Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for cellular adaptation to hypoxia, although its physiological roles and activation mechanisms during normoxia have not been studied sufficiently. The Warburg effect, which is a hallmark of malignant tumors that is characterized by increased activity of aerobic glycolysis, accompanies activation of HIF-1 during normoxia. Besides tumor cells that have multiple genetic and epigenetic alterations, normal macrophages also use glycolysis for ATP production by depending upon elevated HIF-1 activity even during normoxia. We recently found that activity of factor inhibiting HIF-1 (FIH-1) is specifically suppressed in macrophages by a nonproteolytic activity of mem-

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Cells in tissues are surrounded by an extracellular cellular matrix that interacts with cells to regulate their activity (1, 2). Matrix metalloproteinases (MMPs) 3 are endopeptidases responsible for extracellular matrix degradation and thereby regulate turnover of the extracellular matrix. However, recent studies have demonstrated that substrates of MMPs are expanded to a variety of pericellular proteins. MT1-MMP/MMP14 is an integral membrane proteinase that cleaves multiple proteins in the pericellular milieu and thereby regulates various cell functions. Substrates of MT1-MMP identified to date include extracellular matrix proteins (type I collagen, fibronectin, vitronectin, laminin-1 and -5, and others), cell adhesion molecules (CD44, syndecan-1, and ␣v integrin), cytokines (SDF-1 and transforming growth factor-␤ and others), and latent forms of pro-MMPs (pro-MMP-2 and pro-MMP13) (3-5). Processing of these proteins by MT1-MMP alters their activities and thereby regulates a variety of cellular functions, such as motility, invasion, growth, differentiation, and apoptosis. Consistent with these functions, forced expression of MT1-MMP in tumor cells enhances behavior consistent with increased malignancy, such as rapid tumor growth, invasion, and metastasis (6). However, MT1-MMP is normally expressed in various types of cell and mice deficient in MT1-MMP expression (MT1 Ϫ/Ϫ ) display pleiotropic defects (7-10). However, we as yet have only limited knowledge of the physiological substrates of MT1-MMP that could explain such pleiotropic effects.Proteases interact with their substrates at least transiently, but in some cases such interaction is more stable. For instance, type I collagen binds MT1-MMP via a hemopexin-like domain and is cleaved (11, 12). Cleavage of collagen by MT1-MMP regulates cell growth and invasion in a collagen-rich environment (13). CD44, a hyaluronic acid receptor, also binds to the hemopexin of MT1-MMP and is cleaved (14). Expression of CD44 and MT1-MMP in tumor cells promotes cell migration, accompanied by the shedding of CD44 by MT1-MMP (14, 15). pro-MMP-2, which is cleaved by MT1-MMP for activation, forms a tri-molecular complex with MT1-MMP and TIM...