SIRT1 ameliorates age-related senescence of mesenchymal stem cells via modulating telomere shelterin

Abstract

Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD(+)-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.

Keywords: SIRT1; TPP1; aging; mesenchymal stem cells; senescence; shelterin; telomerase.

Figure 1

Characterization of young and aged MSCs. (A) Phenotypic characterization of young and aged MSCs by flow cytometry. Representative results showed that both young and aged MSCs were both positive for CD29, CD44, CD73, CD90, and CD105 and negative for CD31, CD34, and CD45. β-galactosidase (SA-β-gal) staining was performed for young (B) and aged (C) MSCs, with percentage of SA-β-gal positive cells shown in bar graph in (D). Cell proliferation capability was also determined by Cell Counting Kit-8 (CCK-8) method and time-course growth was plotted for both young and aged MSCs (E). SIRT1 mRNA expression level was quantified by reverse-transcription quantitative-PCR for young and aged MSCs with GAPDH used as a control (F). Western blot was performed with a representative protein bands shown in (G) and quantified in bar graph (H). SIRT1 activity was also measured and expressed as fold changes shown in bar graph (I). All the data are expressed as mean ± SD (three independent experiments, n = 5 rats/group). ^*implies P < 0.05, compared to young MSCs.

Figure 2

SIRT1 mitigates age-related senescence of MSCs. SIRT1 protein levels in genetically modified young and aged MSCs were evaluated by Western Blot. Representative protein bands were shown in (A) and quantified in bar graph (B,C). Representative images of SA-β-gal staining for both young MSCs that were transfected with either mock- or shSIRT1-vectors (shown in D(i) and D(ii), respectively) and aged MSC transfected with either empty vector or SIRT1 overexpressing (shown in E(i) and E(ii), respectively), with quantification shown in bar graph D(iii) and E(iii), respectively). Representative time-course growth of young (mock- and sh-SIRT1) (F) and aged (vector- and SIRT1-) (G) MSCs were also shown. Data are expressed as mean ± SD (three independent experiments, n = 5 rats/group). ^*Implies P < 0.05, compared to mock-young MSCs. #, P < 0.05, compared to vector-aged MSCs.

Figure 3

SIRT1 positively regulates P16 and P21 expression in MSCs. SIRT1 knockdown in young MSCs was associated with increases in P16 and P21 expression, with representative protein bands shown in (A) and quantified in a bar graph shown in (B). In contrast, SIRT1 overexpression in aged MSCs decreases P16 and P21 expressions. Representative western blot images (C) and the quantitative analysis results (D) were shown. Data are expressed as mean ± SD (three independent experiments, n = 5 rats/group). ^*Implies P < 0.05, compared to mock-young MSCs. #, P < 0.05, compared to vector-aged MSCs.

Figure 4

SIRT1 protects telomere DNA damage in MSCs. Representative immunofluorescence images of young (mock- and shSIRT1-) and aged MSCs (vector- and SIRT1) stained with γ-H2AX (Green), TRF2 (Red), and DAPI (Blue) (A). Quantitative analysis of γ-H2AX foci in young (mock- and shSIRT1-) (B) and aged MSCs (vector- and SIRT1-) (C) were shown. A cell was considered TIF positive if ≥50% of its γ-H2AX foci were associated with TRF2 (as shown in merged image). Quantitative analysis of TIF positive cells in young (mock- and shSIRT1-) (D) and aged MSCs (vector- and SIRT1-) (E) were shown. Data are expressed as mean ± SD (three independent experiments, n = 5 rats/group). ^*Implies P < 0.05, compared to mock-young MSCs. #, P < 0.05, compared to vector-aged MSCs.

Figure 5

SIRT1 modulates telomerase activity, but not telomere length in MSCs. Quantitative analysis of age-related alterations of telomerase activity (A) and telomere length (B) in MSCs was shown. Quantitative analysis of telomerase activity in young (mock- and shSIRT1-) (C) and aged (vector- and SIRT1-) MSCs (D) was also shown. Quantitative analysis of telomere length in young (mock- and shSIRT1-) (E) and aged (vector- and SIRT1-) (F) MSCs was shown. No siginificant change in telomere length was observed when SIRT1 was manipulated in young or aged MSCs. Data are expressed as mean ± SD (three independent experiments, n = 5 rats/group). ^*Signifies P < 0.05, compared to young MSCs. #, P < 0.05, compared to mock-young MSCs. ^, P < 0.05, compared to vector-aged MSCs.

Figure 6

SIRT1 positively modulates TPP1 expression in MSCs. (A) Age-related changes in mRNA levels of each telomere shelterin components including TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 in MSCs was quantified. The mRNA expression levels were quantified when SIRT1 was manipulated in young (mock- and shSIRT1-) (B) and aged (vector- and SIRT1-) MSCs (C). Representative immunoblot images (D) and the quantitative analysis results (E,F) demonstrated changes in TPP1 protein level in young (mock- and shSIRT1-) and aged (vector- and SIRT1-) MSCs. Data are expressed as mean ± SD (three independent experiments, n = 5 rats/group). ^*Implies P < 0.05, compared to young MSCs. #, P < 0.05, compared to mock-young MSCs. ^, P < 0.05, compared to vector-aged MSCs.