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Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells.

Hou J, Han ZP, Jing YY, Yang X, Zhang SS, Sun K, Hao C, Meng Y, Yu FH, Liu XQ, Shi YF, Wu MC, Zhang L, Wei LX - Cell Death Dis (2013)

Bottom Line: Stem cells were characterized by their stemness: self-renewal and pluripotency.Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs.Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs.

View Article: PubMed Central - PubMed

Affiliation: 1] Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China [2] Department of Pharmacy, Changhai Hospital, The Second Military Medical University, Shanghai, China.

ABSTRACT
Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.

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Inhibition autophagy results in stemness injury and ROS increase in irradiated MSCs. (a) LC3 expression was detected by western blotting assay in irradiated MSCs pretreated with starvation by autophagy inhibitor 3-MA or CQ. (b) Irradiated MSCs pretreated with starvation were transfected with shRNAs to knockdown the autophagy-associated genes ATG7 and Beclin1. The bottom panel is a GAPDH-loading control. (c) CFU-F assays. The number of colonies was determined after 14 days of culture. (d) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation by inhibiting autophagy were measured by real-time PCR and western blotting. (e) Osteogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors was stained with Alizarin Red S. (f) The expression of osteogenic markers ALPL, OGN and RUNX2 were measured by real-time PCR. (g) Adipogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with Oil red-O. (h) The expression of adipogenic markers LPL, CFD and PPAR-γ were measured by real-time PCR. (i) Irradiated MSCs pretreated with starvation by autophagy inhibitors stained with DCF-DA to detect ROS level were measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (j) Irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. The data presented are from three replicates as mean±S.D. *P<0.05
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fig5: Inhibition autophagy results in stemness injury and ROS increase in irradiated MSCs. (a) LC3 expression was detected by western blotting assay in irradiated MSCs pretreated with starvation by autophagy inhibitor 3-MA or CQ. (b) Irradiated MSCs pretreated with starvation were transfected with shRNAs to knockdown the autophagy-associated genes ATG7 and Beclin1. The bottom panel is a GAPDH-loading control. (c) CFU-F assays. The number of colonies was determined after 14 days of culture. (d) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation by inhibiting autophagy were measured by real-time PCR and western blotting. (e) Osteogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors was stained with Alizarin Red S. (f) The expression of osteogenic markers ALPL, OGN and RUNX2 were measured by real-time PCR. (g) Adipogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with Oil red-O. (h) The expression of adipogenic markers LPL, CFD and PPAR-γ were measured by real-time PCR. (i) Irradiated MSCs pretreated with starvation by autophagy inhibitors stained with DCF-DA to detect ROS level were measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (j) Irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. The data presented are from three replicates as mean±S.D. *P<0.05

Mentions: Autophagy was suppressed by chemical inhibitors to detect the role of autophagy in maintaining stemness of irradiated MSCs. The most extensively used autophagy inhibitor is 3-methyladenine (3-MA), which blocks the formation of autophagosomes by inhibiting the class III phosphatidylinositol 3-kinase.28 Chloroquine (CQ) is another autophagy inhibitor, which inhibits autophagy at a later step in the pathway, that caused an additional accumulation of LC3-II. The addition of 3-MA significantly reduced the expression of LC3-II, and CQ accumulated LC3-II expression in irradiated MSCs pretreated with starvation (Figure 5a). After autophagy inhibition with 3-MA and CQ, irradiated MSCs pretreated with starvation demonstrated lower CFU-F efficiency than control (Figure 5c). The expressions of Nanog, Oct4 and Sox2 were downregulated by autophagy inhibitor 3-MA and CQ in irradiated MSCs pretreated with starvation (Figure 5d). When autophagy in irradiated MSCs pretreated with starvation was deleted by autophagy inhibitor, the potential of osteogenesis and adipogenesis differentiation in MSCs decreased significantly (Figures 5e and g). Meanwhile, we also observed downregulated expression of mRNA associated with osteoblastic and adipocytic differentiation in irradiated MSCs pretreated with starvation when autophagy was inhibited (Figures 5f and h). We also inhibited autophagy in irradiated MSCs pretreated with starvation by short hairpin RNA (shRNA)-ATG7 or shRNA-Beclin1. As shown in Figures 5b–h, the expression of ATG7 and Beclin1 was significantly inhibited and the two shRNAs, respectively, diminished the protection of autophagy on MSCs stemness.


Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells.

Hou J, Han ZP, Jing YY, Yang X, Zhang SS, Sun K, Hao C, Meng Y, Yu FH, Liu XQ, Shi YF, Wu MC, Zhang L, Wei LX - Cell Death Dis (2013)

Inhibition autophagy results in stemness injury and ROS increase in irradiated MSCs. (a) LC3 expression was detected by western blotting assay in irradiated MSCs pretreated with starvation by autophagy inhibitor 3-MA or CQ. (b) Irradiated MSCs pretreated with starvation were transfected with shRNAs to knockdown the autophagy-associated genes ATG7 and Beclin1. The bottom panel is a GAPDH-loading control. (c) CFU-F assays. The number of colonies was determined after 14 days of culture. (d) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation by inhibiting autophagy were measured by real-time PCR and western blotting. (e) Osteogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors was stained with Alizarin Red S. (f) The expression of osteogenic markers ALPL, OGN and RUNX2 were measured by real-time PCR. (g) Adipogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with Oil red-O. (h) The expression of adipogenic markers LPL, CFD and PPAR-γ were measured by real-time PCR. (i) Irradiated MSCs pretreated with starvation by autophagy inhibitors stained with DCF-DA to detect ROS level were measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (j) Irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. The data presented are from three replicates as mean±S.D. *P<0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Inhibition autophagy results in stemness injury and ROS increase in irradiated MSCs. (a) LC3 expression was detected by western blotting assay in irradiated MSCs pretreated with starvation by autophagy inhibitor 3-MA or CQ. (b) Irradiated MSCs pretreated with starvation were transfected with shRNAs to knockdown the autophagy-associated genes ATG7 and Beclin1. The bottom panel is a GAPDH-loading control. (c) CFU-F assays. The number of colonies was determined after 14 days of culture. (d) The expression of stemness markers Nanog, Oct4 and Sox2 of irradiated MSCs pretreated with starvation by inhibiting autophagy were measured by real-time PCR and western blotting. (e) Osteogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors was stained with Alizarin Red S. (f) The expression of osteogenic markers ALPL, OGN and RUNX2 were measured by real-time PCR. (g) Adipogenic differentiation of irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with Oil red-O. (h) The expression of adipogenic markers LPL, CFD and PPAR-γ were measured by real-time PCR. (i) Irradiated MSCs pretreated with starvation by autophagy inhibitors stained with DCF-DA to detect ROS level were measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (j) Irradiated MSCs pretreated with starvation by autophagy inhibitors were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. The data presented are from three replicates as mean±S.D. *P<0.05
Mentions: Autophagy was suppressed by chemical inhibitors to detect the role of autophagy in maintaining stemness of irradiated MSCs. The most extensively used autophagy inhibitor is 3-methyladenine (3-MA), which blocks the formation of autophagosomes by inhibiting the class III phosphatidylinositol 3-kinase.28 Chloroquine (CQ) is another autophagy inhibitor, which inhibits autophagy at a later step in the pathway, that caused an additional accumulation of LC3-II. The addition of 3-MA significantly reduced the expression of LC3-II, and CQ accumulated LC3-II expression in irradiated MSCs pretreated with starvation (Figure 5a). After autophagy inhibition with 3-MA and CQ, irradiated MSCs pretreated with starvation demonstrated lower CFU-F efficiency than control (Figure 5c). The expressions of Nanog, Oct4 and Sox2 were downregulated by autophagy inhibitor 3-MA and CQ in irradiated MSCs pretreated with starvation (Figure 5d). When autophagy in irradiated MSCs pretreated with starvation was deleted by autophagy inhibitor, the potential of osteogenesis and adipogenesis differentiation in MSCs decreased significantly (Figures 5e and g). Meanwhile, we also observed downregulated expression of mRNA associated with osteoblastic and adipocytic differentiation in irradiated MSCs pretreated with starvation when autophagy was inhibited (Figures 5f and h). We also inhibited autophagy in irradiated MSCs pretreated with starvation by short hairpin RNA (shRNA)-ATG7 or shRNA-Beclin1. As shown in Figures 5b–h, the expression of ATG7 and Beclin1 was significantly inhibited and the two shRNAs, respectively, diminished the protection of autophagy on MSCs stemness.

Bottom Line: Stem cells were characterized by their stemness: self-renewal and pluripotency.Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs.Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs.

View Article: PubMed Central - PubMed

Affiliation: 1] Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China [2] Department of Pharmacy, Changhai Hospital, The Second Military Medical University, Shanghai, China.

ABSTRACT
Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.

Show MeSH
Related in: MedlinePlus