Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.
Eur J Pharmacol. 2013 Jun 15;710(1-3):100-9. doi: 10.1016/j.ejphar.2013.03.048. Epub 2013 Apr 12.
Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [(3)H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10(-11)M) and α-SMA expression/polymerization at higher concentration (10(-6)M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.
缓激肽可将正常肺成纤维细胞转化为肌成纤维细胞，诱导成纤维细胞增殖并激活丝裂原激活蛋白激酶途径（MAPK），但尚未研究其对哮喘患者支气管成纤维细胞（HBAFb）的影响。我们研究了缓激肽诱导的 HBAFb 与正常支气管成纤维细胞（HNBFb）的增殖和分化以及相关的细胞内机制。通过 Western blot 分析评估了缓激肽刺激的 HBAFb 和 HNBFb 中的缓激肽 B2 受体表达；通过[（3）H]胸苷掺入评估细胞增殖；通过 Western blot 和免疫荧光评估α-平滑肌肌动蛋白（SMA）的表达/聚合；通过免疫沉淀和 Western blot 分别评估表皮生长因子（EGF）受体、细胞外调节激酶（ERK）1/2 和 p38 MAPK 的激活。与 HNBFb 相比，HBAFb 中缓激肽 B2 受体和α-SMA 的组成型表达更高。缓激肽增加了 HBAFb 中的缓激肽 B2 受体表达。缓激肽通过缓激肽 B2 受体，在较低浓度（10（-11）M）下显著增加成纤维细胞增殖，并在两种细胞中更高浓度（10（-6）M）下增加α-SMA 的表达/聚合。缓激肽通过缓激肽 B2 受体增加 ERK1/2 和 p38 的磷酸化；EGF 受体抑制剂 AG1478 和泛金属蛋白酶抑制剂 GM6001 阻断了缓激肽诱导的 ERK1/2 激活，但不阻断 p38 磷酸化。缓激肽通过缓激肽 B2 受体诱导 EGF 受体磷酸化，AG1478 可抑制该磷酸化。在 HBAFb 中，AG1478、GM6001、ERK1/2 抑制剂 U0126 和 p38 抑制剂 SB203580 抑制了缓激肽诱导的细胞增殖，但只有 SB203580 降低了肌成纤维细胞分化。这些数据表明，缓激肽通过 MAPK 途径和 EGF 受体的反式激活，积极参与哮喘患者支气管成纤维细胞的增殖和分化，这可能导致哮喘中的气道重塑，为哮喘患者的潜在治疗意义开辟了新的前景。
