A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells
J. Clin. Invest. Masako Miura, et al. 114:1704 doi:10.1172/JCI20427 [
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Figure 4TGF-β–associated replicative senescence of
caspase-3–deficient BMSSCs. (
A) Replicative senescence assessed by β-gal staining. Representative pictures of β-gal–positive cells induced by TGF-β are shown in the upper panel (arrows). Original magnification, ×200. Replicative senescence was increased in
Casp3–/– BMSSCs compared with WT BMSSCs (lower panel;
n = 6; *
P < 0.001). TGF-β accelerated senescence in
caspase-3–deficient BMSSCs (
P < 0.01;
#P < 0.05) but not in WT mice. (
B) Annexin V staining of BMSSCs. The number of annexin V–positive cells was found to be similar among each genotype under the regular culture condition (–, upper panels). However, TGF-β treatment reduced the number of annexin V–positive cells in
Casp3–/– BMSSCs (+, lower panels). Annexin V–positive cells appear in green. Original magnification, ×200. (
C) Population doubling of BMSSCs. BMSSCs were continuously passaged at the same cell density after confluency. Fifty days after the culture was started,
Casp3–/– BMSSCs stopped proliferating and showed enlarged cell body and nuclei, although WT BMSSCs continued proliferating (upper panels).
Caspase-3–deficient BMSSCs showed decreased population doubling (lower panel) (
n = 6; *
P < 0.001;
P < 0.01). (
D) Western blot analysis of BMSSCs.
Casp3–/– BMSSCs showed upregulated expression of TGF-βRI, Smad2, p21, and p53 along with downregulated expression of Cdc2 compared with WT. After TGF-β treatment, expression of TGF-βRI, Smad2, p-Smad2, p21, and p53 was further upregulated accompanying with downregulated expression of Cdk2 and Cdc2. Smad3 and TGF-βRII expressions were not changed in
Casp3–/– BMSSCs even with TGF-β treatment. Ten micrograms of protein was applied to each lane, and HSP90 was used as an additional control for protein loading.
