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. 2021 Oct 26;10(11):2892.
doi: 10.3390/cells10112892.

Pulmonary Alveolar Stem Cell Senescence, Apoptosis, and Differentiation by p53-Dependent and -Independent Mechanisms in Telomerase-Deficient Mice

Kexiong Zhang  1 , Lihui Wang  1 , Xiaojing Hong  1 , Hao Chen  1 , Yao Shi  1 , Yingying Liu  1 , Jun Liu  1 , Jun-Ping Liu  1   2   3
Affiliations

Pulmonary Alveolar Stem Cell Senescence, Apoptosis, and Differentiation by p53-Dependent and -Independent Mechanisms in Telomerase-Deficient Mice

Kexiong Zhang et al. Cells. .

Abstract

Pulmonary premature ageing and fibrogenesis as in idiopathic pulmonary fibrosis (IPF) occur with the DNA damage response in lungs deficient of telomerase. The molecular mechanism mediating pulmonary alveolar cell fates remains to be investigated. The present study shows that naturally occurring ageing is associated with the DNA damage response (DDR) and activation of the p53 signalling pathway. Telomerase deficiency induced by telomerase RNA component (TERC) knockout (KO) accelerates not only replicative senescence but also altered differentiation and apoptosis of the pulmonary alveolar stem cells (AEC2) in association with increased innate immune natural killer (NK) cells in TERC KO mice. TERC KO results in increased senescence-associated heterochromatin foci (SAHF) marker HP1γ, p21, p16, and apoptosis-associated cleaved caspase-3 in AEC2. However, additional deficiency of the tumour suppressor p53 in the Trp53-/- allele of the late generation of TERC KO mice attenuates the increased senescent and apoptotic markers significantly. Moreover, p53 deficiency has no significant effect on the increased gene expression of T1α (a marker of terminal differentiated AEC1) in AEC2 of the late generation of TERC KO mice. These findings demonstrate that, in natural ageing or premature ageing accelerated by telomere shortening, pulmonary senescence and IPF develop with alveolar stem cell p53-dependent premature replicative senescence, apoptosis, and p53-independent differentiation, resulting in pulmonary senescence-associated low-grade inflammation (SALI). Our studies indicate a natural ageing-associated molecular mechanism of telomerase deficiency-induced telomere DDR and SALI in pulmonary ageing and IPF.

Keywords: apoptosis; differentiation; lung alveolar type 2 cells; p53; senescence; telomerase RNA component; telomere shortening.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1
Age-related pulmonary fibrosis in mice with compromised lung respiratory function and activation of the p53 pathway. (A) Masson’s staining of mouse pulmonary sections of different genders and ages. Arrows indicate pulmonary fibrosis. (B) Quantitation of the pulmonary fibrosis by ImageJ. Three mice per group. (C) RNA was extracted from 2- and 18-month-old lung tissues, and the gene expression of p21, p53, HP1γ, collagen (Col1α), Vimentin (Vim), and α-SMA was determined by real-time PCR and graphed as fold-activation value based on three mice in each group. (D) The mouse respiratory functional analysis of different ages. The 1/2 expiration flux, tidal volume, and minute volume were determined by the eSpira Forced Manoeuvers System. Four mice per group. * and ** indicate statistical significance p < 0.05 and p < 0.01 respectively.
Figure 2
Figure 2
Significant increases in SA-β-gal staining, expression of cell cycle inhibitor p21, lysosomal β-galactosidase-associated GLB1, and natural killer cell marker NK1.1 in G3Terc−/− mouse lungs. (A) Representative SA-β-gal-stained lungs of Terc+/+ and G3Terc−/− (magnification: 20×). Scale bar = 100 µm. (B) Quantification of SA-β-gal-stained cells by cell counting (as in A). Data are mean ± SE values based on the indicated number of lungs. (C) qPCR analysis of the indicated genes with RNA isolated from the indicated number of lungs by determining the relative mRNA level (ratio to β-actin). (D) Western blotting analysis with proteins from the lung tissues. (E) Representative NK1.1-stained lungs of Terc+/+, G3Terc−/− (magnification: 40× and 80×). Scale bar = 50 µm. (F) The analysis of lung weight change by calculating the ratio to body weight based on the indicated number of mice. Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
Significant increases in SA-β-gal staining, expression of cell cycle inhibitor p21, lysosomal β-galactosidase-associated GLB1, and natural killer cell marker NK1.1 in G3Terc−/− mouse lungs. (A) Representative SA-β-gal-stained lungs of Terc+/+ and G3Terc−/− (magnification: 20×). Scale bar = 100 µm. (B) Quantification of SA-β-gal-stained cells by cell counting (as in A). Data are mean ± SE values based on the indicated number of lungs. (C) qPCR analysis of the indicated genes with RNA isolated from the indicated number of lungs by determining the relative mRNA level (ratio to β-actin). (D) Western blotting analysis with proteins from the lung tissues. (E) Representative NK1.1-stained lungs of Terc+/+, G3Terc−/− (magnification: 40× and 80×). Scale bar = 50 µm. (F) The analysis of lung weight change by calculating the ratio to body weight based on the indicated number of mice. Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
Marked increases in p21, p16, and SAHF-associated Hp1γ staining rates in AEC2 cells from late-generation Terc−/− lungs, and p53 deletion significantly reversed increased Hp1γ expression. (A) Representative double immunofluorescence staining of Terc+/+, G3Terc−/−, G2Terc−/−, and G2Terc−/−p53−/− mouse lungs (magnification: 40×) for SPC along with p21, p16, or Hp1γ. Scale bar = 30 µm. (B) Quantification of p21-, p16-, and Hp1γ-stained AEC2 cells by cell counting (as in A). Data are mean ± SE values based on the indicated number of lungs. Statistical significance is indicated by ** p < 0.01, *** p < 0.001.
Figure 4
Figure 4
Marked increases in apoptosis-related cleaved caspase-3 staining rates in the AEC2 cells from late-generation Terc−/− mouse lungs and rescued in p53−/−. (A). Representative double immunofluorescence staining of WT, G3Terc−/−, G2Terc−/−, and G2Terc−/−p53−/− mouse lungs (magnification: 40×) for SPC and cleaved caspase-3. Scale bar = 30 µm. (B). Quantification of cleaved caspase-3 stained AEC2 cells by cell counting (as in (A)). Data are mean ± SE values based on the indicated number of lungs. (C). Marked increases in apoptosis with cleaved caspase-3 in the late-generation Terc−/− mouse lungs by Western blotting analysis. (D). Increases in apoptosis-related Bax mRNA levels in the late-generation Terc−/− mouse lungs by RT-qPCR analysis. Data are mean ± SE values based on the indicated number of lungs. (E). Significant decrease in the AEC2 cell number by cell counting in the slides from late-generation Terc−/− mouse lungs, and rescued in p53−/−. Data are mean ± SE values based on the indicated number of lungs. (F). Representative flow cytometry result showing significant decreases in AEC2 cell number (gated by CD45−EpCAM+) in late-generation Terc−/− mouse lungs. Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
Significant elevation of AEC1 cell marker T1α in both late-generation mouse lungs and isolated AEC2 cells, which was not rescued by p53−/−. (A) qPCR analysis of T1α mRNA levels from the indicated number of lungs. Data are expressed as mean ± SE values. (B) qPCR analysis of T1α mRNA levels with AEC2 cell RNAs isolated from lungs by FACS sorting. Data are expressed as mean ± SE values based on the indicated animal numbers used to isolate lung AEC2 cells. (C) Representative T1α immunofluorescence staining of WT, G3Terc−/−, G2Terc−/−, and G2Terc−/−p53−/− mouse lungs (magnification: 40×). Scale bar = 30 µm. (D) Quantification of T1α stained rate by cell counting (as in (A)). Data are mean ± SE values based on the indicated number of lungs. Statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001.
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