Histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates Toll-like receptor 4 signaling in lipopolysaccharide-stimulated mouse macrophages

Abstract

Objective: We have previously demonstrated that pretreatment and posttreatment of animals with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, can improve survival in a mouse model of lipopolysaccharide (LPS)-induced severe shock. This study was designed to assess whether SAHA affects LPS/Toll-like receptor 4 signaling through acetylation of heat shock protein 90 (HSP90) and degradation of its client protein interleukin-1 receptor-associated kinase 1 (IRAK1).

Methods: RAW264.7 cells were exposed to LPS (1 μg/mL) for 2 h, followed by treatment with SAHA (10 μM) or geldanamycin (3 μM), an inhibitor of HSP90. Sham (no SAHA, no LPS) macrophages served as a control. The cells were harvested at different time points, and time zero served as the reference point.

Results: LPS dramatically increased protein expression of myeloid differentiation factor 88 and IRAK1, and stimulated nuclear translocation of nuclear factor κB, leading to an increases of gene expression and protein production of tumor necrosis factor α and interleukin-6. Treatment with SAHA significantly attenuated these LPS-stimulated alterations. LPS or SAHA did not change the levels of HSP90 protein, but immunoprecipitation studies demonstrated that SAHA treatment enhanced acetylation of HSP90, and increased the dissociation of IRAK1, compared to the LPS control.

Conclusions: SAHA suppresses LPS/Toll-like receptor 4 signaling in LPS-stimulated macrophages through multiple potential mechanisms. It inhibits the function of HSP90 through hyperacetylation of the chaperone protein, which results in dissociation and degradation of the client protein IRAK1 and, at least in part, leads to a decrease in nuclear translocation of nuclear factor κB and attenuation of key proinflammatory cytokine expression.

FIG. 1. Effects of SAHA on protein expression of MyD88 in LPS -stimulated macrophages

Cellular levels of MyD88 protein in RAW264.7 cells at 6, 8 and 10 hours were examined by immunoblots with anti- MyD88 and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). LPS significantly increased and SAHA decreased the MyD88 protein expression, respectively. # denotes a significant difference compared with the sham group (p < 0.05). * denotes a significant difference compared with the LPS group (p < 0.05). Values at time zero used as the reference point.

FIG. 2. Effects of SAHA on protein expression of IRAK1 and HSP90 in LPS-stimulated macrophages

Cellular levels of IRAK1 and HSP90 proteins in RAW264.7 cells at 6, 8, and 10 hours were examined by immunoblots. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). LPS significantly increased and SAHA decreased IRAK1 protein levels, respectively. Neither LPS nor SAHA affected expression of HSP90. # denotes a significant difference compared with the sham group (p < 0.05). * denotes a significant difference compared with the LPS group (p < 0.05). Values at time zero used as the reference point.

FIG. 3. Effects of SAHA on nuclear translocation of NF-κB in LPS-stimulated macrophages

Nuclear fractions were subjected to western blotting with anti- NF-κB p65 and anti-Histone H3 antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). LPS significantly increased and SAHA decreased the nuclear translocation of NF-κB, respectively.# denotes a significant difference compared with the sham group (p < 0.05). * denotes a significant difference compared with the LPS group (p < 0.05). Values at time zero used as the reference point. Histone H3 serves as an internal control for equal sample loading.

FIG. 4. Effects of SAHA on gene expression of TNF-α and IL-6 in LPS-stimulated macrophages

TNF-α and IL-6 mRNA levels were determined by real-time PCR and expressed as mean values ± SD (n = 3). LPS significantly increased and SAHA decreased the expression of TNF-α and IL-6 genes, respectively. # denotes a significant difference compared with the sham group (p < 0.05). *denotes a significant difference compared with the LPS group (p < 0.05). Values at time zero used as the reference point.

FIG. 5. Effects of SAHA on protein secretion of TNF-α and IL-6 in LPS-stimulated macrophages

Concentrations of TNF-α and IL-6 in the cell culture mediums were determined by ELISA. The cytokine concentration was expressed as mean values ± SD (n = 3). LPS significantly increased and SAHA decreased the TNF-α and IL-6 secretion from the macrophages, respectively. # denotes a significant difference compared with the sham group (p < 0.05). * denotes a significant difference compared with the LPS group (p < 0.05). Values at time zero used as the reference point.

FIG. 6. Effects SAHA on acetylation of HSP90 and association of IRAK1 with HSP90 in LPS stimulated macrophages

The IRAK1 and acetylated HSP90 proteins in HSP90 protein complexes were examined by immunoprecipitation and immunoblots. LPS significantly increased and SAHA decreased association of IRAK1 protein with HSP90, respectively. Moreover, SAHA increased HSP90 acetylation compared to the LPS group. Ig G heavy chain, derived from external antibodies, serves as an internal control for equal amount of antibody used in the experiment.