Anti-inflammatory properties of histone deacetylase inhibitors: a mechanistic study

Abstract

Background: We have demonstrated that postshock administration of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, can significantly improve early survival in a highly lethal model of hemorrhagic shock. As the primary insult in hemorrhagic shock is cellular hypoxia, and transcription factor hypoxia-inducible factor-1α (HIF-1α) controls proinflammatory gene expression in macrophages, we hypothesized that SAHA would attenuate the HIF-1α associated proinflammatory pathway in a hypoxic macrophage model.

Methods: Mouse macrophages were exposed to hypoxic conditions (0.5% O2, 10% CO2, and 89.5% N2) at 37°C in the presence or absence of SAHA (10 μmol/L). The cells and culture medium were harvested at 1 hour, 4 hours, and 8 hours. Sham (no hypoxia, no SAHA) served as a control. Western blots were performed to assess protein levels of prolyl hydroxylase 2 (PHD2), HIF-1α, and inducible nitric oxide synthase (iNOS) in the cells. Colorimetric biochemical assay and enzyme-linked immunosorbent assay were used to analyze the release of nitric oxide (NO) and secretion of tumor necrosis factor α (TNF-α), respectively, in the cell culture medium.

Results: Hypoxia significantly increased cellular level of HIF-1α (1 hour and 4 hours), gene transcription of iNOS (4 hours and 8 hours), iNOS protein (8 hours), NO production (8 hours), and TNF-α secretion (4 hours and 8 hours). SAHA treatment attenuated all of the above hypoxia-induced alterations in the macrophages. In addition, SAHA treatment significantly increased cellular level of PHD2, one of the upstream negative regulators of HIF-1α, at 1 hour.

Conclusions: Treatment with SAHA attenuates hypoxia-HIF-1α-inflammatory pathway in macrophages and suppresses hypoxia-induced release of proinflammatory NO and TNF-α. SAHA also causes an early increase in cellular PHD2, which provides, at least in part, a new explanation for the decrease in the HIF-1α protein levels.

Fig. 1. Effects of SAHA on level of HIF-1 α protein in hypoxia-stimulated macrophages (1a) and normal macrophages (1b)

Mouse macrophages RAW264.7 were harvested at 1 and 4 hours after hypoxia in the presence or absence of SAHA. Sham (no SAHA, no hypoxia) macrophages served as a control. Whole cell lysates were subjected to western blotting with anti-HIF-1α and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). # denotes a significant difference compared with the sham group (p ≤ 0.01). * denotes a significant difference compared with the hypoxia groups (p ≤ 0.01). All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 1. Effects of SAHA on level of HIF-1 α protein in hypoxia-stimulated macrophages (1a) and normal macrophages (1b)

Mouse macrophages RAW264.7 were harvested at 1 and 4 hours after hypoxia in the presence or absence of SAHA. Sham (no SAHA, no hypoxia) macrophages served as a control. Whole cell lysates were subjected to western blotting with anti-HIF-1α and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). # denotes a significant difference compared with the sham group (p ≤ 0.01). * denotes a significant difference compared with the hypoxia groups (p ≤ 0.01). All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 2. Effects of SAHA on level of PHD2 protein in hypoxia-stimulated macrophages (2a) and normal macrophages (2b)

Mouse macrophages RAW264.7 were harvested at 1and 4 hours after hypoxia in the presence or absence of SAHA. Sham (no SAHA, no hypoxia) macrophages served as a control. Whole cell lysates were subjected to western blotting with anti-PHD2 and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). * denotes a significant difference compared with the hypoxia group (p ≤ 0.01). All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 2. Effects of SAHA on level of PHD2 protein in hypoxia-stimulated macrophages (2a) and normal macrophages (2b)

Mouse macrophages RAW264.7 were harvested at 1and 4 hours after hypoxia in the presence or absence of SAHA. Sham (no SAHA, no hypoxia) macrophages served as a control. Whole cell lysates were subjected to western blotting with anti-PHD2 and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). * denotes a significant difference compared with the hypoxia group (p ≤ 0.01). All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 3. (a) Effects of SAHA on gene expression of iNOS in hypoxia-stimulated macrophages

Mouse macrophages were harvested at 1, 4 and 8 hours after hypoxia with or without SAHA treatment. iNOS mRNA levels were determined by real-time PCR and expressed as mean values ± SD (n = 3). # denotes a significant difference compared with the sham group (p ≤ 0.05). *denotes a significant difference compared with the hypoxia group (p ≤ 0.001). (b) Effects of SAHA on cellular protein level of iNOS in hypoxia-stimulated macrophages. Mouse macrophages RAW264.7 were harvested 1, 4 and 8 hours after hypoxia in the presence or absence of SAHA. Whole cell lysates were subjected to western blotting with anti- iNOS and anti-actin antibodies. Specific bands were quantified by densitometry and expressed as mean values ± SD (n = 3). # denotes a significant difference compared with the sham group (p ≤ 0.01). * denotes a significant difference compared with the hypoxia group (p ≤ 0.01). Sham (no SAHA, no hypoxia) macrophages served as a control. All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 4. Effects of SAHA on production of NO in hypoxia-stimulated macrophages

Cell culture medium of RAW264.7 macrophages was harvested 4 and 8 hours after hypoxia with or without SAHA treatment. Sham (no SAHA, no hypoxia) macrophages served as a control. Nitrite level was used as a measure of NO released into the cell culture medium. Concentrations of Nitrite in the cell culture mediums were determined by colorimetric biochemical assay. The NO concentration was expressed as mean values ± SD (n = 3). # denotes a significant difference compared with the sham group (p ≤ 0.01). * denotes a significant difference compared with the hypoxia group (p ≤ 0.01). All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 5. (a) Effects of SAHA on expression of TNF-α gene in hypoxia-stimulated macrophages

Mouse macrophages were harvested 1, 4 and 8 hours after hypoxia with or without SAHA treatment. TNF-α mRNA levels were determined by real-time PCR and expressed as mean values ± SD (n = 3). *denotes a significant difference compared with the hypoxia groups (p ≤ 0.01). (b) Effects of SAHA on secretion of TNF-α protein by hypoxia-stimulated macrophages. Cell culture mediums of RAW264.7 macrophages were harvested at 4 and 8 hours after hypoxia stimulation with or without SAHA treatment. Concentrations of TNF-α in the cell culture mediums were determined by ELISA. TNF-α concentration was expressed as mean values ± SD (n = 3). # denotes a significant difference compared with the sham group (p ≤ 0.001). * denotes a significant difference compared with the hypoxia groups (p ≤ 0.001). Sham (no SAHA, no hypoxia) macrophages served as a control. All time points (hours) in this study used start of treatment as the reference point (time zero).

Fig. 6. Schematic depiction of mechanism by which SAHA could attenuate hypoxia- HIF-1α inflammatory pathway in macrophages

SAHA treatment increases cytoplasmic levels of PHD2 protein, decreases HIF-1α protein, inhibits transcription of iNOS and TNF-α genes, decreases iNOS expression, attenuates NO production and TNF-α secretion. SAHA, suberoylanilide hydroxamic acid; PHD2, prolyl hydroxylase 2; HIF-1α, hypoxia-inducible factor-1α; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor α; NO, nitric oxide. Figure numbers in parentheses refer to figures that present more detailed data related to these steps in the pathway.