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. 2017 Nov 3;8(1):1312.
doi: 10.1038/s41467-017-01509-0.

Programmed Cell Senescence in Skeleton During Late Puberty

Affiliations
Free PMC article

Programmed Cell Senescence in Skeleton During Late Puberty

Changjun Li et al. Nat Commun. .
Free PMC article

Abstract

Mesenchymal stem/progenitor cells (MSPCs) undergo rapid self-renewal and differentiation, contributing to fast skeletal growth during childhood and puberty. It remains unclear whether these cells change their properties during late puberty to young adulthood, when bone growth and accrual decelerate. Here we show that MSPCs in primary spongiosa of long bone in mice at late puberty undergo normal programmed senescence, characterized by loss of nestin expression. MSPC senescence is epigenetically controlled by the polycomb histone methyltransferase enhancer of zeste homolog 2 (Ezh2) and its trimethylation of histone H3 on Lysine 27 (H3K27me3) mark. Ezh2 maintains the repression of key cell senescence inducer genes through H3K27me3, and deletion of Ezh2 in early pubertal mice results in premature cellular senescence, depleted MSPCs pool, and impaired osteogenesis as well as osteoporosis in later life. Our data reveals a programmed cell fate change in postnatal skeleton and unravels a regulatory mechanism underlying this phenomenon.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Cellular senescence occurs in primary spongiosa of long bone during late puberty. ae Representative senescence-associated β-galactosidase (SA-βGal) staining (blue) and quantitative analysis of SA-βGal+ cells in femoral metaphysis (ac) and diaphysis (d, e) sections from increasing ages of male mice. 4, 6, 8, and 12W represent 4-, 6-, 8-, and 12-week-old mice, respectively. Images in a are lower power with boxes outlining the area of higher power in b. Numbers of SA-βGal+ cells per mm2 tissue area in primary spongiosa (N. SA-βGal+ cells/PS.Ar) (c) and diaphysis (N. SA-βGal+ cells/DP.Ar) (e). Counterstained with eosin (pink). f, g Representative images of immunofluorescence staining (f) and quantitative analysis of ki67+ (g) cells (red) in femoral primary spongiosa from 4, 6, 8, and 12-week-old male mice. DAPI stains nuclei blue. Images in upper panels in f are lower power with boxes outlining the area of higher power in lower panels. Five mice per group. Data are represented as mean ± s.e.m. Ar tissue area, DP diaphysis, GP growth plate, N number, PS primary spongiosa. *P < 0.01 as determined by ANOVA
Fig. 2
Fig. 2
Senescent MSPCs are characterized by loss of nestin expression. a, b Representative images of GFP immunofluorescence staining (green) and quantitative analysis of GFP+ cells in femoral primary spongiosa from 4, 6, 8, and 12-week-old male Nestin-GFP mice. Images in upper panels in a are lower power with boxes outlining the area of higher power in lower panels. Numbers of GFP+ cells per mm2 tissue area in primary spongiosa (N. GFP+ cells/PS.Ar) b. 4W, 6W, 8W, and 12W represent 4-, 6-, 8-, and 12-week-old mice, respectively. DAPI stains nuclei blue. c, d Representative images of the flow cytometry analysis (c) and the percentage of the CD45GFP+ cells (d) in femoral metaphysis from 4-, 6-, 8-, and 12-week-old male Nestin-GFP mice. e, f Double-immunofluorescence images of femoral metaphysis sections from 4-, 6-, 8-, and 12-week-old male Nestin-GFP mice using antibodies against GFP (green) and Ki67 (red) e. DAPI stains nuclei blue. GP growth plate. PS primary spongiosa. Quantification of the percentage of GFP+ cells that express Ki67 f. Five mice per group. Data are represented as mean ± s.e.m. *P < 0.05 as determined by ANOVA. g Diagram showing isolation of Nestin-GFP+ (red) and Nestin-GFP (purple) mesenchymal stem/progenitor cells (MSPCs) by fluorescence-activated cell sorting. Detailed information on the isolation of MSPCs from femoral metaphysis from Nestin-GFP mice are described in Supplementary Fig. 3 and Methods section. hj The sorted cells were cultured, and the SA-βGal staining (h), BrdU incorporation (i), and p16INK4a immunostaining (j) were performed, and representative images were shown. km Quantification of the percentage of the cells that express SA-βGal (k), BrdU (l), and p16INK4a (m). n = 5. Data are represented as mean ± s.e.m. *P < 0.01 as determined by Student’s t-tests
Fig. 3
Fig. 3
Senescence of MSPCs is regulated by bone formation-regulatory agents. af Four-week-old male Nestin-GFP mice were treated with recombinant mouse-GH (5 mg/kg B.W.) or vehicle (Veh) by daily intraperitoneal injection for 4 weeks. Immunofluorescence staining of femur sections were performed using antibodies against GFP (green) (a) or Ki67 (red) (e). Images in upper panels in a and e are lower power with boxes outlining the area of higher power in lower panels. Quantification of the number of GFP+ (b) or Ki67+ (f) cells in femoral primary spongiosa. Representative images of flow cytometry analysis (c) and the quantification of the percentage of the CD45GFP+ cells isolated from femoral metaphysis (d). n = 5. Data are represented as mean ± s.e.m. *P < 0.01 as determined by Student’s t-tests. gj Four-week-old male Nestin-GFP mice were treated with human PTH1-34 (80 μg/kg B.W.) or Veh by daily subcutaneous injection for 4 weeks. Immunofluorescence staining of femur sections were performed using antibodies against GFP (green) (g). Images in upper panels in g are lower power with boxes outlining the area of higher power in lower panels. Quantification of the number of GFP+ (h) cells in femoral primary spongiosa. Representative images of flow cytometry analysis (i) and the quantification of the percentage of the CD45GFP+ cells isolated from femoral metaphysis (j). n = 5. Data are represented as mean ± s.e.m. *P < 0.01 as determined by Student’s t-tests. kp Two-week-old male Nestin-GFP mice were treated with prednisolone (10 mg/m2/day) or Veh by daily intraperitoneal injection for 2 weeks. Immunofluorescence staining of femur sections were performed using antibodies against GFP (green) (k) or Ki67 (red) (o). Images in upper panels in k, o are lower power with boxes outlining the area of higher power in lower panels. Quantification of the number of GFP+ (l) or Ki67+ (p) cells in femoral primary spongiosa. Representative images of flow cytometry analysis (m) and the quantification of the percentage of the CD45GFP+ cells isolated from femoral metaphysis (n). n = 5. Data are represented as mean ± s.e.m. *P < 0.01 as determined by Student’s t-tests. GP growth plate, PS primary spongiosa
Fig. 4
Fig. 4
Ezh2 represses cell senescence inducer genes in MSPCs via H3K27me3. a CD45GFP+ and CD45GFP mesenchymal stem/progenitor cells (MSPCs) were separately isolated from femoral metaphyses from 4-week-old male Nestin-GFP mice, and the total RNA was subjected for PCR array analysis of the chromatin modification enzymes. b Validation of messenger RNA changes by qRT-PCR analysis. cg Chromatin immunoprecipitation (ChIP)-qPCR assays with H3K27me3 antibody or IgG antibody were performed using CD45GFP+ and CD45GFP MSPCs. Schematic representation of the INK4a-ARF-INK4b locus in mouse c. Rectangles indicate coding exons of the three genes (p15 INK4b , p19 ARF and p16 INK4a) separated by intronic sequences (black horizontal line). The regions chosen for PCR amplification by the primers in ChIP-qPCR assays are also indicated. ChIP and input DNA were measured using RT-qPCR with specific primers targeting the promoter regions of senescence-associated genes (df). ChIP and input DNA were measured using quantitative RT-PCR with specific primers targeting the promoter regions of osteogenic genes (g). hm Quantitative RT-PCR analysis of p16 INK4a (h), p21 CIP1 (i), ki67 (j), p53 (k), Runx2 (l), Osteocalcin (m) expression in the sorted CD45GFP+ and CD45GFP MSPCs. n = 3, Data are represented as mean ± s.e.m. *P < 0.01 as determined by Student’s t-tests
Fig. 5
Fig. 5
Ezh2-H3K27me3 regulates progression of the senescence of MSPCs. Schematic diagram indicating the experimental workflow in different genetic Ezh2 (a, e) or Utx (h) ablation mouse models. ad A single dose of tamoxifen (100 mg/kg B.W.) was injected in 3-week-old male Nestin-Cre ERT2 (WT) and Ezh2 iKO mice and isolated the MSPCs from metaphysis region of femoral bone 1 week later. Co-staining of the SA-βGal and Ezh2 expression indicates that SA-βGal and Ezh2 are mutually expressed in cells (b). White arrows represent SA-βGal+ cells in merged images. The percentage of the cells that express SA-βGal, Ezh2, or both (c). Population doubling time (PDT) of the cells was measured (d). f, g Three-week-old male Nestin-Cre ERT2 ::Ezh2 flox/flox (Ezh2 iKO) mice and Nestin-Cre ERT2 mice (WT) were injected with three doses of tamoxifen (100 mg/kg B.W., every other day). Representative SA-βGal staining and quantitative analysis of SA-βGal+ cells in femoral primary spongiosa (f). Numbers of the SA-βGal+ cells per mm2 tissue area in primary spongiosa (N. SA-βgal+ cells/PS.Ar) (g). il Six-week-old female Nestin-Cre ERT2 ::Utx flox/flox (Utx iKO) mice and Nestin-Cre ERT2 mice (WT) were injected with tamoxifen for 2 weeks (100 mg/kg B.W., three doses during the first week, and one dose during the second week). Representative SA-βGal staining (i) and quantitative analysis of SA-βGal+ cells (j) in femoral primary spongiosa. Number of SA-βGal+ cells per mm2 tissue area in primary spongiosa (N. SA-βgal+ cells/PS.Ar). Representative immunofluorescence staining using antibodies against nestin (k) and quantitative analysis of nestin+ cells (l) in femoral primary spongiosa. Number of nestin+ cells per mm2 tissue area in primary spongiosa (N. nestin+ cells/PS.Ar). mp Six-week-old male Nestin-GFP mice were treated with GSK-J4 (100 mg/kg B.W.) or vehicle by daily intraperitoneal injection for 2 weeks. Immunofluorescence staining of femur sections were performed using antibodies against GFP (green) (m). Quantification of the number of GFP+ (n) cells in femoral primary spongiosa. Representative images of flow cytometry analysis (o) and the quantification of the percentage of the CD45GFP+ cells isolated from femoral metaphysis (p). n = 5. Data are represented as mean ± s.e.m. GP growth plate. PS primary spongiosa. *P < 0.01 as determined by Student’s t-tests
Fig. 6
Fig. 6
Deletion of Ezh2 in nestin+ cells during early puberty impairs osteogenesis. a Schematic diagram indicating the experimental workflow in Nestin-Cre ERT2 ::Ezh2 flox/flox (Ezh2 iKO) mice. In short, three-week-old male Ezh2 iKO mice and Nestin-Cre ERT2 mice (WT) were injected with three doses of tamoxifen (100 mg/kg B.W., every other day. The mice were humanely killed at 4 weeks of age. Double-immunofluorescence staining of femoral metaphysis sections was performed using antibodies against nestin (green) and Ezh2 (red) b. Quantification of the percentage of nestin+ cells that express Ezh2 in primary spongiosa (c). Double-immunofluorescence staining of femur sections was performed using antibodies against CD31 (green) and endomucin (Emcn) (red) (d). Quantitative analysis of relative fluorescence intensities in primary spongiosa (e). Immunofluorescence staining of femur sections using antibodies against osterix (Osx) (f) and osteocalcin (Ocn) (h). DAPI stains nuclei blue. Quantitative analysis of Osx+ and Ocn+ cells in primary spongiosa is shown in g, i, respectively. Representative images of trichrome staining of the metaphyseal trabecular bone (j). Five mice per group. Data are represented as mean ± s.e.m. *P < 0.05 as determined by Student’s t-tests
Fig. 7
Fig. 7
Deletion of Ezh2 in nestin+ cells in early puberty results in bone loss in later life. a Schematic diagram indicating the experimental workflow in Nestin-Cre ERT2 ::Ezh2 flox/flox (Ezh2 iKO) mice. In short, three-week-old male and female Ezh2 iKO mice and Nestin-Cre ERT2 mice (WT) were injected with 3 doses of tamoxifen (100 mg/kg B.W., every other day). The mice were humanely killed at 16 weeks of age. Representative μCT images of distal femur in male mice were shown in b. Quantitative analyses of Trabecular bone volume fraction (BV/TV) (c), trabecular thickness (Tb.Th) (d), trabecular number (Tb.N) (e), and trabecular separation (Tb.Sp) (f). Representative μCT images of cross-sections of femoral mid-diaphysis (g). The ratio of cortical bone area per total area (BA/TA) (h), cortical bone area (BA) (i), and cortical bone thickness (Co.Th) (j). Representative μCT images of distal femur in female mice were shown in k. Quantitative analyses of Trabecular bone volume fraction (BV/TV) (l), trabecular thickness (Tb.Th) (m), trabecular number (Tb.N) (n), and trabecular separation (Tb.Sp) (o). Representative μCT images of cross-sections of femoral mid-diaphysis (p). The ratio of cortical bone area per total area (BA/TA) (q), cortical bone area (BA) (r), and cortical bone thickness (Co.Th) (s). Ten mice per group; GP growth plate. PS primary spongiosa. Data are represented as mean ± s.e.m. *P < 0.05 as determined by Student’s t-tests

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