MECHANISMS OF BIOLOGICAL EFFECTS OF HUMAN AIRWAY TRYPSIN-LIKE PROTEASE (HAT) ON BRONCHIAL CELLS AND PATHOLOGICAL ROLES OF HAT IN THE AIRWAY DISEASES

1.痰液中人气道胰蛋白酶样蛋白酶（HAT）含量：健康志愿者和慢性气道疾病患者痰液中HAT含量无差异。由于患者的支气管灌洗液分泌比健康志愿者丰富，因此认为患者气道HAT的产生增强，HAT导致疾病的进展和扩大2。HAT促进正常人支气管成纤维细胞增殖的机制：HAT在有或无转铁蛋白的无血清培养基中促进支气管成纤维细胞的增殖。蛋白酶激活受体-2激动剂肽（PAR 2AP）促进含转铁蛋白血清培养基中支气管成纤维细胞的增殖。丝氨酸蛋白酶抑制剂Leupeptin和MEK抑制剂U 0126抑制HAT诱导的支气管成纤维细胞增殖。Western blot分析表明HAT促进MAP激酶的磷酸化。结论：HAT通过MAP激酶信号转导促进成纤维细胞增殖，而不是通过PAR 23。HAT促进人支气管上皮细胞（HBEC）合成IL-8：反向PCR检测到PAR 2 mRNA表达。加入HAT和PAR 2激动剂肽可增加HBEC上IL-8 mRNA的诱导和IL-8的释放。结论：HAT对HBEC上IL-8合成的增强是由一个顺序激活过程引起的：HAT激活PAR 2，EGFR激活反式激活和MAP激酶激活4。HAT激活支气管上皮细胞上的PAR 2：PAR与G蛋白偶联激活增加细胞内游离Ca^2+浓度。HAT和PAR 2激动剂肽刺激HBEC后，HBEC内游离Ca^<2+>浓度增加。结论HAT通过激活PAR 2调控HBEC的代谢功能

1. Contents of human airway trypsin-like protease (HAT) in sputum : There was no difference between HAT concentration in sputum from healthy volunteers and that from patients with chronic airway diseases. Because the patients have more abundance of secretion of bronchial lavage fluid than healthy volunteers, it is considered that the production of HAT in airway of patients is enhanced and HAT causes the progress and amplification of disease2. Mechanisms of proliferation enhanced by HAT on normal human bronchial fibroblasts : HAT enhanced proliferation of bronchial fibroblasts in serum-free medium with or without transferrin. Protease-activated receptor-2 agonist peptide (PAR2AP) enhanced proliferation of bronchial fibroblasts in serum-supplemented medium with transferrin. Leupeptin (serine protease inhibitor) and U0126 (MEK inhibitor) inhibited HAT induced proliferation of bronchial fibroblasts. Western blot analysis showed that HAT promoted the phosphorylation of MAP kinase. It was concluded that HAT enhanced the proliferation of fibroblasts through MAP kinase signal transduction, but not through PAR23. Enhanced synthesis of IL-8 by HAT on human bronchial epithelial cells (HBEC) : PAR2 mRNA was detected in HBEC with reverse PCR. Induction of IL-8 mRNA and release of IL-8 were increased by addition of HAT and PAR2 agonist peptide on HBEC. It was concluded that enhancement of IL-8 synthesis by HAT on HBEC was resulted from a sequential activation process : PAR2 activation by HAT, EGFR activation by transactivation and MAP kinase activation4. Activation of PAR2 by HAT on bronchial epithelial cells : Activation of PAR which coupled with G-protein increases the concentration of free Ca^<2+> in cells. Concentration of free Ca^<2+> in HBEC was increased by stimulation of HAT and PAR2 agonist peptide. It was concluded that HAT controlled the metabolic function of HBEC through activation of PAR2