Role of MMP-2 in Alveolar Epithelial Cell Repair after Bleomycin Administration in Rabbits

Abstract

Matrix metalloproteinases (MMPs) have been implicated in the pathological processes of interstitial lung diseases. However, underlying mechanisms, particularly for activity levels and distribution of activated MMP-2 in the disease process, are yet to be elucidated. The present study investigated the immunolocalization of MMP-2, membrane type 1-matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-2, p53, and Ki-67 in a rabbit model of bleomycin-induced pulmonary fibrosis. Gelatin zymography and in situ zymography were used to examine the activity and the localization of MMP-2. Furthermore, we performed Western blot and in situ hybridization for MT1-MMP, an activator for MMP-2. The total MMP-2 level estimated by gelatin zymography increased significantly at 3, 7, and 14 days after bleomycin administration, compared with controls. In the immunohistochemical study, immunoreaction for MMP-2 was strongest in alveolar epithelial cells among the cell populations. Swollen and/or elongated type II alveolar epithelial cells showed strong immunoreactions for MMP-2, MT1-MMP, and TIMP-2. After bleomycin administration, immunoreaction for p53 was observed in bronchiolar and alveolar epithelial cells. The proportion of p53-positive cells was high in epithelial cells from 1 to 14 days as MMP-2 levels were increased, suggesting that p53 may be responsible, at least in part, for the increase of MMP-2. The ratio of activated MMP-2 to total MMP-2 estimated by gelatin zymography increased significantly at 3, 7, 14, and 28 days after bleomycin treatment. In situ zymography revealed that type II alveolar epithelial cells degraded gelatin. An increased expression of MT1-MMP protein was observed by Western blot following administration of bleomycin. In situ hybridization demonstrated that type II alveolar epithelial cells gave intense signal for MT1-MMP mRNA. These results suggest that type II alveolar epithelial cells express MT1-MMP and activate MMP-2 on their cell surfaces, which may lead to the elongation and migration of alveolar epithelial cells in the repair process of bleomycin-induced pulmonary fibrosis.