sir2) regulates cellular lifespan in several organisms. Histone deacetylase 4 (HDAC4) belongs to the class IIa group of HDACs; this class of HDACs is composed of proteins that are important regulators of gene expression that control pleiotropic cellular functions. However, the role of HDAC4 in cellular senescence is still unknown. This study shows that the expression patterns of HDAC4 and Sirtuin 1 (SIRT1; the mammalian homolog of Sir2) are positively correlated during cellular senescence. Moreover, the overexpression of HDAC4 delays senescence, whereas the knockdown of HDAC4 leads to premature senescence in human fibroblasts. Furthermore, it is demonstrated that HDAC4 increases endogenous SIRT1 expression by enhancing its sumoylation modification levels, thereby stabilizing its protein levels. This study, therefore, provides a new molecular mechanism for the regulation of cellular senescence.

hnRNP A1 antagonizes cellular senescence and senescence‐associated secretory phenotype via regulation of SIRT1 mRNA stability

Senescent cells display a senescence‐associated secretory phenotype (SASP) which contributes to tumor suppression, aging, and cancer. However, the underlying mechanisms for SASP regulation are not fully elucidated. SIRT1, a nicotinamide adenosine dinucleotide‐dependent deacetylase, plays multiple roles in metabolism, inflammatory response, and longevity, etc. However, its posttranscriptional regulation and its roles in cellular senescence and SASP regulation are still elusive. Here, we identify the RNA‐binding protein hnRNP A1 as a posttranscriptional regulator of SIRT1, as well as cell senescence and SASP regulator. hnRNP A1 directly interacts with the 3′ untranslated region of SIRT1 mRNA, promotes its stability, and increases SIRT1 expression. hnRNP A1 delays replicative cellular senescence and prevents from Ras OIS via upregulation of SIRT1 expression to deacetylate NF‐κB, thus blunting its transcriptional activity and subsequent IL‐6/IL‐8 induction. hnRNP A1 overexpression promotes cell transformation and tumorigenesis in a SIRT1‐dependent manner. Together, our findings unveil a novel posttranscriptional regulation of SIRT1 by hnRNP A1 and uncover a critical role of hnRNP A1‐SIRT1–NF‐κB pathway in regulating cellular senescence and SASP expression.