Hypoxia-inducible factor-1α mediates TGF-β-induced PAI-1 production in alveolar macrophages in pulmonary fibrosis

Ueno M, Maeno T, Nomura M, Aoyagi-Ikeda K, Matsui H, Hara K, Tanaka T, Iso T, Suga T, Kurabayashi M. Hypoxia-inducible factor-1 alpha mediates TGF-beta-induced PAI-1 production in alveolar macrophages in pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 300: L740-L752, 2011. First published January 14, 2011; doi:10.1152/ajplung.00146.2010.-Hypoxia-inducible factor-1 alpha (HIF-1 alpha), a transcription factor that functions as a master regulator of oxygen homeostasis, has been implicated in fibrinogenesis. Here, we explore the role of HIF-1 alpha in transforming growth factor-beta (TGF-beta) signaling by examining the effects of TGF-beta(1) on the expression of plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of lung tissue from a mouse bleomycin (BLM)-induced pulmonary fibrosis model revealed that expression of HIF-1 alpha and PAI-1 was predominantly induced in alveolar macrophages. Real-time RT-PCR and ELISA analysis showed that PAI-1 mRNA and activated PAI-1 protein level were strongly induced 7 days after BLM instillation. Stimulation of cultured mouse alveolar macrophages (MH-S cells) with TGF-beta(1) induced PAI-1 production, which was associated with HIF-1 alpha protein accumulation. This accumulation of HIF-1 alpha protein was inhibited by SB431542 (type I TGF-beta receptor/ALK receptor inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38 MAP kinase inhibitor). Expression of prolyl-hydroxylase domain (PHD)-2, which is essential for HIF-1 alpha degradation, was inhibited by TGF-beta(1), and this decrease was abolished by SB431542. TGF-beta(1) induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1 alpha silencing. Transcriptome analysis by cDNA microarray of MH-S cells after HIF-1 alpha silencing uncovered several pro-fibrotic genes whose regulation by TGF-beta(1) required HIF-1 alpha, including platelet-derived growth factor-A. Taken together, these findings expand our concept of the role of HIF-1 alpha in pulmonary fibrosis in mediating the effects of TGF-beta(1) on the expression of the pro-fibrotic genes in activated alveolar macrophages.