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HDAC6 deacetylase activity is critical for lipopolysaccharide-induced activation of macrophages.

Yan B, Xie S, Liu Z, Ran J, Li Y, Wang J, Yang Y, Zhou J, Li D, Liu M - PLoS ONE (2014)

Bottom Line: In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation.Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation.These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China.

ABSTRACT
Activated macrophages play an important role in both innate and adaptive immune responses, and aberrant activation of macrophages often leads to inflammatory and immune disorders. However, the molecular mechanisms of how macrophages are activated are not fully understood. In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation. Our data show that suppression of HDAC6 activity significantly restrains LPS-induced activation of macrophages and production of pro-inflammatory cytokines. Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation. These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.

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HDAC6 activity is important for LPS-induced macrophage activation.(A) RAW264.7 cells were treated with tubacin (1 µM), NaB (500 µM) or TSA (5 µM) for 4 hours. Cell lysates were then immunoblotted with antibodies against acetylated α-tubulin, α-tubulin, acetylated histone H4, and histone H4. (B) RAW264.7 cells were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0, 2, 6, 12, and 24 hours. (C) Representative images of resting and activated macrophages. (D) Experiments were performed as in panel B, and the percentage of activated cells was analyzed. (E) Primary BMMs were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0 and 6 hours. (F) Representative images of resting and activated BMMs. (G) Experiments were performed as in panel E, and the percentage of activated BMMs was analyzed. (H) Quantitative RT-PCR analysis of relative HDAC6 mRNA level in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 36 hours. (I) Immunoblotting analysis of α-tubulin and acetylated α-tubulin in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours. (J) RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours were stimulated with LPS (300 ng/ml) for 0 and 24 hours. (K) Experiments were performed as in panel J, and the percentage of activated cells was analyzed. ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant (p≥0.05).
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pone-0110718-g001: HDAC6 activity is important for LPS-induced macrophage activation.(A) RAW264.7 cells were treated with tubacin (1 µM), NaB (500 µM) or TSA (5 µM) for 4 hours. Cell lysates were then immunoblotted with antibodies against acetylated α-tubulin, α-tubulin, acetylated histone H4, and histone H4. (B) RAW264.7 cells were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0, 2, 6, 12, and 24 hours. (C) Representative images of resting and activated macrophages. (D) Experiments were performed as in panel B, and the percentage of activated cells was analyzed. (E) Primary BMMs were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0 and 6 hours. (F) Representative images of resting and activated BMMs. (G) Experiments were performed as in panel E, and the percentage of activated BMMs was analyzed. (H) Quantitative RT-PCR analysis of relative HDAC6 mRNA level in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 36 hours. (I) Immunoblotting analysis of α-tubulin and acetylated α-tubulin in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours. (J) RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours were stimulated with LPS (300 ng/ml) for 0 and 24 hours. (K) Experiments were performed as in panel J, and the percentage of activated cells was analyzed. ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant (p≥0.05).

Mentions: LPS induces macrophage activation through the toll-like receptor 4 (TLR4) signaling and results in cell differentiation towards the M1 phenotype [29], a process involving cytoskeletal reorganization. To investigate the involvement of HDAC6 in LPS-induced macrophage activation, RAW264.7 cells were treated with tubacin, an HDAC6-specific inhibitor [30]. We found that the pan-HDAC inhibitor TSA increased the acetylation of both α-tubulin and histone H4, and the HDAC6-resistant HDAC inhibitor NaB increased histone H4 acetylation with minimal elevation of α-tubulin acetylation; by contrast, tubacin significantly increased α-tubulin acetylation without affecting histone H4 acetylation (Fig. 1A). These results demonstrate the specificity of tubacin in the suppression of HDAC6 activity.


HDAC6 deacetylase activity is critical for lipopolysaccharide-induced activation of macrophages.

Yan B, Xie S, Liu Z, Ran J, Li Y, Wang J, Yang Y, Zhou J, Li D, Liu M - PLoS ONE (2014)

HDAC6 activity is important for LPS-induced macrophage activation.(A) RAW264.7 cells were treated with tubacin (1 µM), NaB (500 µM) or TSA (5 µM) for 4 hours. Cell lysates were then immunoblotted with antibodies against acetylated α-tubulin, α-tubulin, acetylated histone H4, and histone H4. (B) RAW264.7 cells were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0, 2, 6, 12, and 24 hours. (C) Representative images of resting and activated macrophages. (D) Experiments were performed as in panel B, and the percentage of activated cells was analyzed. (E) Primary BMMs were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0 and 6 hours. (F) Representative images of resting and activated BMMs. (G) Experiments were performed as in panel E, and the percentage of activated BMMs was analyzed. (H) Quantitative RT-PCR analysis of relative HDAC6 mRNA level in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 36 hours. (I) Immunoblotting analysis of α-tubulin and acetylated α-tubulin in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours. (J) RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours were stimulated with LPS (300 ng/ml) for 0 and 24 hours. (K) Experiments were performed as in panel J, and the percentage of activated cells was analyzed. ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant (p≥0.05).
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pone-0110718-g001: HDAC6 activity is important for LPS-induced macrophage activation.(A) RAW264.7 cells were treated with tubacin (1 µM), NaB (500 µM) or TSA (5 µM) for 4 hours. Cell lysates were then immunoblotted with antibodies against acetylated α-tubulin, α-tubulin, acetylated histone H4, and histone H4. (B) RAW264.7 cells were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0, 2, 6, 12, and 24 hours. (C) Representative images of resting and activated macrophages. (D) Experiments were performed as in panel B, and the percentage of activated cells was analyzed. (E) Primary BMMs were treated with tubacin (1 µM) for 4 hours and then stimulated with LPS (300 ng/ml) for 0 and 6 hours. (F) Representative images of resting and activated BMMs. (G) Experiments were performed as in panel E, and the percentage of activated BMMs was analyzed. (H) Quantitative RT-PCR analysis of relative HDAC6 mRNA level in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 36 hours. (I) Immunoblotting analysis of α-tubulin and acetylated α-tubulin in RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours. (J) RAW264.7 cells transfected with control or mouse HDAC6 siRNAs for 72 hours were stimulated with LPS (300 ng/ml) for 0 and 24 hours. (K) Experiments were performed as in panel J, and the percentage of activated cells was analyzed. ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant (p≥0.05).
Mentions: LPS induces macrophage activation through the toll-like receptor 4 (TLR4) signaling and results in cell differentiation towards the M1 phenotype [29], a process involving cytoskeletal reorganization. To investigate the involvement of HDAC6 in LPS-induced macrophage activation, RAW264.7 cells were treated with tubacin, an HDAC6-specific inhibitor [30]. We found that the pan-HDAC inhibitor TSA increased the acetylation of both α-tubulin and histone H4, and the HDAC6-resistant HDAC inhibitor NaB increased histone H4 acetylation with minimal elevation of α-tubulin acetylation; by contrast, tubacin significantly increased α-tubulin acetylation without affecting histone H4 acetylation (Fig. 1A). These results demonstrate the specificity of tubacin in the suppression of HDAC6 activity.

Bottom Line: In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation.Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation.These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China.

ABSTRACT
Activated macrophages play an important role in both innate and adaptive immune responses, and aberrant activation of macrophages often leads to inflammatory and immune disorders. However, the molecular mechanisms of how macrophages are activated are not fully understood. In this study, we identify a novel role for histone deacetylse 6 (HDAC6) in lipopolysaccharide (LPS)-induced macrophage activation. Our data show that suppression of HDAC6 activity significantly restrains LPS-induced activation of macrophages and production of pro-inflammatory cytokines. Further study reveals that the regulation of macrophage activation by HDAC6 is independent of F-actin polymerization and filopodium formation; instead, it is mediated by the effects of HDAC6 on cell adhesion and microtubule acetylation. These data thus suggest that HDAC6 is an important regulator of LPS-induced macrophage activation and might be a potential target for the management of inflammatory disorders.

Show MeSH
Related in: MedlinePlus