Background and purpose: Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation. Experimental approach: Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng·mL -1 ) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators. Key results: Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC50 = 520 Ϯ 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 Ϯ 8%) and asthmatic subjects (57 Ϯ 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21 Waf/Cip-1 , p27 Kip1 , cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC50 = 450 Ϯ 100 pM) PDGF-induced cell cycle progression. Conclusions and implications:Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.

Background and purpose: Chemokines play a critical role in the pathogenesis of asthma and facilitate the recruitment of inflammatory cells in the airways. Evidence now suggests that airway smooth muscle (ASM) may serve as a source of chemokines in inflamed airways. Although vitamin D has potent anti-inflammatory properties in vitro in some cell types, its effects on ASM cells remain unclear. Here, we investigated whether 1a, 25-dihydroxy vitamin D 3 (calcitriol) modulated chemokine production in ASM. Experimental approach: Human ASM cell cultures were derived from tracheal samples taken during surgery. ASM cells were treated with tumour necrosis factor alpha (TNFa) and/or interferon gamma (IFNg) for 24 h in the presence of calcitriol and/or the glucocorticoid fluticasone added 2 h before. RANTES (regulated upon activation, normal T-cell expressed and secreted), interferon-inducible protein 10 (IP-10) and fractalkine (FKN) levels in cell supernatants were measured by ELISA. Key results: In TNFa-treated cells, calcitriol inhibited RANTES and IP-10 secretion in a concentration-dependent manner. FKN levels were negligible. In TNFa/IFNg-treated cells, whereas fluticasone or calcitriol alone partially inhibited RANTES secretion (by 38 and 20%, respectively), the combination of both drugs additively inhibited RANTES secretion (by 60%). No effect was observed on IP-10 secretion. Whereas fluticasone enhanced FKN secretion (by 50%), calcitriol significantly decreased FKN levels (by 50%). Interestingly, calcitriol blocked the stimulatory effect of fluticasone on FKN secretion, which was inhibited by 60% with the combination of calcitriol and fluticasone. Conclusions and implications: These findings suggest that vitamin D uniquely modulates human ASM expression of chemokines and may exert some beneficial effects in the treatment of steroid-resistant patients with asthma.

We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF-a and IFN-g impaired steroid anti-inflammatory action through the up-regulation of glucocorticoid receptor beta isoform (GRb) (Mol Pharmacol 2006;69:588-596). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF-a and IFN-g (6 h) as shown by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly, neither GRa nuclear translocation nor GRb expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF-a and IFN-g combination but not with cytokine alone, was also completely insensitive to steroid pretreatment. Chromatin-immunoprecipitation (IP) and siRNA strategies revealed not only increased binding of interferon regulatory factor 1 (IRF-1) transcription factor to CD38 promoter, but also its implication in regulating CD38 gene transcription. Interestingly, the capacity of fluticasone to completely inhibit TNF-a-induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities was completely lost in cells exposed to TNF-a and IFN-g in combination. This early steroid dysfunction seen with cytokine combination could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1, which dose-dependently inhibited GRE-dependent gene transcription. Consistently, reducing IRF-1 cellular levels using siRNA approach significantly restored steroid transactivation activities. Collectively, our findings demonstrate for the first time that IRF-1 is a novel alternative GRb-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GRa activities.

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (R L ) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSAtreated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca 21 . The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in R L and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca 21 in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca 21 in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.Keywords: HDAC; asthma; allergen; mice; trichostatin A Asthma manifests as reversible airway obstruction, hyperresponsiveness, and inflammation (1). Although most patients respond to conventional therapy, some exhibit severe or refractory asthma associated with frequent exacerbations, irreversible airflow limitation, and airway inflammation, despite maximal medical therapy (2). Patients with severe asthma also use healthcare resources disproportionately and experience more adverse effects from high doses of glucocorticoids (3). The need for improved therapeutic approaches to severe asthma continues, and the pursuit of epigenetic modifications of gene expression in asthma offers unique therapeutic opportunities. Accordingly, the acetylation and deacetylation of histone represent a potential therapeutic target (4).Histone acetyl transferases (HATs) induce the acetylation of histone, whereas histone deacetylases (HDACs) remove the acetyl groups from histones to modulate gene transcription. Currently, 11 HDACs have been identified and grouped into three major categories by their sequence similarity to the Saccharomyces cerevisiae reduced potassium dependency-3 (RPD3) or histone-deacetylase ...

Glucocorticoid (GC) insensitivity represents a profound challenge in managing patients with asthma. The mutual inhibition of transcriptional activity between GC receptor (GR) and other regulators is one of the mechanisms contributing to GC resistance in asthma. We recently reported that interferon regulatory factor (IRF)-1 is a novel transcription factor that promotes GC insensitivity in human airway smooth muscle (ASM) cells by interfering with GR signaling (Tliba et al., Am J Respir Cell Mol Biol 2008;38:463-472). Here, we sought to determine whether the inhibition of GR function by IRF-1 involves its interaction with the transcriptional co-regulator GR-interacting protein 1 (GRIP-1), a known GR transcriptional co-activator. We here found that siRNA-mediated GRIP-1 depletion attenuated IRF-1-dependent transcription of the luciferase reporter construct and the mRNA expression of an IRF-1-dependent gene, CD38. In parallel experiments, GRIP-1 silencing significantly reduced GR-mediated transactivation activities. Co-immunoprecipitation and GST pull-down assays showed that GRIP-1, through its repression domain, physically interacts with IRF-1 identifying GRIP-1 as a bona fide transcriptional co-activator for IRF-1. Interestingly, the previously reported inhibition of GR-mediated transactivation activities by either TNF-alpha and IFN-gamma treatment or IRF-1 overexpression was fully reversed by increasing cellular levels of GRIP-1. Together, these data suggest that the cellular accumulation of IRF-1 may represent a potential molecular mechanism mediating altered cellular response to GC through the depletion of GRIP-1 from the GR transcriptional regulatory complexes.