Lixiang Xue

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.

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.

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.

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.

Loss of CARM1 is linked to reduced HuR function in replicative senescence

BackgroundThe co-activator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of HuR. However, the functional impact of this modification is not fully understood. Here, we investigated the influence of HuR methylation by CARM1 upon the turnover of HuR target mRNAs encoding senescence-regulatory proteins.ResultsChanging the methylation status of HuR in HeLa cells by either silencing CARM1 or mutating the major methylation site (R217K) greatly diminished the effect of HuR in regulating the turnover of mRNAs encoding cyclin A, cyclin B1, c-fos, SIRT1, and p16. Although knockdown of CARM1 or HuR individually influenced the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16, joint knockdown of both CARM1 and HuR did not show further effect. Methylation by CARM1 enhanced the association of HuR with the 3′UTR of p16 mRNA, but not with the 3′UTR of cyclin A, cyclin B1, c-fos, or SIRT1 mRNAs. In senescent human diploid fibroblasts (HDFs), reduced CARM1 was accompanied by reduced HuR methylation. In addition, knockdown of CARM1 or mutation of the major methylation site of HuR in HDF markedly impaired the ability of HuR to regulate the expression of cyclin A, cyclin B1, c-fos, SIRT1, and p16 as well to maintain a proliferative phenotype.ConclusionCARM1 represses replicative senescence by methylating HuR and thereby enhancing HuR’s ability to regulate the turnover of cyclin A, cyclin B1, c-fos, SIRT1, and p16 mRNAs.

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.

The tumor suppressor p33ING1b upregulates p16INK4a expression and induces cellular senescence.

ING1 protein is a tumor suppressor which plays significant roles in multiple cellular activities. p47ING1a and p33ING1b are major splice isoforms of ING1 and their roles in senescence need further investigations. Here we studied the functions of ING1 isoforms in cellular senescence and gene regulation, with focus on p16INK4a. We observe that p33ING1b protein is the major ING1 isoform expressed in 2BS human diploid fibroblasts. Overexpression of p33ING1b induces cellular senescence and upregulates p16INK4a expression in 2BS fibroblasts. p33ING1b upregulates p16INK4a transcription. p33ING1b and p300 bind to the p16INK4a promoter. p300/CBP‐specific inhibitor curcumin can reverse the induction of p16INK4a by p33ING1b. These results help to better understand the function of ING1.

The Retinoblastoma Protein Selectively Represses E2F1 Targets via a TAAC DNA Element during Cellular Senescence

Background: Expression patterns of E2F1 target genes differ during cellular senescence. Results: Rb protein selectively represses specific E2F1 target genes via a TAAC element in senescent cells. Conclusion: Cellular senescence is influenced by selective repression of E2F1 target transcription by Rb. Significance: An understanding of how E2F1 target genes that participate in proliferation are regulated is crucial for elucidating the mechanisms of cellular senescence. The retinoblastoma (Rb) protein mediates heterochromatin formation at the promoters of E2 transcription factor 1 (E2F1) target genes, such as proliferating cell nuclear antigen and cyclin A2 (CCNA2), and represses these genes during cellular senescence. However, the selectivity of Rb recruitment is still not well understood. Here, we demonstrate that a senescence-associated gene is a direct target of E2F1 and is also repressed by heterochromatin in senescent cells. In contrast, ARF and p27KIP1, which are also E2F1 targets, are not repressed by Rb and heterochromatin formation. By comparing the promoter sequences of these genes, we found a novel TAAC element that is present in the cellular senescence-inhibited gene, proliferating cell nuclear antigen, and CCNA2 promoters but absent from the ARF and p27KIP1 promoters. This TAAC element associates with Rb and is required for Rb recruitment. We further determined that TAAC element-mediated Rb association requires the E2F1 binding site, but not E2F1 protein. These results provide a novel molecular mechanism for the different expression patterns of E2F1 targets and afford new mechanistic insight regarding the selectivity of Rb-mediated heterochromatin formation and gene repression during cellular senescence.

Senescence as a novel mechanism involved in β-adrenergic receptor mediated cardiac hypertrophy

Pathological cardiac hypertrophy used to be elucidated by biomechanical, stretch-sensitive or neurohumoral mechanisms. However, a series of hints have indicated that hypertrophy process simulates senescence program. However, further evidence need to be pursued. To verify this hypothesis and examine whether cardiac senescence is a novel mechanism of hypertrophy induced by isoproterenol, 2-month-old male Sprague Dawley rats were subjected to isoproterenol infusion (0.25mg/kg/day) for 7 days by subcutaneous injection). Key characteristics of senescence (senescence-associated β-galactosidase activity, lipofuscin, expression of cyclin-dependent kinase inhibitors) were examined in cardiac hypertrophy model. Senescence-like phenotype, such as increased senescence-associated β-galactosidase activity, accumulation of lipofuscin and high levels of cyclin-dependent kinase inhibitors (e.g. p16, p19, p21 and p53) was found along the process of cardiac hypertrophy. Cardiac-specific transcription factor GATA4 increased in isoproterenol-treated cardiomyocytes as well. We further found that myocardial hypertrophy could be inhibited by resveratrol, an anti-aging compound, in a dose-dependent manner. Our results showed for the first time that cardiac senescence is involved in the process of pathological cardiac hypertrophy induced by isoproterenol.