RESUMO
OBJECTIVE: Lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, stimulates proliferation of vascular smooth muscle cells (VSMC). We investigated the direct impact of 17beta-estradiol (E2) on the proliferation of VSMC from rat aorta. RESULTS: VSMC derived from both female and male rats expressed estrogen receptors alpha and beta. Treatments with 1% fetal bovine serum or 5 microM lysoPC increased the incorporation of [3H]-thymidine in VSMC obtained from female rats. 17Beta-E2 did not alter the response to fetal bovine serum, but significantly suppressed the enhanced deoxyribonucleic acid synthesis which had been induced by lysoPC in a dose-dependent manner (10(-4)-10(-6) M). Estrogen also inhibited the proliferation of VSMC from male animals. ICI 182,780, a specific estrogen receptor antagonist, and 17alpha-E2, an inactive form of estradiol, also decreased the mitogenic response to lysoPC in VSMC. In addition, N-acetyl-L-cysteine, a potent antioxidant, inhibited the lysoPC effect. Flow cytometric analysis using the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate revealed that elevated intracellular formation of reactive oxygen species elicited with lysoPC was depressed significantly by 17beta-E2, ICI 182,780, or 17alpha-E2 as well as by N-acetyl-L-cysteine. CONCLUSION: 17Beta-E2 inhibits in vitro VSMC proliferation induced by lysoPC via a nongenomic antioxidant mechanism.
Assuntos
Antioxidantes/farmacologia , Divisão Celular/efeitos dos fármacos , Estradiol/farmacologia , Lisofosfatidilcolinas/farmacologia , Músculo Liso Vascular/citologia , Animais , Aorta Torácica , Células Cultivadas , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Sangue Fetal , Citometria de Fluxo , Masculino , Músculo Liso Vascular/química , Músculo Liso Vascular/efeitos dos fármacos , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The pregnant uterus undergoes dramatic changes of tissue remodelling during the labour and post-partum period. We studied the production of matrix metalloproteinase 9 (MMP-9), as a major contributor of tissue remodelling, in human myometrium at parturition. The regulation of proMMP-9 by interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) was also investigated in human myometrial smooth muscle cells. MMP-9 was present in myometrial smooth muscle cells, interstitial fibroblasts and inflammatory cells. The gelatinolytic activities of proMMP-9 in myometrium increased dramatically during labour. IL-1beta and TNF-alpha induced proMMP-9 in myometrial smooth muscle cells, but these effects did not seem to be mediated by protein kinase C. On the other hand, neither 17beta-oestradiol nor progesterone itself affected proMMP-9 production in myometrial smooth muscle cells. Moreover, progesterone, which is known as a physiological suppressor of MMP-9 in other species, did not decrease the IL-1beta- and TNF-alpha-induced production of proMMP-9. These results suggest that IL-1beta and TNF-alpha are effective up-regulators of proMMP-9 in the tissue remodelling of human myometrium during labour.
Assuntos
Interleucina-1/metabolismo , Trabalho de Parto/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Músculo Liso/enzimologia , Miométrio/enzimologia , Fator de Necrose Tumoral alfa/metabolismo , Útero/enzimologia , Células Cultivadas , Precursores Enzimáticos/biossíntese , Feminino , Humanos , Músculo Liso/citologia , Gravidez , Regulação para Cima , Útero/citologiaRESUMO
To elucidate the endocrine mechanism of human parturition, the expression of c-Jun and c-Fos mRNA were examined in relation to estrogen receptor (ER) and progesterone receptor (PR) in human myometrium. c-Jun mRNA was detected in all myometrial tissues (n=5) during labor but not before labor (n=5) and in oxytocin-resistant postterm pregnancy (n=3). c-Fos mRNA was detected in only one myometrial tissue from a woman in labor. The distribution and intensity of immunostaining for ER and PR were semiquantitatively scored. During the late pregnancies, no significant difference was seen in the receptor scores for myometrial ER and PR between the patients who experienced labor and those who did not. Receptor scores for ER and PR were significantly lower in postterm pregnancy than in late pregnancy, regardless of the labor status. These data suggest that there are no changes in ER and PR in human myometrium during parturition. On the other hand, postterm pregnancy is associated with low ER and PR. c-Jun, induced during labor without changes in ER and PR, may play a role as a signaling mechanism in human myometrium.
Assuntos
Genes jun/genética , Trabalho de Parto/metabolismo , Miométrio/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Northern Blotting , Feminino , Humanos , Imuno-Histoquímica , Miométrio/citologia , Gravidez , RNA Mensageiro/análise , Valores de ReferênciaRESUMO
Wilson disease (WD), an autosomal recessive disorder of copper transport, is marked by impaired biliary excretion and incorporation of copper into ceruloplasmin. Molecular mechanism regulating the expression of the WD gene was studied. We isolated, sequenced, and characterized approximately 1.3 kb of the 5'-flanking region of the WD gene from the human genomic library. The approximately 1.3 kb of the WD sequence directed high level of luciferase activity in HepG2 cells. Interestingly, the 5'-flanking region contained four metal response elements (MREs) and six MRE-like sequences (MLSs), usually found in the metallothionein genes. It also contained a number of putative regulatory elements such as Sp1, AP-1, AP-2, and E-box, but lacked TATA box. The transcription start site was located at 335 base pairs upstream of the translation initiation site. Successive 5'-deletion analyses suggested that the 159-base pair region from -811 to -653, which includes MLS2 (-802 to -796) and MLS3 (-785 to -779), contained one or more positive regulatory element(s). A negative element was also identified at region -1038 to -812. A protein-MLS complex was identified through electrophoretic mobility shift and competition assay using MLS2/MLS3 and HepG2 cell nuclear proteins.
Assuntos
Degeneração Hepatolenticular/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Primers do DNA/genética , Proteínas de Ligação a DNA/análise , Regulação da Expressão Gênica/genética , Genes Reporter/genética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
Ceramide acts as a mediator of programmed cell death in various cell types, but its molecular mechanisms linked to the cell cycle are poorly understood. In this study, we investigated the expression of the p21 gene and its relationship to apoptosis induced by ceramide. In SK-HEP-1 cells, the addition of C6-ceramide resulted in a dose- and time-dependent growth suppression and DNA fragmentation characteristics of apoptosis. p21 protein was induced during that process, while the protein level of p53, known as a transcriptional activator of p21, was not elevated under the same condition. This apoptotic cell death with p21 induction was also observed in the Hep 3B cells lacking functional p53 after exposure to C6-ceramide. These findings suggest that ceramide-induced apoptosis is associated with the upregulation of p21 mRNA and protein in a p53-independent pathway.
Assuntos
Apoptose , Carcinoma Hepatocelular/patologia , Ciclinas/genética , Neoplasias Hepáticas/patologia , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/farmacologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Fragmentação do DNA , Regulação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fosforilação , Proteína do Retinoblastoma/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
Treatment of cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, was found to inhibit the proliferation of mouse fibroblast LP1-1 cells and the p34cdc2 kinase activity of M-phase promoting factor (MPF). However, it showed relatively little effect on expression of the cyclin B1 and cdc2 genes. On the other hand, when the nuclear extracts obtained from the cells at early G2 phase were treated with cAMP analogs, the kinase activity was significantly decreased as compared to the untreated control. Furthermore, the inhibitory effect of cAMP analogs could be reversed upon treating with okadaic acid even in the presence of the cAMP analogs, implying that cdc25 remains in an active form. In addition, the treatment of okadaic acid stimulated the cell progression. These results suggest that down-regulation of MPF activity through protein kinase A-mediated pathway is under post-translational control and cdc25 activation pathway involving okadaic acid-sensitive phosphatase play a role in the regulation of MPF activity.