Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
Department of Pharmaceutical Sciences, Thomas Jefferson University, Jefferson School of Pharmacy, Philadelphia, PA, USA.
J Immunol. 2013 Sep 1;191(5):2624-2636. doi: 10.4049/jimmunol.1300104. Epub 2013 Jul 31.
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 we see in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles that can be modeled in vitro by exposing cultured ASM cells to TNF-α/IFN-γ. This action drives GC insensitivity via protein phosphatase 5-dependent impairment of GC receptor phosphorylation. In this study, 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 localization of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNF-α/IFN-γ was assessed using complementary inhibitory strategies, including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific small hairpin RNA 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 GC receptor-α phosphorylation at Ser(211) and transactivation properties via the suppression of cytokine-induced protein phosphatase 5 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.
确定导致严重哮喘患者糖皮质激素（GC）相对抵抗的因素，并寻找逆转这种抵抗的方法至关重要。在哮喘中，我们在气道平滑肌（ASM）束中观察到体内 GC 抵抗途径的证据，这些证据可以通过将培养的 ASM 细胞暴露于 TNF-α/IFN-γ 中来在体外模拟。这种作用通过蛋白磷酸酶 5 依赖性 GC 受体磷酸化的损害导致 GC 不敏感。在这项研究中，我们使用我们的 GC 不敏感 ASM 模型研究了 KCa3.1 离子通道是否调节 GC 抵抗途径的活性。支气管活检的免疫组织化学染色显示，KCa3.1 通道定位于健康对照者和哮喘患者的 ASM 质膜和核，无论疾病严重程度如何。Western blot 分析和免疫荧光染色证实了 ASM 细胞中 KCa3.1 通道的核定位。使用互补的抑制策略（包括 KCa3.1 阻滞剂（TRAM-34 和 ICA-17043）或通过腺病毒传递的 KCa3.1 特异性短发夹 RNA）评估了 KCa3.1 通道在 TNF-α/IFN-γ 诱导的 GC 抵抗趋化因子中的调节作用。KCa3.1 通道阻断导致 fluticasone 抵抗的 CX3CL1、CCL5 和 CCL11 基因和蛋白表达显著减少。KCa3.1 通道阻断还通过抑制细胞因子诱导的蛋白磷酸酶 5 表达，恢复了 fluticasone 诱导的 GC 受体-α Ser(211)磷酸化和转导活性。KCa3.1 阻断的作用在健康对照者和哮喘患者的 ASM 细胞中均可见。总之，KCa3.1 通道有助于调节 ASM 细胞中的 GC 抵抗炎症途径：阻断 KCa3.1 通道可能增强严重哮喘中的皮质类固醇活性。
