RESUMO
Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.
Assuntos
Epitélio/metabolismo , Mesoderma/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Masculino , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multiproteicos , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100 , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteína 28 com Motivo TripartidoRESUMO
BACKGROUND: Fibroblasts can be misidentified as macrophages because both cell types share antigens that are associated with popular antibodies targeting the monocyte/macrophage lineage. With the recent description of fibroblast-specific protein 1 (FSP1), we revisited the specificity of antibodies directed against macrophages to determine systematically which antibodies best distinguish both cell types in fibrotic tissues. METHODS: Tissue fibrosis was produced in mice carrying the GFP transgene encoding green fluorescent protein under the control of the FSP1 promoter. Single cell suspensions from these marked tissues were submitted for flow cytometry using antibodies against Mac-1, Mac-2, Mac-3, F4/80, CD68, major histocompatibility complex (MHC) class II, and CD45, and cDNA amplification of mRNA encoding the above target antigens was performed using specific primer sets in sorted pools of cells. Fibrotic tissues were also stained by immunohistochemistry with the same antibodies and examined under confocal microscopy. RESULTS: Comparison overlap between FSP1(+) fibroblasts with each of the macrophage markers demonstrated that all antimacrophage antibodies (Mac-1, Mac-2, Mac-3, CD68, MHC class II, and CD45) except one (F4/80) recognize both cell types. CONCLUSION: Antibodies directed against F4/80 clearly distinguish macrophages from FSP1(+) fibroblasts in fibrotic tissues and is the preferred antibody in mice.