[Frontiers in Bioscience 10, 2534-2547, September 1, 2005] 2534 Characterization of alpha-enolase as an interferon-alpha 2 alpha 1 regulated gene Lirlândia P. Sousa 1,2 , Bruno S. A. F. Brasil 1,2 , Breno De M. Silva 1,2 , Sarah V. Nogueira 1,2 , Anderson A. Andrade 1,2 , Paulo C. P. Ferreira 2 , Santuza M. R. Teixeira 3 , Kenneth J. Gollob 3 , Erna G. Kroon 2 , Kanefusa Kato 4 , and Cláudio A. Bonjardim 1,2 1 Grupo de Transdução de Sinal, 2 Laboratório de Vírus, Departamento de Microbiologia, 3 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-90, Belo Horizonte, Minas Gerais, Brazil, 4 Department of Biochemistry, Institute for Developmental Research, Aichi, Japan TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Material and Methods 3.1. Cell culture, probes, chemicals, and antibodies 3.2. Cell treatment 3.3. RNA isolation and Northern blot Analysis 3.4. Electrophoretic mobility shift assay (EMSA) 3.5. Lysates preparation and Western Blot Analysis 3.6. Flow Cytometry Analysis of Peripheral Blood Mononuclear Cells 3.7. Cell Surface-generated plasmin Assay 4. Results 4.1. Interferons-alpha and.gamma stimulate alpha-enolase mRNA accumulation 4.2. Early response genes associated with alpha-ENO expression 4.3. DNA-protein complexes formed upon IFN-alpha 2 alpha 1 stimulation 4.4. IFN-alpha 2 alpha 1 stimulates both ERK1/2 and CREB/ATF-1 phosphorylation 4.5. IFN-alpha 2 alpha 1. upregulated α-enolase expression at the surface of monocytes 4.6. IFN-alpha increased cell surface plasmin generation 5. Discussion 8. Acknowledgements 9. References 1. ABSTRACT Interferons (IFNs) are multifunctional cytokines that after binding to the cell surface receptor induce the expression of a large number of genes, which in turn, mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Here we show that IFN-alpha activates the mitogen-activated protein kinases (MAPK) ERK1/2 and the transcription factor CREB/ATF-1, which lead to the alpha-enolase (alpha- ENO) gene expression in fibroblasts. Alpha-ENO mRNA accumulation was apparent 6 h post-IFN stimulation and required both de novo protein synthesis and active gene transcription, which is typical of a secondary response gene. Alpha-ENO expression does not appear to be restricted to fibroblasts, since it was equally verified in peripheral blood mononuclear cells (PBMC). Furthermore, IFN-alpha stimulates the expression of the primary response genes c-fos and egr-1, which was followed by an increase in DNA binding activity of c-FOS and EGR-1 proteins, as verified by shift assays using the cis-acting elements AP-1 and EGR-1 localized at the alpha-ENO promoter. Finally, we also demonstrated that IFN treatment of PBMC cause an increase in both, alpha-ENO expression on the cell surface and plasmin generation followed addition of exogenous plasminogen. 2. INTRODUCTION Interferons (IFNs) are a heterogenic family of cytokines that in vertebrates exert a broad spectrum of biological functions, such as, growth inhibition, regulation of the immune system and antimicrobial activity (1, 2). The recognition of pathogen-associated molecular patterns (PAMPs) by the Toll-like receptors (TLRs), engages downstream signaling pathways resulting in IFN production and secretion (3, 4). Humans IFNs, type I (mainly alpha, beta, and tau) and type II (IFN-gamma), once secreted, bind to specific cell surface receptors and activate a cascade of intracellular signaling pathways that results in the induction of hundreds of IFN-stimulated genes (ISGs), whose gene products are responsible for their biological activities (1, 5, 6). The function of some of the IFN-stimulated proteins is well described, whereas that of many others remains poorly characterized (7). Although the classical JAK/STAT pathway is well characterized for IFN signaling (1), recent studies indicate that IFN activates several other protein kinases, including the MAP kinase family and downstream transcription factors (8). Alpha enolase (alpha-ENO) is a multifunctional protein firstly characterized by its enzymatic activity in the glycolytic pathway. Unlike other glycolytic enzyme genes,