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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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Caspase-resistant HDAC4 mutant extends the duration of inflammatory cytokine production. (A) FLAG-tagged wild-type (WT) and D289E mutant HDAC4 were transfected into BV2 cells by electroporation, followed by LPS treatment for 36 h. The ectopic HDAC4 was detected by an FLAG antibody. Note that caspase-3–resistant mutant HDAC4 (D289E) is resistant to LPS-induced degradation and caused elevated p70 S6K (T389) phosphorylation at 24 and 36 h after LPS treatment. (B, C) Media from WT and D289E mutant HDAC4–expressing BV2 cells were analyzed for secreted IL-6 (B) and TNF-α (C) by ELISA at indicated time points after LPS treatment. Note that both IL-6 and TNF-α levels were markedly higher in D289E mutant HDAC4–expressing BV2 cells than in control and WT HDAC4–expressing cells at later but not early time points (**p < 0.01 vs. HDAC4 WT). (D, E) Real-time PCR showed that mRNA expression of IL-6 and TNF-α was not affected by overexpressing caspase-3–resistant HDAC4 (D289E).
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Figure 5: Caspase-resistant HDAC4 mutant extends the duration of inflammatory cytokine production. (A) FLAG-tagged wild-type (WT) and D289E mutant HDAC4 were transfected into BV2 cells by electroporation, followed by LPS treatment for 36 h. The ectopic HDAC4 was detected by an FLAG antibody. Note that caspase-3–resistant mutant HDAC4 (D289E) is resistant to LPS-induced degradation and caused elevated p70 S6K (T389) phosphorylation at 24 and 36 h after LPS treatment. (B, C) Media from WT and D289E mutant HDAC4–expressing BV2 cells were analyzed for secreted IL-6 (B) and TNF-α (C) by ELISA at indicated time points after LPS treatment. Note that both IL-6 and TNF-α levels were markedly higher in D289E mutant HDAC4–expressing BV2 cells than in control and WT HDAC4–expressing cells at later but not early time points (**p < 0.01 vs. HDAC4 WT). (D, E) Real-time PCR showed that mRNA expression of IL-6 and TNF-α was not affected by overexpressing caspase-3–resistant HDAC4 (D289E).

Mentions: The degradation of HDAC4 occurred late during the LPS response, when the production of inflammatory cytokines has begun to decline (Figure 2A). As HDAC4 is required for a robust production of IL-6 and TNF-α (Figure 1, A and B), these observations suggest that HDAC4 degradation might contribute to the attenuation of inflammatory cytokine production. To test this hypothesis, we mutated the caspase-3 cleavage site in HDAC4 by converting aspartic acid (D289) to glutamic acid (E). The HDAC4-D289E mutant was previously shown to be resistant to caspase-3 cleavage (Liu et al., 2004; Paroni et al., 2004). We electroporated wild-type and D289E mutant HDAC4 into BV2 cells and subjected them to LPS treatment. As shown in Figure 5A, ectopic wild-type HDAC4, similar to endogenous HDAC4, was clearly degraded after prolonged LPS treatment. In contrast, the level of caspase-3–resistant HDAC4-D289 mutant remained steady, supporting the importance of caspase-3–mediated cleavage in promoting HDAC4 degradation. Of importance, BV2 cells expressing HDAC4-D289E mutant, but not wild-type HDAC4, produced markedly more IL-6 and TNF-α than control cells at the later time points of LPS treatment (Figure 5, B and C, 12–36 h). Consistent with HDAC4 regulating inflammatory cytokine posttranscriptionally (Figure 1), IL-6 and TNF-α mRNA levels were not affected by HDAC4-D289E mutant (Figure 5, D and E). Furthermore, phosphorylation of the mTOR target, p70 S6 kinase, was also elevated in HDAC4-D289E–expressing cells at 24 and 36 h compared with control BV2 (Figure 5A). These results indicate that HDAC4 degradation contributes to the attenuation of inflammatory cytokine production after prolonged LPS activation.


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)

Caspase-resistant HDAC4 mutant extends the duration of inflammatory cytokine production. (A) FLAG-tagged wild-type (WT) and D289E mutant HDAC4 were transfected into BV2 cells by electroporation, followed by LPS treatment for 36 h. The ectopic HDAC4 was detected by an FLAG antibody. Note that caspase-3–resistant mutant HDAC4 (D289E) is resistant to LPS-induced degradation and caused elevated p70 S6K (T389) phosphorylation at 24 and 36 h after LPS treatment. (B, C) Media from WT and D289E mutant HDAC4–expressing BV2 cells were analyzed for secreted IL-6 (B) and TNF-α (C) by ELISA at indicated time points after LPS treatment. Note that both IL-6 and TNF-α levels were markedly higher in D289E mutant HDAC4–expressing BV2 cells than in control and WT HDAC4–expressing cells at later but not early time points (**p < 0.01 vs. HDAC4 WT). (D, E) Real-time PCR showed that mRNA expression of IL-6 and TNF-α was not affected by overexpressing caspase-3–resistant HDAC4 (D289E).
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Figure 5: Caspase-resistant HDAC4 mutant extends the duration of inflammatory cytokine production. (A) FLAG-tagged wild-type (WT) and D289E mutant HDAC4 were transfected into BV2 cells by electroporation, followed by LPS treatment for 36 h. The ectopic HDAC4 was detected by an FLAG antibody. Note that caspase-3–resistant mutant HDAC4 (D289E) is resistant to LPS-induced degradation and caused elevated p70 S6K (T389) phosphorylation at 24 and 36 h after LPS treatment. (B, C) Media from WT and D289E mutant HDAC4–expressing BV2 cells were analyzed for secreted IL-6 (B) and TNF-α (C) by ELISA at indicated time points after LPS treatment. Note that both IL-6 and TNF-α levels were markedly higher in D289E mutant HDAC4–expressing BV2 cells than in control and WT HDAC4–expressing cells at later but not early time points (**p < 0.01 vs. HDAC4 WT). (D, E) Real-time PCR showed that mRNA expression of IL-6 and TNF-α was not affected by overexpressing caspase-3–resistant HDAC4 (D289E).
Mentions: The degradation of HDAC4 occurred late during the LPS response, when the production of inflammatory cytokines has begun to decline (Figure 2A). As HDAC4 is required for a robust production of IL-6 and TNF-α (Figure 1, A and B), these observations suggest that HDAC4 degradation might contribute to the attenuation of inflammatory cytokine production. To test this hypothesis, we mutated the caspase-3 cleavage site in HDAC4 by converting aspartic acid (D289) to glutamic acid (E). The HDAC4-D289E mutant was previously shown to be resistant to caspase-3 cleavage (Liu et al., 2004; Paroni et al., 2004). We electroporated wild-type and D289E mutant HDAC4 into BV2 cells and subjected them to LPS treatment. As shown in Figure 5A, ectopic wild-type HDAC4, similar to endogenous HDAC4, was clearly degraded after prolonged LPS treatment. In contrast, the level of caspase-3–resistant HDAC4-D289 mutant remained steady, supporting the importance of caspase-3–mediated cleavage in promoting HDAC4 degradation. Of importance, BV2 cells expressing HDAC4-D289E mutant, but not wild-type HDAC4, produced markedly more IL-6 and TNF-α than control cells at the later time points of LPS treatment (Figure 5, B and C, 12–36 h). Consistent with HDAC4 regulating inflammatory cytokine posttranscriptionally (Figure 1), IL-6 and TNF-α mRNA levels were not affected by HDAC4-D289E mutant (Figure 5, D and E). Furthermore, phosphorylation of the mTOR target, p70 S6 kinase, was also elevated in HDAC4-D289E–expressing cells at 24 and 36 h compared with control BV2 (Figure 5A). These results indicate that HDAC4 degradation contributes to the attenuation of inflammatory cytokine production after prolonged LPS activation.

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