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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 depletion stabilized p53 protein through dephosphorylating MDM2. (a) GADD45β+/+ and GADD45β−/− cells were exposed to arsenite as indicated dose for 12 h. The cell extracts were subjected to western blotting with specific antibodies against p-p53 Ser15, p53, GADD45α, Bax, and PUMA. (b) After arsenite treatment for 6 h, total RNA was extracted, and reverse transcription was performed as described in Materials and Methods. Bax, puma, and p53 mRNA level in GADD45β+/+ and GADD45β−/− cells with or without arsenite treatment was analyzed by PCR. (c) GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate and were then pretreated with 10 μM of MG132 for 4 h. The cell culture medium was replaced by fresh medium containing 10 μM CHX with or without arsenite as indicated. The cells were extracted for determination of p53 and p21 protein levels at time points as indicated. (d) GADD45β+/+ and GADD45β−/− cells were treated by 10 μM of arsenite for indicated time, and cell extracts were subjected to western blotting with specific antibodies against p-MDM2 Ser166 or MDM2. (e and f) The cells of HCT116, HCT116/Bax−/− and HCT116/PUMA−/− were identified (e), and the expressions of caspase3 and cleaved caspase3 in these three cells with or without arsenite treatment as indicated dose were detected using western blotting assay (f)
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fig3: GADD45β depletion stabilized p53 protein through dephosphorylating MDM2. (a) GADD45β+/+ and GADD45β−/− cells were exposed to arsenite as indicated dose for 12 h. The cell extracts were subjected to western blotting with specific antibodies against p-p53 Ser15, p53, GADD45α, Bax, and PUMA. (b) After arsenite treatment for 6 h, total RNA was extracted, and reverse transcription was performed as described in Materials and Methods. Bax, puma, and p53 mRNA level in GADD45β+/+ and GADD45β−/− cells with or without arsenite treatment was analyzed by PCR. (c) GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate and were then pretreated with 10 μM of MG132 for 4 h. The cell culture medium was replaced by fresh medium containing 10 μM CHX with or without arsenite as indicated. The cells were extracted for determination of p53 and p21 protein levels at time points as indicated. (d) GADD45β+/+ and GADD45β−/− cells were treated by 10 μM of arsenite for indicated time, and cell extracts were subjected to western blotting with specific antibodies against p-MDM2 Ser166 or MDM2. (e and f) The cells of HCT116, HCT116/Bax−/− and HCT116/PUMA−/− were identified (e), and the expressions of caspase3 and cleaved caspase3 in these three cells with or without arsenite treatment as indicated dose were detected using western blotting assay (f)

Mentions: Our most recent study has shown that arsenite-induced p53 protein induction via p50 (NFκB1)-mediated miR-190/PH-domain and leucine-rich repeat protein phosphatase 1/Akt pathway is essential for apoptotic response.21 To test whether GADD45β participated in the regulation of p53 protein expression upon arsenite exposure, we evaluated p53 protein induction in both GADD45β+/+ and GADD45β−/− cells. As shown in Figure 3a, arsenite-induced p53 protein level was remarkably increased in GADD45β−/− cells compared with that in GADD45β+/+ cells. It has been known that p53 phosphorylation at Ser15 attenuated its binding with MDM2 and enhanced p53 protein accumulation.22 Thus, we also determined p53 phosphorylation at Ser15 in both cell lines. The results showed that consistent with total p53 protein expression, p53 phosphorylation at Ser15 was also elevated in GADD45β−/− cells. The results obtained from determination of p53 mRNA levels in both cell lines strongly revealed that p53 mRNA level was regulated by neither arsenite treatment nor GADD45β expression (Figure 3b), suggesting that GADD45β might mediate p53 protein expression at either protein degradation or translation. We therefore compared p53 protein-degradation rates between GADD45β+/+ and GADD45β−/− cells. The data showed that p53 protein-degradation rate in GADD45β−/− cells was much slower than that in GADD45β+/+ cells (Figure 3c), and arsenite treatment could delay p53 protein degradation in both cell lines (Figure 3c). In contrast to p53 protein, p21 protein degradation was faster in GADD45β−/− cells as compared with that in GADD45β+/+ cells (Figure 3c). It has been well known that MDM2 phosphorylation at Ser166 increases its binding activity to p53 protein and mediates p53 protein degradation.22, 23 So we compared MDM2 phosphorylation at Ser166 between the GADD45β+/+ and GADD45β−/− cells following arsenite treatment. The results indicated that MDM2 phosphorylation at Ser166 was much lower in GADD45β−/− cells in comparison with that in GADD45β+/+ cells (Figure 3d), however, GADD45β deletion did not affect total MDM2 expression (Figure 3d), suggesting that GADD45β regulated p53 protein degradation via mediating MDM2 protein phosphorylation at Ser166, rather than affecting total MDM2 expression.


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 depletion stabilized p53 protein through dephosphorylating MDM2. (a) GADD45β+/+ and GADD45β−/− cells were exposed to arsenite as indicated dose for 12 h. The cell extracts were subjected to western blotting with specific antibodies against p-p53 Ser15, p53, GADD45α, Bax, and PUMA. (b) After arsenite treatment for 6 h, total RNA was extracted, and reverse transcription was performed as described in Materials and Methods. Bax, puma, and p53 mRNA level in GADD45β+/+ and GADD45β−/− cells with or without arsenite treatment was analyzed by PCR. (c) GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate and were then pretreated with 10 μM of MG132 for 4 h. The cell culture medium was replaced by fresh medium containing 10 μM CHX with or without arsenite as indicated. The cells were extracted for determination of p53 and p21 protein levels at time points as indicated. (d) GADD45β+/+ and GADD45β−/− cells were treated by 10 μM of arsenite for indicated time, and cell extracts were subjected to western blotting with specific antibodies against p-MDM2 Ser166 or MDM2. (e and f) The cells of HCT116, HCT116/Bax−/− and HCT116/PUMA−/− were identified (e), and the expressions of caspase3 and cleaved caspase3 in these three cells with or without arsenite treatment as indicated dose were detected using western blotting assay (f)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: GADD45β depletion stabilized p53 protein through dephosphorylating MDM2. (a) GADD45β+/+ and GADD45β−/− cells were exposed to arsenite as indicated dose for 12 h. The cell extracts were subjected to western blotting with specific antibodies against p-p53 Ser15, p53, GADD45α, Bax, and PUMA. (b) After arsenite treatment for 6 h, total RNA was extracted, and reverse transcription was performed as described in Materials and Methods. Bax, puma, and p53 mRNA level in GADD45β+/+ and GADD45β−/− cells with or without arsenite treatment was analyzed by PCR. (c) GADD45β+/+ and GADD45β−/− cells were seeded into six-well plate and were then pretreated with 10 μM of MG132 for 4 h. The cell culture medium was replaced by fresh medium containing 10 μM CHX with or without arsenite as indicated. The cells were extracted for determination of p53 and p21 protein levels at time points as indicated. (d) GADD45β+/+ and GADD45β−/− cells were treated by 10 μM of arsenite for indicated time, and cell extracts were subjected to western blotting with specific antibodies against p-MDM2 Ser166 or MDM2. (e and f) The cells of HCT116, HCT116/Bax−/− and HCT116/PUMA−/− were identified (e), and the expressions of caspase3 and cleaved caspase3 in these three cells with or without arsenite treatment as indicated dose were detected using western blotting assay (f)
Mentions: Our most recent study has shown that arsenite-induced p53 protein induction via p50 (NFκB1)-mediated miR-190/PH-domain and leucine-rich repeat protein phosphatase 1/Akt pathway is essential for apoptotic response.21 To test whether GADD45β participated in the regulation of p53 protein expression upon arsenite exposure, we evaluated p53 protein induction in both GADD45β+/+ and GADD45β−/− cells. As shown in Figure 3a, arsenite-induced p53 protein level was remarkably increased in GADD45β−/− cells compared with that in GADD45β+/+ cells. It has been known that p53 phosphorylation at Ser15 attenuated its binding with MDM2 and enhanced p53 protein accumulation.22 Thus, we also determined p53 phosphorylation at Ser15 in both cell lines. The results showed that consistent with total p53 protein expression, p53 phosphorylation at Ser15 was also elevated in GADD45β−/− cells. The results obtained from determination of p53 mRNA levels in both cell lines strongly revealed that p53 mRNA level was regulated by neither arsenite treatment nor GADD45β expression (Figure 3b), suggesting that GADD45β might mediate p53 protein expression at either protein degradation or translation. We therefore compared p53 protein-degradation rates between GADD45β+/+ and GADD45β−/− cells. The data showed that p53 protein-degradation rate in GADD45β−/− cells was much slower than that in GADD45β+/+ cells (Figure 3c), and arsenite treatment could delay p53 protein degradation in both cell lines (Figure 3c). In contrast to p53 protein, p21 protein degradation was faster in GADD45β−/− cells as compared with that in GADD45β+/+ cells (Figure 3c). It has been well known that MDM2 phosphorylation at Ser166 increases its binding activity to p53 protein and mediates p53 protein degradation.22, 23 So we compared MDM2 phosphorylation at Ser166 between the GADD45β+/+ and GADD45β−/− cells following arsenite treatment. The results indicated that MDM2 phosphorylation at Ser166 was much lower in GADD45β−/− cells in comparison with that in GADD45β+/+ cells (Figure 3d), however, GADD45β deletion did not affect total MDM2 expression (Figure 3d), suggesting that GADD45β regulated p53 protein degradation via mediating MDM2 protein phosphorylation at Ser166, rather than affecting total MDM2 expression.

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