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Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production.

Wang B, Liu TY, Lai CH, Rao YH, Choi MC, Chi JT, Dai JW, Rathmell JC, Yao TP - Mol. Biol. Cell (2014)

Bottom Line: Inhibition of GSK3β or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation.We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation.Of importance, a caspase-3-resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710 Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

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Glycolysis-coupled caspase-3 activation leads to LPS-induced HDAC4 degradation. (A) LPS promoted glucose uptake in BV2 cells at 12 and 24 h (**p < 0.01 and p < 0.001 vs. LPS 0h). (B) Glycolytic rate was determined in activated BV2 cells. LPS dramatically increased glycolytic rate at 12 and 24 h (***p < 0.001 vs. LPS 0h). (C) HDAC4 KD had no apparent effect on glucose uptake in BV2 cells upon LPS treatment at 12 h. (D, E) BV2 cells were pretreated with 2-dexoy-d-glucose (2DG; 1 μM) or oxamic acid (30 mM) for 4 h, followed by LPS treatment for 24 h. Both 2DG and oxamic acid pretreatment markedly blunted LPS-induced HDAC4 degradation. (F) HDAC4 and active caspase-3 protein levels in BV2 cells were determined by immunoblotting at indicated time points after LPS treatment. Note that the appearance and abundance of active caspase-3 coincided with the loss of HDAC4. (G) BV2 cells were pretreated with pan-caspase inhibitor Z-VAD-FMK for 4 h at indicated concentrations, followed by LPS treatment for 24 h. High dose (50 μM) of Z-VAD-FMK effectively abolished caspase-3 activation and prevented HDAC4 degradation. (H) BV2 cells were transfected with a siRNA for caspase-3, followed by 24 h LPS treatment. Caspase-3 KD effectively abolished LPS-induced HDAC4 degradation. (I) BV2 cells were untreated or pretreated with Z-VAD-FMK for 4 h, followed by LPS treatment. At the indicated time points, apoptosis was analyzed by fluorescence-activated cell sorting using Annexin V-PE and 7-AAD Apoptosis Detection Kit. Pretreatment of Z-VAD-FMK did not suppress LPS-induced apoptosis (***p < 0.001 vs. control).
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Figure 3: Glycolysis-coupled caspase-3 activation leads to LPS-induced HDAC4 degradation. (A) LPS promoted glucose uptake in BV2 cells at 12 and 24 h (**p < 0.01 and p < 0.001 vs. LPS 0h). (B) Glycolytic rate was determined in activated BV2 cells. LPS dramatically increased glycolytic rate at 12 and 24 h (***p < 0.001 vs. LPS 0h). (C) HDAC4 KD had no apparent effect on glucose uptake in BV2 cells upon LPS treatment at 12 h. (D, E) BV2 cells were pretreated with 2-dexoy-d-glucose (2DG; 1 μM) or oxamic acid (30 mM) for 4 h, followed by LPS treatment for 24 h. Both 2DG and oxamic acid pretreatment markedly blunted LPS-induced HDAC4 degradation. (F) HDAC4 and active caspase-3 protein levels in BV2 cells were determined by immunoblotting at indicated time points after LPS treatment. Note that the appearance and abundance of active caspase-3 coincided with the loss of HDAC4. (G) BV2 cells were pretreated with pan-caspase inhibitor Z-VAD-FMK for 4 h at indicated concentrations, followed by LPS treatment for 24 h. High dose (50 μM) of Z-VAD-FMK effectively abolished caspase-3 activation and prevented HDAC4 degradation. (H) BV2 cells were transfected with a siRNA for caspase-3, followed by 24 h LPS treatment. Caspase-3 KD effectively abolished LPS-induced HDAC4 degradation. (I) BV2 cells were untreated or pretreated with Z-VAD-FMK for 4 h, followed by LPS treatment. At the indicated time points, apoptosis was analyzed by fluorescence-activated cell sorting using Annexin V-PE and 7-AAD Apoptosis Detection Kit. Pretreatment of Z-VAD-FMK did not suppress LPS-induced apoptosis (***p < 0.001 vs. control).

Mentions: At the time when HDAC4 protein degradation became apparent, 18–24 h after LPS treatment, we noticed that the culture medium had turned visibly yellow, indicative of acidification. Because LPS-challenged macrophages activate glycolysis and produce lactate (Rodriguez-Prados et al., 2010), whose accumulation would acidify the medium, we asked whether HDAC4 degradation was linked to glycolysis. We confirmed that glucose uptake and glycolysis were indeed elevated in BV2 cells upon LPS treatment, and knockdown of HDAC4 had no apparent effect on this activity (Figure 3, A–C). Of importance, inhibition of glycolysis by a nonmetabolizable glucose analogue, 2-deoxyglucose (2-DG), or a lactate dehydrogenase inhibitor, oxamic acid, effectively blunted LPS-induced HDAC4 degradation in BV2 cells (Figure 3, D and E, top). These results indicate that active glycolysis is required for LPS-induced HDAC4 degradation.


Glycolysis-dependent histone deacetylase 4 degradation regulates inflammatory cytokine production.

Wang B, Liu TY, Lai CH, Rao YH, Choi MC, Chi JT, Dai JW, Rathmell JC, Yao TP - Mol. Biol. Cell (2014)

Glycolysis-coupled caspase-3 activation leads to LPS-induced HDAC4 degradation. (A) LPS promoted glucose uptake in BV2 cells at 12 and 24 h (**p < 0.01 and p < 0.001 vs. LPS 0h). (B) Glycolytic rate was determined in activated BV2 cells. LPS dramatically increased glycolytic rate at 12 and 24 h (***p < 0.001 vs. LPS 0h). (C) HDAC4 KD had no apparent effect on glucose uptake in BV2 cells upon LPS treatment at 12 h. (D, E) BV2 cells were pretreated with 2-dexoy-d-glucose (2DG; 1 μM) or oxamic acid (30 mM) for 4 h, followed by LPS treatment for 24 h. Both 2DG and oxamic acid pretreatment markedly blunted LPS-induced HDAC4 degradation. (F) HDAC4 and active caspase-3 protein levels in BV2 cells were determined by immunoblotting at indicated time points after LPS treatment. Note that the appearance and abundance of active caspase-3 coincided with the loss of HDAC4. (G) BV2 cells were pretreated with pan-caspase inhibitor Z-VAD-FMK for 4 h at indicated concentrations, followed by LPS treatment for 24 h. High dose (50 μM) of Z-VAD-FMK effectively abolished caspase-3 activation and prevented HDAC4 degradation. (H) BV2 cells were transfected with a siRNA for caspase-3, followed by 24 h LPS treatment. Caspase-3 KD effectively abolished LPS-induced HDAC4 degradation. (I) BV2 cells were untreated or pretreated with Z-VAD-FMK for 4 h, followed by LPS treatment. At the indicated time points, apoptosis was analyzed by fluorescence-activated cell sorting using Annexin V-PE and 7-AAD Apoptosis Detection Kit. Pretreatment of Z-VAD-FMK did not suppress LPS-induced apoptosis (***p < 0.001 vs. control).
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Figure 3: Glycolysis-coupled caspase-3 activation leads to LPS-induced HDAC4 degradation. (A) LPS promoted glucose uptake in BV2 cells at 12 and 24 h (**p < 0.01 and p < 0.001 vs. LPS 0h). (B) Glycolytic rate was determined in activated BV2 cells. LPS dramatically increased glycolytic rate at 12 and 24 h (***p < 0.001 vs. LPS 0h). (C) HDAC4 KD had no apparent effect on glucose uptake in BV2 cells upon LPS treatment at 12 h. (D, E) BV2 cells were pretreated with 2-dexoy-d-glucose (2DG; 1 μM) or oxamic acid (30 mM) for 4 h, followed by LPS treatment for 24 h. Both 2DG and oxamic acid pretreatment markedly blunted LPS-induced HDAC4 degradation. (F) HDAC4 and active caspase-3 protein levels in BV2 cells were determined by immunoblotting at indicated time points after LPS treatment. Note that the appearance and abundance of active caspase-3 coincided with the loss of HDAC4. (G) BV2 cells were pretreated with pan-caspase inhibitor Z-VAD-FMK for 4 h at indicated concentrations, followed by LPS treatment for 24 h. High dose (50 μM) of Z-VAD-FMK effectively abolished caspase-3 activation and prevented HDAC4 degradation. (H) BV2 cells were transfected with a siRNA for caspase-3, followed by 24 h LPS treatment. Caspase-3 KD effectively abolished LPS-induced HDAC4 degradation. (I) BV2 cells were untreated or pretreated with Z-VAD-FMK for 4 h, followed by LPS treatment. At the indicated time points, apoptosis was analyzed by fluorescence-activated cell sorting using Annexin V-PE and 7-AAD Apoptosis Detection Kit. Pretreatment of Z-VAD-FMK did not suppress LPS-induced apoptosis (***p < 0.001 vs. control).
Mentions: At the time when HDAC4 protein degradation became apparent, 18–24 h after LPS treatment, we noticed that the culture medium had turned visibly yellow, indicative of acidification. Because LPS-challenged macrophages activate glycolysis and produce lactate (Rodriguez-Prados et al., 2010), whose accumulation would acidify the medium, we asked whether HDAC4 degradation was linked to glycolysis. We confirmed that glucose uptake and glycolysis were indeed elevated in BV2 cells upon LPS treatment, and knockdown of HDAC4 had no apparent effect on this activity (Figure 3, A–C). Of importance, inhibition of glycolysis by a nonmetabolizable glucose analogue, 2-deoxyglucose (2-DG), or a lactate dehydrogenase inhibitor, oxamic acid, effectively blunted LPS-induced HDAC4 degradation in BV2 cells (Figure 3, D and E, top). These results indicate that active glycolysis is required for LPS-induced HDAC4 degradation.

Bottom Line: Inhibition of GSK3β or iNOS suppresses nitric oxide (NO) production, glycolysis, and HDAC4 degradation.We present evidence that sustained glycolysis induced by LPS treatment activates caspase-3, which cleaves HDAC4 and triggers its degradation.Of importance, a caspase-3-resistant mutant HDAC4 escapes LPS-induced degradation and prolongs inflammatory cytokine production.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710 Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

Show MeSH
Related in: MedlinePlus