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ATM regulates insulin-like growth factor 1-secretory clusterin (IGF-1-sCLU) expression that protects cells against senescence.

Luo X, Suzuki M, Ghandhi SA, Amundson SA, Boothman DA - PLoS ONE (2014)

Bottom Line: In contrast, administration of an IGF-1 inhibitor caused apoptosis of senescent cells.Thus, IGF-1 signaling is required for survival, whereas sCLU appears to protect cells from premature senescence, as IMR-90 cells with sCLU knockdown undergo senescence faster than control cells.Thus, the ATM-IGF-1-sCLU pathway protects cells from lethality and suspends senescence.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Radiation Oncology, Laboratory of Molecular Cell Stress Responses, Program in Cell Stress and Cancer Nanomedicine, Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
Downstream factors that regulate the decision between senescence and cell death have not been elucidated. Cells undergo senescence through three pathways, replicative senescence (RS), stress-induced premature senescence (SIPS) and oncogene-induced senescence. Recent studies suggest that the ataxia telangiectasia mutant (ATM) kinase is not only a key protein mediating cellular responses to DNA damage, but also regulates cellular senescence induced by telomere end exposure (in RS) or persistent DNA damage (in SIPS). Here, we show that expression of secretory clusterin (sCLU), a known pro-survival extracellular chaperone, is transcriptionally up-regulated during both RS and SIPS, but not in oncogene-induced senescence, consistent with a DNA damage-inducible mechanism. We demonstrate that ATM plays an important role in insulin-like growth factor 1 (IGF-1) expression, that in turn, regulates downstream sCLU induction during senescence. Loss of ATM activity, either by genomic mutation (ATM-deficient fibroblasts from an ataxia telangiectasia patient) or by administration of a chemical inhibitor (AAI, an inhibitor of ATM and ATR), blocks IGF-1-sCLU expression in senescent cells. Downstream, sCLU induction during senescence is mediated by IGF-1R/MAPK/Egr-1 signaling, identical to its induction after DNA damage. In contrast, administration of an IGF-1 inhibitor caused apoptosis of senescent cells. Thus, IGF-1 signaling is required for survival, whereas sCLU appears to protect cells from premature senescence, as IMR-90 cells with sCLU knockdown undergo senescence faster than control cells. Thus, the ATM-IGF-1-sCLU pathway protects cells from lethality and suspends senescence.

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Related in: MedlinePlus

sCLU induction during senescence is mediated through IGF-1R/MAPK/Erk/Egr-1 signaling pathway.(A) IGF-1 expression was increased in senescent IMR-90 cells. IGF-1 levels in media from young or senescent IMR-90 cells were measured using ELISAs as described above, and normalized to media volume and adjusted cell number (4×104 cells). (B) Total IGF-1 receptor (IGF-1R), phosphorylated IGF-1 receptor (p-IGF-1R), Src, phosphorylated Src, Erk-1/2, phosphorylated-Erk-1/2, and Egr-1 levels in young (Y), middle-age (M), premature-senescent (PS), and senescent (S) IMR-90 cells were determined by western blot analyses. β-Actin levels were measured to monitor loading. (C) Exposure to the indicated doses of the IGF-1R tyrosine kinase inhibitor (AG1024) repressed sCLU levels in senescent IMR-90 cells. psCLU and sCLU were detected by western blot analysis. β-Actin levels were monitored to show equal loading. Senescent IMR-90 cells exposed to varying doses of AG1024 (µM as indicated for 24 h) showed dose-dependent increases in apoptosis (monitored by TUNEL + staining). Only senescent, and not young or middle-aged IMR-90, cells were hypersensitive to AG1024. In the experiment outlined below the western blot analyses cells were exposed to AG1024 (5 µM) for 48 h and therefore higher levels of apoptotic cells were noted in senescent IMR-90 cells compared to cells exposed for 24 h above. Note that only senescent cells were hypersensitive to these inhibitor treatments. All experiments were repeated three times in triplicate.
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pone-0099983-g004: sCLU induction during senescence is mediated through IGF-1R/MAPK/Erk/Egr-1 signaling pathway.(A) IGF-1 expression was increased in senescent IMR-90 cells. IGF-1 levels in media from young or senescent IMR-90 cells were measured using ELISAs as described above, and normalized to media volume and adjusted cell number (4×104 cells). (B) Total IGF-1 receptor (IGF-1R), phosphorylated IGF-1 receptor (p-IGF-1R), Src, phosphorylated Src, Erk-1/2, phosphorylated-Erk-1/2, and Egr-1 levels in young (Y), middle-age (M), premature-senescent (PS), and senescent (S) IMR-90 cells were determined by western blot analyses. β-Actin levels were measured to monitor loading. (C) Exposure to the indicated doses of the IGF-1R tyrosine kinase inhibitor (AG1024) repressed sCLU levels in senescent IMR-90 cells. psCLU and sCLU were detected by western blot analysis. β-Actin levels were monitored to show equal loading. Senescent IMR-90 cells exposed to varying doses of AG1024 (µM as indicated for 24 h) showed dose-dependent increases in apoptosis (monitored by TUNEL + staining). Only senescent, and not young or middle-aged IMR-90, cells were hypersensitive to AG1024. In the experiment outlined below the western blot analyses cells were exposed to AG1024 (5 µM) for 48 h and therefore higher levels of apoptotic cells were noted in senescent IMR-90 cells compared to cells exposed for 24 h above. Note that only senescent cells were hypersensitive to these inhibitor treatments. All experiments were repeated three times in triplicate.

Mentions: Our data strongly suggested that functional ATM could regulate IGF-1 expression during RS or SIPS pathways. Indeed, IGF-1 expression was approximately two-fold higher in conditioned media from senescent compared to young IMR-90 cells (Figure 4A). Analyses of differential signal transduction responses in senescent versus young IMR-90 cells showed that IGF-1 receptor (IGF-1R) levels were activated (elevated P-IGF-1R/t-IGF-1R) in senescent cells; antibodies to human IGF-1R are not ideal. Src and Erk-1/2 kinase levels were also elevated in middle-aged and senescent compared to young IMR-90 cells. The transcription factor, Egr-1, which binds the hCLU promoter and mediates sCLU expression [28], was significantly increased during senescence in IMR-90 cells (Figure 4B). To further confirm the specificity of IGF-1/IGF-1R/MAPK/ERK-1/2/Egr-1 signaling in sCLU induction during senescence, we treated senescent IMR-90 cells with the IGF-1R inhibitor, AG1024, which effectively blocked IR-induced sCLU expression [27], [28] and also suppressed sCLU expression in senescent IMR-90 cells in a dose-dependent manner (Figure 4C). While significantly suppressing sCLU expression, we also noted that senescent IMR-90 cells exposed to the IGF-1R inhibitor (AG1024) underwent significant programmed cell death (apoptotic) responses in a dose-dependent manner (Figure 4C). Interestingly, young and middle-aged cells did not respond to AG1024 in the same manner with no significant apoptosis noted after exposure to 5 µM of the drug (Figure 4C). Collectively, these results strongly suggest that when cell undergo RS or SIPS, but not oncogene-induced senescence, ATM-dependent DDR leads to increased IGF-1 expression that stimulates IGF-1R/MAPK/Erk-1/2/Egr-1 signaling that leads to concomitant elevated sCLU levels. The data further suggest that the IGF-1/IGF-1R-sCLU pathway is required for the overall survival of senescent cells, whereby suppressing this pathway induces an intracellular death decision.


ATM regulates insulin-like growth factor 1-secretory clusterin (IGF-1-sCLU) expression that protects cells against senescence.

Luo X, Suzuki M, Ghandhi SA, Amundson SA, Boothman DA - PLoS ONE (2014)

sCLU induction during senescence is mediated through IGF-1R/MAPK/Erk/Egr-1 signaling pathway.(A) IGF-1 expression was increased in senescent IMR-90 cells. IGF-1 levels in media from young or senescent IMR-90 cells were measured using ELISAs as described above, and normalized to media volume and adjusted cell number (4×104 cells). (B) Total IGF-1 receptor (IGF-1R), phosphorylated IGF-1 receptor (p-IGF-1R), Src, phosphorylated Src, Erk-1/2, phosphorylated-Erk-1/2, and Egr-1 levels in young (Y), middle-age (M), premature-senescent (PS), and senescent (S) IMR-90 cells were determined by western blot analyses. β-Actin levels were measured to monitor loading. (C) Exposure to the indicated doses of the IGF-1R tyrosine kinase inhibitor (AG1024) repressed sCLU levels in senescent IMR-90 cells. psCLU and sCLU were detected by western blot analysis. β-Actin levels were monitored to show equal loading. Senescent IMR-90 cells exposed to varying doses of AG1024 (µM as indicated for 24 h) showed dose-dependent increases in apoptosis (monitored by TUNEL + staining). Only senescent, and not young or middle-aged IMR-90, cells were hypersensitive to AG1024. In the experiment outlined below the western blot analyses cells were exposed to AG1024 (5 µM) for 48 h and therefore higher levels of apoptotic cells were noted in senescent IMR-90 cells compared to cells exposed for 24 h above. Note that only senescent cells were hypersensitive to these inhibitor treatments. All experiments were repeated three times in triplicate.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4061041&req=5

pone-0099983-g004: sCLU induction during senescence is mediated through IGF-1R/MAPK/Erk/Egr-1 signaling pathway.(A) IGF-1 expression was increased in senescent IMR-90 cells. IGF-1 levels in media from young or senescent IMR-90 cells were measured using ELISAs as described above, and normalized to media volume and adjusted cell number (4×104 cells). (B) Total IGF-1 receptor (IGF-1R), phosphorylated IGF-1 receptor (p-IGF-1R), Src, phosphorylated Src, Erk-1/2, phosphorylated-Erk-1/2, and Egr-1 levels in young (Y), middle-age (M), premature-senescent (PS), and senescent (S) IMR-90 cells were determined by western blot analyses. β-Actin levels were measured to monitor loading. (C) Exposure to the indicated doses of the IGF-1R tyrosine kinase inhibitor (AG1024) repressed sCLU levels in senescent IMR-90 cells. psCLU and sCLU were detected by western blot analysis. β-Actin levels were monitored to show equal loading. Senescent IMR-90 cells exposed to varying doses of AG1024 (µM as indicated for 24 h) showed dose-dependent increases in apoptosis (monitored by TUNEL + staining). Only senescent, and not young or middle-aged IMR-90, cells were hypersensitive to AG1024. In the experiment outlined below the western blot analyses cells were exposed to AG1024 (5 µM) for 48 h and therefore higher levels of apoptotic cells were noted in senescent IMR-90 cells compared to cells exposed for 24 h above. Note that only senescent cells were hypersensitive to these inhibitor treatments. All experiments were repeated three times in triplicate.
Mentions: Our data strongly suggested that functional ATM could regulate IGF-1 expression during RS or SIPS pathways. Indeed, IGF-1 expression was approximately two-fold higher in conditioned media from senescent compared to young IMR-90 cells (Figure 4A). Analyses of differential signal transduction responses in senescent versus young IMR-90 cells showed that IGF-1 receptor (IGF-1R) levels were activated (elevated P-IGF-1R/t-IGF-1R) in senescent cells; antibodies to human IGF-1R are not ideal. Src and Erk-1/2 kinase levels were also elevated in middle-aged and senescent compared to young IMR-90 cells. The transcription factor, Egr-1, which binds the hCLU promoter and mediates sCLU expression [28], was significantly increased during senescence in IMR-90 cells (Figure 4B). To further confirm the specificity of IGF-1/IGF-1R/MAPK/ERK-1/2/Egr-1 signaling in sCLU induction during senescence, we treated senescent IMR-90 cells with the IGF-1R inhibitor, AG1024, which effectively blocked IR-induced sCLU expression [27], [28] and also suppressed sCLU expression in senescent IMR-90 cells in a dose-dependent manner (Figure 4C). While significantly suppressing sCLU expression, we also noted that senescent IMR-90 cells exposed to the IGF-1R inhibitor (AG1024) underwent significant programmed cell death (apoptotic) responses in a dose-dependent manner (Figure 4C). Interestingly, young and middle-aged cells did not respond to AG1024 in the same manner with no significant apoptosis noted after exposure to 5 µM of the drug (Figure 4C). Collectively, these results strongly suggest that when cell undergo RS or SIPS, but not oncogene-induced senescence, ATM-dependent DDR leads to increased IGF-1 expression that stimulates IGF-1R/MAPK/Erk-1/2/Egr-1 signaling that leads to concomitant elevated sCLU levels. The data further suggest that the IGF-1/IGF-1R-sCLU pathway is required for the overall survival of senescent cells, whereby suppressing this pathway induces an intracellular death decision.

Bottom Line: In contrast, administration of an IGF-1 inhibitor caused apoptosis of senescent cells.Thus, IGF-1 signaling is required for survival, whereas sCLU appears to protect cells from premature senescence, as IMR-90 cells with sCLU knockdown undergo senescence faster than control cells.Thus, the ATM-IGF-1-sCLU pathway protects cells from lethality and suspends senescence.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Radiation Oncology, Laboratory of Molecular Cell Stress Responses, Program in Cell Stress and Cancer Nanomedicine, Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
Downstream factors that regulate the decision between senescence and cell death have not been elucidated. Cells undergo senescence through three pathways, replicative senescence (RS), stress-induced premature senescence (SIPS) and oncogene-induced senescence. Recent studies suggest that the ataxia telangiectasia mutant (ATM) kinase is not only a key protein mediating cellular responses to DNA damage, but also regulates cellular senescence induced by telomere end exposure (in RS) or persistent DNA damage (in SIPS). Here, we show that expression of secretory clusterin (sCLU), a known pro-survival extracellular chaperone, is transcriptionally up-regulated during both RS and SIPS, but not in oncogene-induced senescence, consistent with a DNA damage-inducible mechanism. We demonstrate that ATM plays an important role in insulin-like growth factor 1 (IGF-1) expression, that in turn, regulates downstream sCLU induction during senescence. Loss of ATM activity, either by genomic mutation (ATM-deficient fibroblasts from an ataxia telangiectasia patient) or by administration of a chemical inhibitor (AAI, an inhibitor of ATM and ATR), blocks IGF-1-sCLU expression in senescent cells. Downstream, sCLU induction during senescence is mediated by IGF-1R/MAPK/Egr-1 signaling, identical to its induction after DNA damage. In contrast, administration of an IGF-1 inhibitor caused apoptosis of senescent cells. Thus, IGF-1 signaling is required for survival, whereas sCLU appears to protect cells from premature senescence, as IMR-90 cells with sCLU knockdown undergo senescence faster than control cells. Thus, the ATM-IGF-1-sCLU pathway protects cells from lethality and suspends senescence.

Show MeSH
Related in: MedlinePlus