We have shown that TSA, an inhibitor of HDAC activity, abrogates Mch-induced increases in airway resistance in both naive and antigen-exposed mice. Moreover, we show that TSA inhibits basal and antigen-induced sensitivity to Mch without altering numbers of leukocytes or concentrations of cytokines in BALF. Our experiments in human PCLS show a decrease in the carbachol-induced contraction after treatment with TSA, and a decrease in the agonist-induced intracellular release of Ca2+, with no effect on Ca2+ sensitization. Collectively, these data suggest that the inhibition of Mch responsiveness by TSA is not attributable to the anti-inflammatory activity of TSA, but is rather a direct effect on agonist-induced smooth muscle contraction. Further, the effects of TSA on AHR appear distinct from those of glucocorticoids, which modulate airway inflammation in response to antigen. Therefore, glucocorticoids and the inhibition of HDAC may modulate AHR by disparate mechanisms.
Our results diverge from those of Choi and colleagues (12
), who reported that in antigen-exposed mice, TSA inhibited airway inflammation, including numbers of eosinophils and concentrations of IL-4 in BAL. The dose of TSA used and/or the timing of TSA treatment may explain the differential effects of TSA on these parameters of inflammation. Choi and colleagues treated mice with 1 mg/kg TSA on the first day of sensitization, and the mice received 11 total doses of TSA over 22 days, whereas in our study, mice were treated with 0.6 mg/kg TSA on the day of antigen challenge, 26 days after the first sensitization treatment, and they received three doses of TSA over 3 days. A higher dose of TSA and/or a longer course of treatment may exert a direct effect on airway inflammation as well as smooth muscle contraction. However, our conclusion, that TSA can abrogate airway sensitivity to Mch in naive mice that exhibit no associated changes in inflammation, supports the hypothesis that TSA may directly inhibit bronchoconstriction, and our study of ASM showing a decrease in the intracellular release of Ca2+
after TSA treatment further supports this hypothesis. Our findings are in agreement with studies indicating that the contraction of isolated guinea pig tracheal rings in response to histamine, carbachol, and 5-hydroxytryptamine is abrogated by inhibitors of HDAC (11
Contraction in ASM is regulated by multiple mechanisms, including release of Ca2+
stored in the sarcoplasmic reticulum, as well as the modulation of Ca2+
sensitivity by the activation of RhoA (29
). Our finding that treatment with TSA inhibits the agonist-induced mobilization of calcium in human ASM in a dose dependent manner is novel, and elucidates a potential mechanism by which TSA inhibits contractile, agonist-induced AHR. Although our results demonstrate that TSA inhibits the activity of HDAC in the lung, the effects on chromatin structure and gene expression remain unclear. Expression profiling studies suggest that 2–10% of genes are modulated by HDACs (30
), and that the inhibition of HDAC can both up-regulate and down-regulate gene expression (31
). Mechanisms unrelated to chromatin remodeling and gene expression may also contribute to the effects of TSA on agonist-induced airway contraction. For example, the inhibition of HDAC hyperacetylates tubulin, decreases tubulin function, and impairs lysosome exocytosis (7
). HDAC8, a Class I HDAC, associates with α-actin, and the inhibition of HDAC8 inhibits in turn the contraction, size, and spreading of smooth muscle (32
). Further studies evaluating the inhibition of specific HDACs or the activity of specific genes after treatment with TSA will elucidate other mechanisms by which TSA inhibits ASM contraction in response to contractile agonists. We previously showed that augmenting the activity of HDAC by stimulating ASM cells with a combination of TNF-α and IFN-γ diminishes the acetylation of NF-κB (33
). Thus, TSA may inhibit AHR in mice and human ASM by modulating gene expression or by altering the acetylation of nonhistone targets, as demonstrated in the case of NF-κB.
ASM has long been a target in asthma therapy (34
), and newer therapies that disrupt ASM were recently approved (35
). Patients with asthma demonstrate increased airway constriction in response to challenge with methacholine, which is commonly used to diagnose patients with suspected asthma (36
). Our finding that the administration of TSA inhibits the activity of HDAC as well as the mobilization of calcium in response to contractile agonists in ASM suggests that the increased sensitivity to methacholine in patients with asthma may be an epigenetically regulated phenomenon, and that further elucidation of the mechanism by which inhibiting HDAC decreases the agonist-induced contraction of ASM could lead to novel asthma therapies.
In conclusion, our experiments show that in both human and murine models, TSA effectively inhibits lung HDAC activity and decreases agonist-induced lung resistance in both naive and antigen-challenged mice, and in human PCLS. Moreover, inhibitors of HDAC appear to modulate airway resistance by decreasing the release of Ca2+ in response to contractile agonists, a mechanism unrelated to any effects on inflammation. Thus, the inhibition of HDAC may offer a therapeutic approach that inhibits bronchoconstriction, apart from effects on airway inflammation.