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A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

Guerra L, Carr HS, Richter-Dahlfors A, Masucci MG, Thelestam M, Frost JA, Frisan T - PLoS ONE (2008)

Bottom Line: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR.Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress.The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

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Summary of the Net1-regulated survival signals upon exposure to genotoxic agents.Upon intoxication or irradiation, Net1 is dephosphorylated and induces activation of RhoA, leading to a RhoA dependent phosphorylation of p38 MAPK and its downstream effector protein MK2. This signalling pathway can be blocked at different levels by: i) iRNA knock down of endogenous Net1 levels or expression of the dominant negative Net1ΔDH; ii) C3-mediated RhoA inhibition or iRNA knock down of endogenous RhoA levels; iii) p38 specific inhibitors. In each case, the effect of this interference results in enhanced cell death in response to genotoxic agents.
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pone-0002254-g011: Summary of the Net1-regulated survival signals upon exposure to genotoxic agents.Upon intoxication or irradiation, Net1 is dephosphorylated and induces activation of RhoA, leading to a RhoA dependent phosphorylation of p38 MAPK and its downstream effector protein MK2. This signalling pathway can be blocked at different levels by: i) iRNA knock down of endogenous Net1 levels or expression of the dominant negative Net1ΔDH; ii) C3-mediated RhoA inhibition or iRNA knock down of endogenous RhoA levels; iii) p38 specific inhibitors. In each case, the effect of this interference results in enhanced cell death in response to genotoxic agents.

Mentions: The current literature identifies JNK as the main MAPK induced by irradiation [17]. Consistent with our previous results showing that CDT or IR do not activate the JNK regulators Rac and Cdc42 [6], we detected only low levels of JNK phosphorylation and AP1 activation 4h and 12h after intoxication in HeLa cells (data not shown). In contrast, up to 10 fold increase of p38 MAPK phosphorylation was consistently observed both in HeLa and HCT116 cells (Figures 5 and 6). This result is not surprising since, depending on the experimental models, p38 MAPK was shown to contribute to either survival or death signals in response to DNA damage [17]. The downstream signals involved in the survival response remain unclear. Reinhardt at al. recently showed that the survival in p53-deficient fibroblasts exposed to cisplatin and doxorubicin is enhanced by ATM-dependent activation of p38 MAPK and its downstream effector MK2 [28]. This pathway has been defined as the third cell cycle-dependent checkpoint, in addition to the well-characterized ATM/Chk2 and ATR/Chk1 responses [28]. Our findings demonstrate that these signals operate also in tumor cells, such as HeLa and HCT116, since blockage of p38 MAPK activation by specific inhibitor abrogates their capacity to survive irradiation or intoxication (Figures 5 and 6). Furthermore, we have identified Net1 and RhoA as key molecules controlling the activation of this novel checkpoint pathway (Figures 7 and 8). A schematic illustration of the Net1-regulated signal cascade identified in this work is shown in Figure 11.


A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

Guerra L, Carr HS, Richter-Dahlfors A, Masucci MG, Thelestam M, Frost JA, Frisan T - PLoS ONE (2008)

Summary of the Net1-regulated survival signals upon exposure to genotoxic agents.Upon intoxication or irradiation, Net1 is dephosphorylated and induces activation of RhoA, leading to a RhoA dependent phosphorylation of p38 MAPK and its downstream effector protein MK2. This signalling pathway can be blocked at different levels by: i) iRNA knock down of endogenous Net1 levels or expression of the dominant negative Net1ΔDH; ii) C3-mediated RhoA inhibition or iRNA knock down of endogenous RhoA levels; iii) p38 specific inhibitors. In each case, the effect of this interference results in enhanced cell death in response to genotoxic agents.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2386254&req=5

pone-0002254-g011: Summary of the Net1-regulated survival signals upon exposure to genotoxic agents.Upon intoxication or irradiation, Net1 is dephosphorylated and induces activation of RhoA, leading to a RhoA dependent phosphorylation of p38 MAPK and its downstream effector protein MK2. This signalling pathway can be blocked at different levels by: i) iRNA knock down of endogenous Net1 levels or expression of the dominant negative Net1ΔDH; ii) C3-mediated RhoA inhibition or iRNA knock down of endogenous RhoA levels; iii) p38 specific inhibitors. In each case, the effect of this interference results in enhanced cell death in response to genotoxic agents.
Mentions: The current literature identifies JNK as the main MAPK induced by irradiation [17]. Consistent with our previous results showing that CDT or IR do not activate the JNK regulators Rac and Cdc42 [6], we detected only low levels of JNK phosphorylation and AP1 activation 4h and 12h after intoxication in HeLa cells (data not shown). In contrast, up to 10 fold increase of p38 MAPK phosphorylation was consistently observed both in HeLa and HCT116 cells (Figures 5 and 6). This result is not surprising since, depending on the experimental models, p38 MAPK was shown to contribute to either survival or death signals in response to DNA damage [17]. The downstream signals involved in the survival response remain unclear. Reinhardt at al. recently showed that the survival in p53-deficient fibroblasts exposed to cisplatin and doxorubicin is enhanced by ATM-dependent activation of p38 MAPK and its downstream effector MK2 [28]. This pathway has been defined as the third cell cycle-dependent checkpoint, in addition to the well-characterized ATM/Chk2 and ATR/Chk1 responses [28]. Our findings demonstrate that these signals operate also in tumor cells, such as HeLa and HCT116, since blockage of p38 MAPK activation by specific inhibitor abrogates their capacity to survive irradiation or intoxication (Figures 5 and 6). Furthermore, we have identified Net1 and RhoA as key molecules controlling the activation of this novel checkpoint pathway (Figures 7 and 8). A schematic illustration of the Net1-regulated signal cascade identified in this work is shown in Figure 11.

Bottom Line: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR.Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress.The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

Show MeSH
Related in: MedlinePlus