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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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Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.(A) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (B) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (C and D) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: p value < 0.05.
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pone-0073206-g003: Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.(A) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (B) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (C and D) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: p value < 0.05.

Mentions: Total body irradiation leads to a net bone loss, an effect that coincides with the presence of senescent cells and a diminution in the absolute number of stromal and hematopoietic stem/progenitor cells [4,29]. However, whether the bone mineralization potential of MSCs and osteoblasts is differently affected by the senescent phenotype is currently unknown. Therefore, we exposed MSC and OB–SC populations to 10 Gy IR in vitro and investigated their ability to mineralize and upregulate key osteogenic transcription factors when placed in differentiation media. As we observed for adipogenesis, we found the osteogenic potential of MSC progenitors to be severely abrogated post IR, as no mineralized nodules were observed in senescent cultures when compared to control non-irradiated populations (Figure 3A). This contrasts to what we observed in the OB–SC population which surprisingly could still efficiently form mineralized nodules after irradiation, although the process was reduced compared to non-irradiated cultures. Solubilisation and quantification of Alizarin Red by spectrometry allowed us to precisely quantify the mineralization process (Figure 3B).


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)

Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.(A) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (B) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (C and D) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: p value < 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756945&req=5

pone-0073206-g003: Abrogation of osteogenic differentiation potential following irradiation is limited to stromal progenitor cells.(A) MSC and osteoblasts (OB–SC) were exposed (IR) or not (CTRL) to 10 Gy IR and one week later placed in osteogenic differentiation media for 14 to 21 days. Representative photographs showing mineralization nodules accumulation stained with Alizarin Red S is shown for each population. Scale bar: 2mm. Phase contrast photograph showing the presence of senescent MSC in absence of mineralization is also shown. (B) Quantification of mineralization was determined by the extraction of Alizarin Red S and detection by spectrophotometry. (C and D) Expression of Runx2 and Osx was determined by quantitative real-time PCR using RNA extracted from control and IR-induced senescent MSC and OB–SC populations cultured or not in osteogenic differentiation media. Mean ± standard error; *: p value < 0.05.
Mentions: Total body irradiation leads to a net bone loss, an effect that coincides with the presence of senescent cells and a diminution in the absolute number of stromal and hematopoietic stem/progenitor cells [4,29]. However, whether the bone mineralization potential of MSCs and osteoblasts is differently affected by the senescent phenotype is currently unknown. Therefore, we exposed MSC and OB–SC populations to 10 Gy IR in vitro and investigated their ability to mineralize and upregulate key osteogenic transcription factors when placed in differentiation media. As we observed for adipogenesis, we found the osteogenic potential of MSC progenitors to be severely abrogated post IR, as no mineralized nodules were observed in senescent cultures when compared to control non-irradiated populations (Figure 3A). This contrasts to what we observed in the OB–SC population which surprisingly could still efficiently form mineralized nodules after irradiation, although the process was reduced compared to non-irradiated cultures. Solubilisation and quantification of Alizarin Red by spectrometry allowed us to precisely quantify the mineralization process (Figure 3B).

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