Limits...
A p53-dependent response limits epidermal stem cell functionality and organismal size in mice with short telomeres.

Flores I, Blasco MA - PLoS ONE (2009)

Bottom Line: Telomere maintenance is essential to ensure proper size and function of organs with a high turnover.In particular, a dwarf phenotype as well as phenotypes associated to premature loss of tissue regeneration, including the skin (hair loss, hair graying, decreased wound healing), are found in mice deficient for telomerase, the enzyme responsible for maintaining telomere length.Together, these findings indicate the existence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres that is actively limiting their contribution to tissue regeneration, thereby impinging on tissue fitness.

View Article: PubMed Central - PubMed

Affiliation: Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain.

ABSTRACT
Telomere maintenance is essential to ensure proper size and function of organs with a high turnover. In particular, a dwarf phenotype as well as phenotypes associated to premature loss of tissue regeneration, including the skin (hair loss, hair graying, decreased wound healing), are found in mice deficient for telomerase, the enzyme responsible for maintaining telomere length. Coincidental with the appearance of these phenotypes, p53 is found activated in several tissues from these mice, where is thought to trigger cellular senescence and/or apoptotic responses. Here, we show that p53 abrogation rescues both the small size phenotype and restitutes the functionality of epidermal stem cells (ESC) of telomerase-deficient mice with dysfunctional telomeres. In particular, p53 ablation restores hair growth, skin renewal and wound healing responses upon mitogenic induction, as well as rescues ESCmobilization defects in vivo and defective ESC clonogenic activity in vitro. This recovery of ESC functions is accompanied by a downregulation of senescence markers and an increased proliferation in the skin and kidney of telomerase-deficient mice with critically short telomeres without changes in apoptosis rates. Together, these findings indicate the existence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres that is actively limiting their contribution to tissue regeneration, thereby impinging on tissue fitness.

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p53 loss increases the proliferation rate and ameliorates a senescence-associated response in late-generation telomerase-deficient mice without changes in apotosis rates.Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67 (A) and p21 (B) before and after TPA-treatment. Quantification of Ki-67-positive cells in interfollicular epidermis size (C) and p21-positive cells at the bulge region of hair follicles (D). (E) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67, p21 and assessed for senescence-associated ß-galactosidase (SA ß-gal) activity. Inserts: high magnification images showing renal cells assessed for SA-ß-gal activity. (F) Percentage of renal cells positive for Ki-67, p21 and senescence-associated ß-galactosidase activity. n = number of sections used for quantification. (G) Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom) before and after TPA-treatment. (H) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom).
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pone-0004934-g006: p53 loss increases the proliferation rate and ameliorates a senescence-associated response in late-generation telomerase-deficient mice without changes in apotosis rates.Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67 (A) and p21 (B) before and after TPA-treatment. Quantification of Ki-67-positive cells in interfollicular epidermis size (C) and p21-positive cells at the bulge region of hair follicles (D). (E) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67, p21 and assessed for senescence-associated ß-galactosidase (SA ß-gal) activity. Inserts: high magnification images showing renal cells assessed for SA-ß-gal activity. (F) Percentage of renal cells positive for Ki-67, p21 and senescence-associated ß-galactosidase activity. n = number of sections used for quantification. (G) Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom) before and after TPA-treatment. (H) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom).

Mentions: The fact that G4 Terc−/−p53−/− newborns reach standard body size and weight suggests that p53 deficiency enables G4 Terc−/−p53−/− cells to enter into additional rounds of cell proliferation in the presence of critically short telomeres, a situation that resembles the known effects of p53 ablation in bypassing cellular senescence both in vivo and in vitro [24], [39], [40]. However, whether a p53-dependent cellular senescence and/or apoptosis response contributes to set organ and organismal size during embryonic development, or to limit stem cell functionality in adult tissues, remains unaddressed to date. To further analyze the consequences of increased p53 in mice with critically short telomeres, we examine whether tissues from telomerase-deficient mice with a reduced body size (G3 Terc−/−) enter senescence or undergo apoptosis in a p53-dependent manner. Senescence is associated with a decline in proliferation that culminates in a permanent arrest of the cell cycle [41]. Therefore, we first compared the ability of G3 Terc−/− keratinocytes to proliferate in the presence or absence of p53. As shown in Fig 6a,b, we detected slightly fewer Ki67-positive keratinocytes in G3 Terc−/−p53+/+ untreated interfollicular (IFE) skin compared to G3 Terc−/−p53−/− or wild type skin, although the differences did not reach statistical significance. In contrast, a marked reduction in proliferation (Ki67-positive cells) is observed in TPA-treated G3 Terc−/−p53+/+ skin when compared with TPA-treated G3 Terc−/−p53−/− and TPA-treated wild type controls (Fig. 6a,b), suggesting that the defective proliferation response to TPA of G3 Terc−/− keratinocytes is mediated by p53. In addition to growth arrest, senescence is associated at the molecular level with enhanced expression of the p53-transcriptional target p21WAF1 [41]. In resting skin conditions, a faint p21 expression was observed in G3 Terc−/−p53+/+ epidermis, which becomes overtly visible after TPA treatment (Fig. 6c,d). Similarly to that observed for p53 expression in G3 Terc−/−p53+/+ epidermis, positive-p21 cells located at the hair bulge and its close proximity, suggesting that the p53 transcriptionaly target p21 becomes active in stem/progenitor cells with short telomeres upon enforced proliferation. In addition, p21 expression was barely detectable at the bulge of wild type and G3 Terc−/−p53−/− hair follicles further (Fig. 6c,d), indicating that in the hair bulge the induction of p21 is mediated by p53. In other epidermal compartments, such as the hair bulb, infundibulum and IFE, a TPA-mediated p21 induction was observed regardless of p53 gene status (Fig. 6c,d), which suggest a minor role of p53 on the p21 upregulation in the above compartments in agreement with previous reports [42]. Next, we examined the kidney, an organ in which p53 loss partially rescues a senescence-associated phenotype induced by nuclear damage [43]. Similarly to the results obtained in keratinocytes, we detected a marked reduction in Ki67-positive cells, which was accompanied by p21-upregulation in G3 Terc−/− renal cells compared to wild types (Fig. 6e,f). In addition, we observed numerous G3 Terc−/− renal cells harboring senescence-associated ß-galactosidase activity (Fig 6e,f). Importantly, both proliferative defects and senescence markers are rescued in the absence of p53 (Fig. 6e,f), indicating the importance of p53 in mediating cellular arrest and senescence in response to short telomeres. Finally, absence of active caspase 3- and TUNEL-positive cells both in skin and kidney rules out an involvement of apoptosis in the observed phenotypes, in agreement with previously published data [9] (Fig. 6g,h). Taken together, these findings strongly suggest the presence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres, which in turn limits their contribution to tissue size and fitness.


A p53-dependent response limits epidermal stem cell functionality and organismal size in mice with short telomeres.

Flores I, Blasco MA - PLoS ONE (2009)

p53 loss increases the proliferation rate and ameliorates a senescence-associated response in late-generation telomerase-deficient mice without changes in apotosis rates.Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67 (A) and p21 (B) before and after TPA-treatment. Quantification of Ki-67-positive cells in interfollicular epidermis size (C) and p21-positive cells at the bulge region of hair follicles (D). (E) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67, p21 and assessed for senescence-associated ß-galactosidase (SA ß-gal) activity. Inserts: high magnification images showing renal cells assessed for SA-ß-gal activity. (F) Percentage of renal cells positive for Ki-67, p21 and senescence-associated ß-galactosidase activity. n = number of sections used for quantification. (G) Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom) before and after TPA-treatment. (H) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom).
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pone-0004934-g006: p53 loss increases the proliferation rate and ameliorates a senescence-associated response in late-generation telomerase-deficient mice without changes in apotosis rates.Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67 (A) and p21 (B) before and after TPA-treatment. Quantification of Ki-67-positive cells in interfollicular epidermis size (C) and p21-positive cells at the bulge region of hair follicles (D). (E) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for Ki-67, p21 and assessed for senescence-associated ß-galactosidase (SA ß-gal) activity. Inserts: high magnification images showing renal cells assessed for SA-ß-gal activity. (F) Percentage of renal cells positive for Ki-67, p21 and senescence-associated ß-galactosidase activity. n = number of sections used for quantification. (G) Representative tail-skin sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom) before and after TPA-treatment. (H) Representative kidney sections from wild-type, G3 Terc−/− and G3 Terc−/−p53−/− littermates stained for active caspase 3 (top) and TUNEL (bottom).
Mentions: The fact that G4 Terc−/−p53−/− newborns reach standard body size and weight suggests that p53 deficiency enables G4 Terc−/−p53−/− cells to enter into additional rounds of cell proliferation in the presence of critically short telomeres, a situation that resembles the known effects of p53 ablation in bypassing cellular senescence both in vivo and in vitro [24], [39], [40]. However, whether a p53-dependent cellular senescence and/or apoptosis response contributes to set organ and organismal size during embryonic development, or to limit stem cell functionality in adult tissues, remains unaddressed to date. To further analyze the consequences of increased p53 in mice with critically short telomeres, we examine whether tissues from telomerase-deficient mice with a reduced body size (G3 Terc−/−) enter senescence or undergo apoptosis in a p53-dependent manner. Senescence is associated with a decline in proliferation that culminates in a permanent arrest of the cell cycle [41]. Therefore, we first compared the ability of G3 Terc−/− keratinocytes to proliferate in the presence or absence of p53. As shown in Fig 6a,b, we detected slightly fewer Ki67-positive keratinocytes in G3 Terc−/−p53+/+ untreated interfollicular (IFE) skin compared to G3 Terc−/−p53−/− or wild type skin, although the differences did not reach statistical significance. In contrast, a marked reduction in proliferation (Ki67-positive cells) is observed in TPA-treated G3 Terc−/−p53+/+ skin when compared with TPA-treated G3 Terc−/−p53−/− and TPA-treated wild type controls (Fig. 6a,b), suggesting that the defective proliferation response to TPA of G3 Terc−/− keratinocytes is mediated by p53. In addition to growth arrest, senescence is associated at the molecular level with enhanced expression of the p53-transcriptional target p21WAF1 [41]. In resting skin conditions, a faint p21 expression was observed in G3 Terc−/−p53+/+ epidermis, which becomes overtly visible after TPA treatment (Fig. 6c,d). Similarly to that observed for p53 expression in G3 Terc−/−p53+/+ epidermis, positive-p21 cells located at the hair bulge and its close proximity, suggesting that the p53 transcriptionaly target p21 becomes active in stem/progenitor cells with short telomeres upon enforced proliferation. In addition, p21 expression was barely detectable at the bulge of wild type and G3 Terc−/−p53−/− hair follicles further (Fig. 6c,d), indicating that in the hair bulge the induction of p21 is mediated by p53. In other epidermal compartments, such as the hair bulb, infundibulum and IFE, a TPA-mediated p21 induction was observed regardless of p53 gene status (Fig. 6c,d), which suggest a minor role of p53 on the p21 upregulation in the above compartments in agreement with previous reports [42]. Next, we examined the kidney, an organ in which p53 loss partially rescues a senescence-associated phenotype induced by nuclear damage [43]. Similarly to the results obtained in keratinocytes, we detected a marked reduction in Ki67-positive cells, which was accompanied by p21-upregulation in G3 Terc−/− renal cells compared to wild types (Fig. 6e,f). In addition, we observed numerous G3 Terc−/− renal cells harboring senescence-associated ß-galactosidase activity (Fig 6e,f). Importantly, both proliferative defects and senescence markers are rescued in the absence of p53 (Fig. 6e,f), indicating the importance of p53 in mediating cellular arrest and senescence in response to short telomeres. Finally, absence of active caspase 3- and TUNEL-positive cells both in skin and kidney rules out an involvement of apoptosis in the observed phenotypes, in agreement with previously published data [9] (Fig. 6g,h). Taken together, these findings strongly suggest the presence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres, which in turn limits their contribution to tissue size and fitness.

Bottom Line: Telomere maintenance is essential to ensure proper size and function of organs with a high turnover.In particular, a dwarf phenotype as well as phenotypes associated to premature loss of tissue regeneration, including the skin (hair loss, hair graying, decreased wound healing), are found in mice deficient for telomerase, the enzyme responsible for maintaining telomere length.Together, these findings indicate the existence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres that is actively limiting their contribution to tissue regeneration, thereby impinging on tissue fitness.

View Article: PubMed Central - PubMed

Affiliation: Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain.

ABSTRACT
Telomere maintenance is essential to ensure proper size and function of organs with a high turnover. In particular, a dwarf phenotype as well as phenotypes associated to premature loss of tissue regeneration, including the skin (hair loss, hair graying, decreased wound healing), are found in mice deficient for telomerase, the enzyme responsible for maintaining telomere length. Coincidental with the appearance of these phenotypes, p53 is found activated in several tissues from these mice, where is thought to trigger cellular senescence and/or apoptotic responses. Here, we show that p53 abrogation rescues both the small size phenotype and restitutes the functionality of epidermal stem cells (ESC) of telomerase-deficient mice with dysfunctional telomeres. In particular, p53 ablation restores hair growth, skin renewal and wound healing responses upon mitogenic induction, as well as rescues ESCmobilization defects in vivo and defective ESC clonogenic activity in vitro. This recovery of ESC functions is accompanied by a downregulation of senescence markers and an increased proliferation in the skin and kidney of telomerase-deficient mice with critically short telomeres without changes in apoptosis rates. Together, these findings indicate the existence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres that is actively limiting their contribution to tissue regeneration, thereby impinging on tissue fitness.

Show MeSH
Related in: MedlinePlus