Functional KCa3.1 Channels Regulate Steroid Insensitivity in Bronchial Smooth Muscle Cells

Abstract

Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma there is in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles which can be modelled in vitro by exposing cultured ASM cells to TNFα/IFNγ. This drives GC insensitivity via protein phosphatase-5 (PP5)-dependent impairment of GC receptor (GR) phosphorylation. Here, we investigated whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and immunofluorescence staining confirmed the nuclear localisation of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNFα/IFNγ was assessed using complementary inhibitory strategies including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific shRNA delivered by adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5 and CCL11 gene and protein expression. KCa3.1 channel blockade also restored fluticasone-induced GRα phosphorylation at ser211 and transactivation properties via the suppression of cytokine-induced PP5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary KCa3.1 channels contribute to the regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in severe asthma.

Effect of the Plant Derivative Compound A on the Production of Corticosteroid-Resistant Chemokines in Airway Smooth Muscle Cells

“…We have previously reported that ASM cells exposed to TNF-a/IFN-g combination become refractory to the antiinflammatory action of corticosteroids (23,25,29,32,33 (19,20), and experimental autoimmune neuritis (17). These studies highlighted the lack of typical side effects associated with GC treatment and the equal efficacy of CpdA when compared with dexamethasone.…”

“…The concentrations of CX3CL1, CXCL10, and CCL5 in the supernatants were measured as described previously (23).…”

“…Quantitative PCR was performed as described previously (23) and as described in the online supplement.…”

Ca2+ signalling in fibroblasts and the therapeutic potential of KCa3.1 channel blockers in fibrotic diseases

“…K Ca 3.1 is expressed widely in the airways of both healthy controls and people with asthma (Arthur et al, ; Chachi et al, ). Immunostaining is evident in the airway epithelial cells, goblet cells, airway smooth muscle cells, airway fibroblasts, submucosal glands, inflammatory cells and endothelial cells (Arthur et al, ; Chachi et al, ).…”

“…K Ca 3.1 is expressed widely in the airways of both healthy controls and people with asthma (Arthur et al, ; Chachi et al, ). Immunostaining is evident in the airway epithelial cells, goblet cells, airway smooth muscle cells, airway fibroblasts, submucosal glands, inflammatory cells and endothelial cells (Arthur et al, ; Chachi et al, ). Functional K Ca 3.1 channels are also expressed by cultured primary human airway epithelial cells and airway smooth muscle cells (Arthur et al, ; Shepherd et al, ).…”