Homocysteine (Hcy) has recently been recognized as an integral component of several disorders. However, the association between hyperhomocysteinemia (HHcy) and pulmonary disease is not well understood. ]The combination of two-dimensional electrophoresis and tandem mass spectrometry detected and identified proteins that are differentially expressed in human type 11 alveolar epithelial cells (A549 cells) treated by Hey. We found that aldose reductase (AR) showed more abundant expression in the cells, Further, Hey (100500 mu M) could induce a time- and dose-dependent upregulation of AR protein levels. Immunohistochemical staining of cross-sections from HHcy mice lungs also revealed increased expression of AR protein. Intracellular levels of reactive oxygen species (ROS) were remarkably elevated in A549 cells treated with Hey. Pretreatment of A549 cells with catalase and SOD significantly suppressed the Hcy-induced AR expression, which suggests the involvement of ROS in this process. The major signaling pathway mediating the upregulation of AR was demonstrated to be the Ras/Raf/ERK1/2 pathway. In addition, Hey might reduce surfactant protein B (SP-B) expression in the cells, which could be significantly attenuated by Alrestatin, an AR inhibitor, indicating a damaging role of Hcy-induced AR elevation in the lung. These results show a novel and unanticipated link between HHcy and AR upregulation that may be a risk factor in pulmonary disease of patients with HHcy. (c) 2005 Elsevier Inc. All rights reserved.