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Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

Despars G, Carbonneau CL, Bardeau P, Coutu DL, Beauséjour CM - PLoS ONE (2013)

Bottom Line: Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules.This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes.Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche du CHU Ste-Justine, Montréal, Québec, Canada.

ABSTRACT
DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB-SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner.

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Senescence of multipotent and committed stromal lineages following exposure to IR.(A) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (B) Quantification of the proportion of SAβ-gal positive cells in each population. (C) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (D) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.
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pone-0073206-g001: Senescence of multipotent and committed stromal lineages following exposure to IR.(A) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (B) Quantification of the proportion of SAβ-gal positive cells in each population. (C) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (D) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.

Mentions: Whether cells undergo cellular senescence or apoptosis in response to IR is cell type specific and needs to be determined. Bone homeostasis is believed to rely, at least in part, on bone marrow-derived MSC through their ability to differentiate in osteoblasts and/or adipocytes. Using defined purification procedures [29], we isolated multipotent MSC and more committed osteoblasts (OB–SC) and exposed these populations, along with the pre-adipocyte 3T3-L1 cell line, to 10 Gy IR. While irradiated cell populations showed no sign of apoptosis (data not shown), they all expressed markers of senescence (Figure 1). Indeed, irradiated populations displayed an enlarged cytoplasm and a flattened appearance, two morphological characteristics of senescent cells. Moreover, with the exception of the OB–SC population, the majority of cells stained positive for the senescence-associated β-galactosidase (SAβ-gal) (Figure 1A and 1B). Persistent DNA damage, as detected by 53BP1 foci, was also observed in all senescent populations, a phenotype almost completely absent in control non irradiated cell populations (Figure 1B). Finally, the expected loss in the proliferation potential of all three irradiated cell populations was confirmed using a colony forming unit (CFU) assay (Figure 1C). These results demonstrate that bone stromal progenitor cells and more differentiated lineages undergo senescence in response to IR-induced DNA damage.


Loss of the osteogenic differentiation potential during senescence is limited to bone progenitor cells and is dependent on p53.

Despars G, Carbonneau CL, Bardeau P, Coutu DL, Beauséjour CM - PLoS ONE (2013)

Senescence of multipotent and committed stromal lineages following exposure to IR.(A) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (B) Quantification of the proportion of SAβ-gal positive cells in each population. (C) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (D) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756945&req=5

pone-0073206-g001: Senescence of multipotent and committed stromal lineages following exposure to IR.(A) Murine bone marrow-derived multipotent stromal cells (MSC), osteoblasts (OB–SC) and pre-adiopocytes (3T3-L1) were exposed (IR) or not (CTRL) to 10 Gy IR and 7 days later stained for the expression of the senescence-associated β-galactosidase (SAβ-gal). (B) Quantification of the proportion of SAβ-gal positive cells in each population. (C) Sustained activation of the DNA damage response in stromal populations was measured by staining for the presence of 53BP1 DNA damage foci (in red) one week post exposure to IR. Nuclei were counterstained with DAPI. (D) The proliferation capacity of MSC, OB–SC and 3T3-L1 cell population was determined using a CFU assay one week post-exposure or not to IR. Mean ± standard error of at least three individual experiments is shown. p values were obtained by performing a Student’s t-test.
Mentions: Whether cells undergo cellular senescence or apoptosis in response to IR is cell type specific and needs to be determined. Bone homeostasis is believed to rely, at least in part, on bone marrow-derived MSC through their ability to differentiate in osteoblasts and/or adipocytes. Using defined purification procedures [29], we isolated multipotent MSC and more committed osteoblasts (OB–SC) and exposed these populations, along with the pre-adipocyte 3T3-L1 cell line, to 10 Gy IR. While irradiated cell populations showed no sign of apoptosis (data not shown), they all expressed markers of senescence (Figure 1). Indeed, irradiated populations displayed an enlarged cytoplasm and a flattened appearance, two morphological characteristics of senescent cells. Moreover, with the exception of the OB–SC population, the majority of cells stained positive for the senescence-associated β-galactosidase (SAβ-gal) (Figure 1A and 1B). Persistent DNA damage, as detected by 53BP1 foci, was also observed in all senescent populations, a phenotype almost completely absent in control non irradiated cell populations (Figure 1B). Finally, the expected loss in the proliferation potential of all three irradiated cell populations was confirmed using a colony forming unit (CFU) assay (Figure 1C). These results demonstrate that bone stromal progenitor cells and more differentiated lineages undergo senescence in response to IR-induced DNA damage.

Bottom Line: Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules.This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes.Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche du CHU Ste-Justine, Montréal, Québec, Canada.

ABSTRACT
DNA damage can lead to the induction of cellular senescence. In particular, we showed that exposure to ionizing radiation (IR) leads to the senescence of bone marrow-derived multipotent stromal cells (MSC) and osteoblast-like stromal cells (OB-SC), a phenotype associated with bone loss. The mechanism by which IR leads to bone dysfunction is not fully understood. One possibility involves that DNA damage-induced senescence limits the regeneration of bone progenitor cells. Another possibility entails that bone dysfunction arises from the inability of accumulating senescent cells to fulfill their physiological function. Indeed, we show here that exposure to IR prevented the differentiation and mineralization functions of MSC, an effect we found was limited to this population as more differentiated OB-SC could still form mineralize nodules. This is in contrast to adipogenesis, which was inhibited in both IR-induced senescent MSC and 3T3-L1 pre-adipocytes. Furthermore, we demonstrate that IR-induced loss of osteogenic potential in MSC was p53-dependent, a phenotype that correlates with the inability to upregulate key osteogenic transcription factors. These results are the first to demonstrate that senescence impacts osteogenesis in a cell type dependent manner and suggest that the accumulation of senescent osteoblasts is unlikely to significantly contribute to bone dysfunction in a cell autonomous manner.

Show MeSH
Related in: MedlinePlus