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GADD45β mediates p53 protein degradation via Src/PP2A/MDM2 pathway upon arsenite treatment.

Yu Y, Huang H, Li J, Zhang J, Gao J, Lu B, Huang C - Cell Death Dis (2013)

Bottom Line: We demonstrated here that GADD45β mediated its anti-apoptotic effect via promoting p53 protein degradation following arsenite treatment.Collectively, our results demonstrate a novel molecular mechanism responsible for GADD45β protection of arsenite-exposed cells from cell death, which provides insight into our understanding of GADD45β function and a unique compound arsenite as both a cancer therapeutic reagent and an environmental carcinogen.Those novel findings may also enable us to design more effective strategies for utilization of arsenite for the treatment of cancers.

View Article: PubMed Central - PubMed

Affiliation: Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA.

ABSTRACT
Growth arrest and DNA-damage-inducible, beta (GADD45β) has been reported to inhibit apoptosis via attenuating c-Jun N-terminal kinase (JNK) activation. We demonstrated here that GADD45β mediated its anti-apoptotic effect via promoting p53 protein degradation following arsenite treatment. We found that p53 protein expression was upregulated in GADD45β-/- cells upon arsenite exposure as compared with those in GADD45β+/+ cells. Further studies showed that GADD45β attenuated p53 protein expression through Src/protein phosphatase 2A/murine double minute 2-dependent p53 protein-degradation pathway. Moreover, we identified that GADD45β-mediated p53 protein degradation was crucial for its anti-apoptotic effect due to arsenite exposure, whereas increased JNK activation was not involved in the increased cell apoptotic response in GADD45β-/- cells under same experimental conditions. Collectively, our results demonstrate a novel molecular mechanism responsible for GADD45β protection of arsenite-exposed cells from cell death, which provides insight into our understanding of GADD45β function and a unique compound arsenite as both a cancer therapeutic reagent and an environmental carcinogen. Those novel findings may also enable us to design more effective strategies for utilization of arsenite for the treatment of cancers.

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GADD45β protected arsenite-treated cells from death. GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate till 80% confluent. The cell culture medium was replaced with 0.1% FBS DMEM for 12 h and then subjected to arsenite treatment at indicated doses. (a) The cells were extracted, and protein samples were subjected to western blotting with specific antibodies as indicated. (b) Cell morphology images were taken under microscope. (c) Cells were subjected to flow-cytometry analysis as described in Materials and Methods
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fig1: GADD45β protected arsenite-treated cells from death. GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate till 80% confluent. The cell culture medium was replaced with 0.1% FBS DMEM for 12 h and then subjected to arsenite treatment at indicated doses. (a) The cells were extracted, and protein samples were subjected to western blotting with specific antibodies as indicated. (b) Cell morphology images were taken under microscope. (c) Cells were subjected to flow-cytometry analysis as described in Materials and Methods

Mentions: GADD45β has been reported to protect hematopoietic cells from UV-induced apoptosis in JNK-dependent pathway,2 and our previous study shows that arsenite treatment induces GADD45β protein expression.6 To evaluate potential role and molecular basis of GADD45β induction in arsenite response, GADD45β+/+ and GADD45β−/− cells were employed. As shown in Figure 1a, arsenite treatment increased GADD45β protein expression in GADD45β+/+ cells, whereas as expected, there was no detectable GADD45β in GADD45β−/− cells. Our results also showed that arsenite exposure resulted in markedly cell death in a dose-dependent manner in GADD45β−/− cells, whereas GADD45β+/+ cells only showed a slightly morphological alteration and inhibition of cell growth under same experimental conditions (Figure 1b). Consistent with cell morphological alterations, flow-cytometry analysis suggested that arsenite treatment led to significant cell death in GADD45β−/− cells, whereas there was no detectable cell death in GADD45β+/+ cells under the same treatment (Figure 1c). This cell death was due to apoptosis because the cell death was consistent with the results obtained from detection of two well-characterized cell apoptotic markers, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) (Figure 1a). Our results strongly suggested that GADD45β induction by arsenite did exhibit a protection from cell death. As published studies have shown that GADD45β suppressed cell apoptosis through directly binding to MKK7 and inhibiting JNK activation,2, 8, 11 we compared MAPKs activation between GADD45β+/+ and GADD45β−/− cells following arsenite treatment. Consistent with previous reports, GADD45β deficiency (GADD45β−/−) resulted in increased JNK activation by arsenite in comparison with that in GADD45β+/+ cells (Figure 2a), and p38 and extracellular signal-regulated kinase (Erk) activation was also slightly elevated in GADD45β−/− cells (Figure 2a). To determine whether elevation of JNK activation had a role in the increased apoptosis upon arsenite treatment, a specific JNK inhibitor SP600125 was employed. The results showed that inhibition of JNK activation by SP600125 pretreatment did not show observable reduction of arsenite-induced apoptosis indicated by cleaved caspase 3 and cleaved PARP (Figure 2b). These results suggested that although JNK activation was elevated in GADD45β−/− cells, it did not contribute to increased sensitivity of GADD45β−/− cells to arsenite-induced apoptotic responses.


GADD45β mediates p53 protein degradation via Src/PP2A/MDM2 pathway upon arsenite treatment.

Yu Y, Huang H, Li J, Zhang J, Gao J, Lu B, Huang C - Cell Death Dis (2013)

GADD45β protected arsenite-treated cells from death. GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate till 80% confluent. The cell culture medium was replaced with 0.1% FBS DMEM for 12 h and then subjected to arsenite treatment at indicated doses. (a) The cells were extracted, and protein samples were subjected to western blotting with specific antibodies as indicated. (b) Cell morphology images were taken under microscope. (c) Cells were subjected to flow-cytometry analysis as described in Materials and Methods
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3674369&req=5

fig1: GADD45β protected arsenite-treated cells from death. GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate till 80% confluent. The cell culture medium was replaced with 0.1% FBS DMEM for 12 h and then subjected to arsenite treatment at indicated doses. (a) The cells were extracted, and protein samples were subjected to western blotting with specific antibodies as indicated. (b) Cell morphology images were taken under microscope. (c) Cells were subjected to flow-cytometry analysis as described in Materials and Methods
Mentions: GADD45β has been reported to protect hematopoietic cells from UV-induced apoptosis in JNK-dependent pathway,2 and our previous study shows that arsenite treatment induces GADD45β protein expression.6 To evaluate potential role and molecular basis of GADD45β induction in arsenite response, GADD45β+/+ and GADD45β−/− cells were employed. As shown in Figure 1a, arsenite treatment increased GADD45β protein expression in GADD45β+/+ cells, whereas as expected, there was no detectable GADD45β in GADD45β−/− cells. Our results also showed that arsenite exposure resulted in markedly cell death in a dose-dependent manner in GADD45β−/− cells, whereas GADD45β+/+ cells only showed a slightly morphological alteration and inhibition of cell growth under same experimental conditions (Figure 1b). Consistent with cell morphological alterations, flow-cytometry analysis suggested that arsenite treatment led to significant cell death in GADD45β−/− cells, whereas there was no detectable cell death in GADD45β+/+ cells under the same treatment (Figure 1c). This cell death was due to apoptosis because the cell death was consistent with the results obtained from detection of two well-characterized cell apoptotic markers, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) (Figure 1a). Our results strongly suggested that GADD45β induction by arsenite did exhibit a protection from cell death. As published studies have shown that GADD45β suppressed cell apoptosis through directly binding to MKK7 and inhibiting JNK activation,2, 8, 11 we compared MAPKs activation between GADD45β+/+ and GADD45β−/− cells following arsenite treatment. Consistent with previous reports, GADD45β deficiency (GADD45β−/−) resulted in increased JNK activation by arsenite in comparison with that in GADD45β+/+ cells (Figure 2a), and p38 and extracellular signal-regulated kinase (Erk) activation was also slightly elevated in GADD45β−/− cells (Figure 2a). To determine whether elevation of JNK activation had a role in the increased apoptosis upon arsenite treatment, a specific JNK inhibitor SP600125 was employed. The results showed that inhibition of JNK activation by SP600125 pretreatment did not show observable reduction of arsenite-induced apoptosis indicated by cleaved caspase 3 and cleaved PARP (Figure 2b). These results suggested that although JNK activation was elevated in GADD45β−/− cells, it did not contribute to increased sensitivity of GADD45β−/− cells to arsenite-induced apoptotic responses.

Bottom Line: We demonstrated here that GADD45β mediated its anti-apoptotic effect via promoting p53 protein degradation following arsenite treatment.Collectively, our results demonstrate a novel molecular mechanism responsible for GADD45β protection of arsenite-exposed cells from cell death, which provides insight into our understanding of GADD45β function and a unique compound arsenite as both a cancer therapeutic reagent and an environmental carcinogen.Those novel findings may also enable us to design more effective strategies for utilization of arsenite for the treatment of cancers.

View Article: PubMed Central - PubMed

Affiliation: Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, NY 10987, USA.

ABSTRACT
Growth arrest and DNA-damage-inducible, beta (GADD45β) has been reported to inhibit apoptosis via attenuating c-Jun N-terminal kinase (JNK) activation. We demonstrated here that GADD45β mediated its anti-apoptotic effect via promoting p53 protein degradation following arsenite treatment. We found that p53 protein expression was upregulated in GADD45β-/- cells upon arsenite exposure as compared with those in GADD45β+/+ cells. Further studies showed that GADD45β attenuated p53 protein expression through Src/protein phosphatase 2A/murine double minute 2-dependent p53 protein-degradation pathway. Moreover, we identified that GADD45β-mediated p53 protein degradation was crucial for its anti-apoptotic effect due to arsenite exposure, whereas increased JNK activation was not involved in the increased cell apoptotic response in GADD45β-/- cells under same experimental conditions. Collectively, our results demonstrate a novel molecular mechanism responsible for GADD45β protection of arsenite-exposed cells from cell death, which provides insight into our understanding of GADD45β function and a unique compound arsenite as both a cancer therapeutic reagent and an environmental carcinogen. Those novel findings may also enable us to design more effective strategies for utilization of arsenite for the treatment of cancers.

Show MeSH
Related in: MedlinePlus