Mitochondrion-processed TERC regulates senescence without affecting telomerase activities

Mitochondrial dysfunctions play major roles in ageing. How mitochondrial stresses invoke downstream responses and how specificity of the signaling is achieved, however, remains unclear. We have previously discovered that the RNA component of Telomerase TERC is imported into mitochondria, processed to a shorter form TERC-53, and then exported back to the cytosol. Cytosolic TERC-53 levels respond to mitochondrial functions, but have no direct effect on these functions, suggesting that cytosolic TERC-53 functions downstream of mitochondria as a signal of mitochondrial functions. Here, we show that cytosolic TERC-53 plays a regulatory role on cellular senescence and is involved in cognition decline in 10 months old mice, independent of its telomerase function. Manipulation of cytosolic TERC-53 levels affects cellular senescence and cognition decline in 10 months old mouse hippocampi without affecting telomerase activity, and most importantly, affects cellular senescence in terc cells. These findings uncover a senescence-related regulatory pathway with a non-coding RNA as the signal in mammals.

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

17β-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.

Molecular Mechanism Study on Different Isoforms of ING1 Family Inhibiting HeLa Cells Proliferation / 生物化学与生物物理进展

ING1 family is a candidate for tumor suppressor,which has three splicing isoforms named p47ING1a,p33ING1b,and p24ING1c. Study of the effect of different isoforms of ING1 on HeLa cells proliferation and its molecular mechanism would help further identifying the functional relationship of ING1 isoforms,and finding important genes regulated by ING1. Cell growth curve and cell cycle analysis were used to observe the effect of ING1a,ING1b,and ING1c on HeLa cells growth,and the result indicated that they could all inhibit HeLa cells growth by arresting cell cycle at G0/G1 phase. PCR method was used to construct the PHD domain deletions of ING1a and ING1b. ING1a,ING1b,ING1c and the PHD domain deletions 1a?C and 1b?C were then overexpressed in HeLa cells. p16INK4a,PTEN/p27Kip1 and p53/p21Waf1 protein levels were detected by Western blot. The result showed that ING1a,ING1b,ING1c,and 1a?C except for 1b?C induced p16INK4a protein expression,in which ING1c had the most powerful effect. Luciferase assay identified that overexpression of pcDNA3.1(+)-1a?C facilitated p16INK4a transcription through enhancing p16INK4a promoter activity,while pcDNA3.1(+)-1b?C repressed the p16INK4a promoter activity . In a word,it was found for the first time that except for the p53/p21Waf1 pathway,three splicing isoforms of ING1 family could also inhibit HeLa cells proliferation though upregulation of p16INK4a and PTEN,and the PHD domain deletion of ING1a enhanced p16INK4a transcription. These findings provide new clews to further study on the mechanisms of ING1 family suppressing cancer cells growth.

Phosphatidylinositol 3-kinase inhibitor, LY294002, induced senescence-like changes in human diploid fibroblasts / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.</p><p><b>METHODS</b>Using a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells.</p><p><b>RESULTS</b>Both 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines.</p><p><b>CONCLUSIONS</b>PI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.</p>

Functions of ?-2-macroglobulin in human diploid fibroblasts during aging / 中华老年医学杂志

Objective To investigate the effects of ?-2-macroglobulin on the aging process of human diploid fibroblasts. Methods pIRES-A2M sense and antisense vectors were constructed and transferred into 2BS cells mediated by lipofectamine.2BS/A2Ms and 2BS/A2Ma cell lines were verified by Southern and Northern blot analysis respectively.Cell growth curve,the population doublings,cell cycle analysis,staining of senescent-associated-?-galactosidase and expression of p16 and p21 in transfected cells were measured. Results Southern and Northern blot analysis verified that the exogenous cDNAs were integrated into genomic DNA in the transfected cells.The ultmost population doublings of 2BS/A2Ms cells were slightly higher than normal 2BS cells.Cell growth curve,cell cycle analysis,staining of senescent-associated-?-galactosidase and the population doublings all revealed that 2BS/A2Ms cells demonstrated obvious difference compared with 2BS/A2Ma cells((P0.05). Conclusions The aging process of 2BS cells is influenced slightly by expression of A2M.

The role of p16 methylation in the aging of human fetal lung diploid fibroblasts / 中华老年医学杂志

Objective　The relationship between DNA methylation and the overexpression of cell cycle negative regulator p16MTS1/INK4a in senescent cells was studied. 　Methods　PCR amplification of p16 exon I following digestion with Sma I , a methylation sensitive DNA endonuclease, was adapted to determine the methylation status at specific site. 　Results　 T-he increased expression of p16 in the aging process of human fetal lung diploid fibroblasts (2BS) was observed. In middle-aged and old cells, the p16 level was about 3 folds and 10 folds respectively as that in young cells. The methylation level of the Sma I site in p16 exon I tended to decline with aging, being about 64% and 41% in young and middle-aged cells respectively, but still maintain relatively as high as about 24% in senescent cells. 　Conclusions　 The overexpression of p16 in senescent human fibroblasts might be related to the alteration of methylation level of exon I, its mechanisms need to be defined further.

Identifying cellular senescence-associated genes by suppression subtractive hybridization / 中华老年医学杂志

Objective The differentially expressed genes in the progress of cellular senescence were studied. Methods Suppression subtractive hybridization was used to young and senescent human diploid fibroblast 2BS cells, then screening and analyzing the subtractive libraries. Results Two kinds of subtraction libraries were obtained, respectively representing the differential genes with higher expression in young cells or senescent cells. Screened by dot blot, thirty of genes with differential ratio larger than 2 were sequenced, showing changes in much of cell functions. Some of them also had expression change between blood samples from newborn or old people. Conclusions The differential expression of RBM4?FBXO7?TOM1 is reported during 2BS cell senescence for the first time. There are the similarities during the senescence of culture cell and organism.

Cellular response to DNA damage caused by methyl methanesulfonate (MMS) in human senescent diploid fibroblasts / 中华老年医学杂志

55 population doubling, PD) were compared with those of the young cells(

Cell cycle negative regulator p16 induces senescence-like alterations in human fibroblasts (2BS) / 中华老年医学杂志

Objective　To investigate the involvement of cell cycle negative regulator p16 in the replicative senescence of normal human diploid fibroblasts. 　Methods　 p16 cDNA and retroviral vector was introduced into normal human fibroblast 2BS cells by transfection technique. Then the effects of p16 on replicative cellular senescence of 2BS cells were examined. 　Results　 Compared with the control cells, 2BS cells transfected with p16 cDNA showed significant suppression of growth rate (decrease 50%) with cell cycle arrested at G1 phase. This phenomenon similar to that of the senescent cells, with the cellular response to stimulation by growth factor decreased 79.4%, showing the characteristics in morphology of senescent fibroblasts. 　Conclusions　 The over expression of p16 gene contributes to the process of cellular senescence of normal human diploid fibroblasts.

Chinese herbs improve transcription of the gene for albumin in nephrotic syndrome rats / 中华肾脏病杂志

; 3. therapy with A&A (A&A); 4. Astragali alone (A); 5. Astragali polysac-charide Ⅰ (APⅠ); 6. AP Ⅱ ; 7. Astragali glucoside (AG), Results The level of serum albumin,albumin mRNA and albumin gene transcription were measured by biochemistry, Northern blot hybridization, nuclear run-on assay and quantity in laser density screening. The level of serum albumin in N was significantly lower than C. The serum albumin concentration in each therapy group was higher than N group. The transcription of the albumin gene was higher in N than in C and highest in A&A. The alterations of northern blot hybridization were same as the transcription results. But both the level of albumin mRNA and the transcription of albumin gene in A, AP I , AP I and AG were no change compared with N. Conclusion A&A increases albumin mRNA expression at least in part by improving the rate of gene transcription, which participate the protection of the decrease of serum albumin in NS. But the effect of A, AP 1 , AP I and AG may mediated by other unknown mechanisms.

The effect of EGF on the expresstions of EGFR and HER-2 genes in human senescent cells / 中华老年医学杂志

Abstract The 3H-thymidine (3H-TdR) incorporation rate of diploid human embryo lung fibroblast cell (2BS) in old cells was significantly lower than that in middle age cells and young cells. EPidermal growth factor (EGF) stimulated tha 3H-TdR incorporation rates of all three kinds of cells with different age. The results of Northern and dot hybridization showed that the level of HER-2 in old cells was lower than those in young cells. EGF could induce expression of HER-2 gene in young cells but not in old cells. As the cells reached 100% of their life span, the EGFR mRNA decreased obviously.