RESUMO
OBJECTIVE: Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein, is a potent bioactive phospholipid. Early growth response gene-1 (Egr-1), an important transcription factor, regulates expression of an array of genes involved in vascular diseases. Whether and how LPA regulates the transcriptional machinery of Egr-1 gene is unknown and is addressed in this study. METHOD AND RESULTS: We found that LPA markedly induces Egr-1 mRNA and protein in aortic smooth muscle cells (SMCs). RNA stability and nuclear run-on assays reveal that LPA-induced Egr-1 gene expression is controlled at the transcriptional level. Reporter gene analyses have shown that the -141 to +20 nt region of the Egr-1 promoter contains regulatory elements. Electrophoretic mobility shift assays reveal that the DNA-binding activities of both CREB and SRF to the CRE and SRE motifs of the Egr-1 promoter are markedly elevated in response to LPA. The increased binding activity depends on the phosphorylation of CREB and SRF. Luciferase assays of a series of deleted or mutated Egr-1 promoter-reporter gene constructs, along with dominant negative CREB transfection analysis revealed that the 2 CRE sites and the 2 proximal SRE sites in the Egr-1 promoter are required for maximal LPA-induced Egr-1 gene expression. CONCLUSIONS: Our data reveal that LPA regulates Egr-1 expression via transcription factors CREB and SRF. These results establish a novel role for CREB in mediating LPA-induced gene expression. Our results imply that elevated LPA levels may, through activation of Egr-1, which regulates an array of atherogenic genes, exacerbate atheromatous lesions.
Assuntos
AMP Cíclico/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Elementos de Resposta/fisiologia , Elemento de Resposta Sérica/fisiologia , Transcrição Gênica , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Músculo Liso Vascular/citologia , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Ratos , Fator de Resposta Sérica/fisiologia , Transdução de SinaisRESUMO
OBJECTIVE: Tissue factor (TF), the initiator of the coagulation cascade, is expressed by cells in atherosclerotic lesions. Lysophosphatidic acid (LPA) is a component of oxidized lipoproteins and an agent released by activated platelets. The present study investigated whether and how TF expression is regulated by LPA. METHODS AND RESULTS: Northern blotting, Western blotting, and TF activity assays demonstrated that LPA markedly induced TF mRNA, protein, and activity in vascular smooth muscle cells. LPA-induced TF expression is primarily controlled at the transcriptional level. Phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signaling-regulated kinases (ERK1/2) was rapidly and markedly induced by LPA. MEK inhibitors U0126 and PD98059 blocked both ERK activation and the increase in TF mRNA. In contrast, the specific p38 MAP kinase inhibitor SB203580 had no effect on LPA-induced TF mRNA increase. The Galpha(i) protein inhibitor, pertussis toxin, abolished LPA-induced phosphorylation of MEKs and ERKs, as well as the induction of TF mRNA. CONCLUSIONS: Our data demonstrate that a Galpha(i) protein and activation of MEKs and ERKs mediate LPA-induced TF expression. Our data suggest that elevated LPA could be a thrombogenic risk factor by upregulating TF expression. These results may have important implications in vascular remodeling and vascular diseases.
Assuntos
Aorta/citologia , Aorta/metabolismo , Lisofosfolipídeos/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Tromboplastina/biossíntese , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Células Cultivadas , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Humanos , Lisofosfolipídeos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Tromboplastina/genética , Tromboplastina/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The primary cilium is a microtubule-based organelle implicated as an essential component of a number of signaling pathways. It is present on cells throughout the mammalian body; however, its functions in most tissues remain largely unknown. Herein we demonstrate that primary cilia are present on cells in murine skin and hair follicles throughout morphogenesis and during hair follicle cycling in postnatal life. Using the Cre-lox system, we disrupted cilia assembly in the ventral dermis and evaluated the effects on hair follicle development. Mice with disrupted dermal cilia have severe hypotrichosis (lack of hair) in affected areas. Histological analyses reveal that most follicles in the mutants arrest at stage 2 of hair development and have small or absent dermal condensates. This phenotype is reminiscent of that seen in the skin of mice lacking Shh or Gli2. In situ hybridization and quantitative RT-PCR analysis indicates that the hedgehog pathway is downregulated in the dermis of the cilia mutant hair follicles. Thus, these data establish cilia as a critical signaling component required for normal hair morphogenesis and suggest that this organelle is needed on cells in the dermis for reception of signals such as sonic hedgehog.