RESUMO
Bacterial lipopolysaccharide (LPS) stably induced the protein kinase C substrate, MacMARCKS, in murine resident peritoneal macrophages; initial induction of MacMARCKS mRNA was detected within 15 min and was protein synthesis-independent. This response was observed in the macrophage cell line RAW264, and occurred also in response to plasmid DNA, a partial mimetic of other responses to LPS. In murine bone marrow-derived macrophages, MacMARCKS was expressed constitutively due to induction by macrophage colony-stimulating factor. Nuclear run-on transcription revealed that, like tumor necrosis factor alpha (TNF-alpha), MacMARCKS was transcribed constitutively in RAW264 cells. The MacMARCKS promoter was sequenced to -1.7 kb and the transcription start site determined. Transient transfections of RAW264 cells revealed that the 113-bp GC-rich proximal promoter contained all the elements required for both high basal activity and 15- to 20-fold activation by LPS.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Proteínas de Membrana/genética , Animais , Sequência de Bases , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Proteínas de Ligação a Calmodulina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Especificidade de Órgãos , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
We investigate the regulation of plasminogen activator inhibitor-2 (PAI-2) in murine macrophages. PAI-2 mRNA was inducible by bacterial lipopolysaccharide (LPS) in primary cells and macrophage-like cell lines. Evidence is presented for a role for autocrine factors, including cyclooxygenase products but not the cytokines tumor necrosis factor alpha or interferon-beta (IFN-beta). PAI-2 mRNA levels generally varied inversely from those of its target, urokinase-type plasminogen activator (uPA), and the macrophage growth factor CSF-1, which induces uPA, inhibited PAI-2 expression in cells treated subsequently with LPS. Expression of PAI-2 was distinct from that of other LPS-inducible genes in terms of induction time course, LPS dose response, and sensitivity to co-stimulation with IFN-gamma. Induction of PAI-2 mRNA in subclones of the cell line RAW 264 was not uniform, reflecting heterogeneous expression in the parent line. The expression pattern of PAI-2 is discussed in terms of a possible role in LPS-induced pathology such as septicemia.
Assuntos
Toxinas Bacterianas/metabolismo , Regulação da Expressão Gênica , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética , Animais , Toxinas Bacterianas/farmacologia , Northern Blotting , Linhagem Celular , Células Cultivadas , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA MensageiroRESUMO
The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A variant mRNA was identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount in situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.
Assuntos
Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/metabolismo , Proteínas Tirosina Fosfatases/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Diferenciação Celular , Divisão Celular , Linhagem da Célula , Clonagem Molecular , DNA Complementar/genética , Olho/embriologia , Olho/metabolismo , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Técnica Indireta de Fluorescência para Anticorpo , Hibridização In Situ , Macrófagos/classificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Nervos Periféricos/embriologia , Nervos Periféricos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a ReceptoresRESUMO
Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-alpha, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.