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
When carcinoma cells metastasize, they change their phenotype to enhance motility. Cells making this switch selectively express S100A4, a p53-associated, calcium-binding protein known in the fibroblast literature as fibroblast-specific protein-1 (FSP1). FSP1 normally acts as a conversion signal for the local formation of tissue fibroblasts by epithelial-mesenchymal transition. We describe here a novel connection between the process of fibroblast development and the acquisition of a metastatic phenotype in genetically engineered mice with mammary carcinoma. More frequent lung metastases were observed in naïve recipients given purified populations of green fluorescent protein (GFP)(+) tumor cells harvested from PyV-mT x FSP1(+/+.GFP) F1 mice compared with GFP(-) tumor cells (P < or = 0.01), where GFP expression is under the control of the FSP1 promoter. The expression of GFP in these metastases reversibly attenuates with the establishment of secondary tumor nodules. Reduced numbers of metastases were also observed in PyV-mT x FSP1(GFP/GFP) F1 mice carrying null alleles for FSP1 (P < or = 0.04) and in PyV-mT x FSP1.Delta TK(+) F1 mice rescued with nucleoside analogues while expressing thymidine kinase under the control of the FSP1 promoter (P < or = 0.01). We propose that epithelial-mesenchymal transition associated with the expression of FSP1 in tumor cells has a functional role in determining the latency of tumor dispersion and may be a convenient therapeutic target for controlling a key initiating event in metastatic progression.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Carcinoma/secundário , Células Epiteliais/fisiologia , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Mesoderma/fisiologia , Metástase Neoplásica/fisiopatologia , Transferência Adotiva , Alelos , Animais , Proteínas de Ligação ao Cálcio/genética , Caseínas/genética , Diferenciação Celular , Movimento Celular , Feminino , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Transplante de Neoplasias , Fenótipo , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100 , Células Estromais/patologia , Timidina Quinase/biossíntese , Timidina Quinase/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.
Assuntos
Retículo Endoplasmático/fisiologia , Infecções por Herpesviridae/fisiopatologia , Alvéolos Pulmonares/ultraestrutura , Fibrose Pulmonar/fisiopatologia , Proteína C Associada a Surfactante Pulmonar/fisiologia , Estresse Fisiológico/fisiopatologia , Antígenos Virais/biossíntese , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Chaperona BiP do Retículo Endoplasmático , Glicoproteínas/biossíntese , Proteínas de Choque Térmico/biossíntese , Infecções por Herpesviridae/complicações , Humanos , Imuno-Histoquímica , Chaperonas Moleculares/biossíntese , Proteínas Nucleares/biossíntese , Dobramento de Proteína , Fibrose Pulmonar/complicações , Proteína C Associada a Surfactante Pulmonar/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição , alfa-Manosidase/biossínteseRESUMO
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.