RESUMO
BACKGROUND: Matrix metalloproteinase-26 (MMP-26), one of the main mediators of extracellular matrix (ECM) degradation, has been shown to exist in trophoblasts of human placenta and to play a role in trophoblast cell invasion. However, little is known about the regulation of MMP-26 expression in human trophoblasts. Recently, gonadotropin-releasing hormone I (GnRH I) and GnRH II have been shown to regulate the expression of MMP-2, MMP-9/tissue inhibitor of metalloproteinases 1 (TIMP-1), and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor (PAI) in human trophoblasts, suggesting that these two hormones may work as paracrine and/or autocrine regulators in modulating the activities of various protease systems at the feto-maternal interface. In this study, we determined the regulatory effects of GnRH I and GnRH II on the expression of MMP-26 in human immortalized cytotrophoblast-like cell line, B6Tert-1. METHODS: Real-time PCR was used to quantify mRNA levels of MMP-26 in human trophoblast-like cell line, B6Tert-1 and primary cultured cytotrophoblasts. Western blotting was used to characterize the expression of MMP-26 and the phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) in B6Tert-1 cells after treatment with GnRH I and GnRH II. RESULTS: We found that GnRH I increased MMP-26 expression in B6Tert-1 cells after 12 h of treatment at both the mRNA and protein level, while GnRH II increased MMP-26 expression beginning at 3 h of treatment. Treatment of GnRH I at 1 nM resulted in maximal increase of MMP-26 mRNA and protein levels, whereas GnRH II treatment at a concentration of 100 nM was required to induce maximal increase in MMP-26 expression. In addition, we demonstrated that the activation of JNK, but not ERK1/2, was required for GnRH I and II-stimulated MMP-26 production in B6Tert-1 cells and primary cytotrophoblasts. CONCLUSIONS: These novel findings indicated that GnRH I and II could up-regulate MMP-26 expression through the JNK signaling pathway in human trophoblast-like/trophoblast cells.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz/genética , Precursores de Proteínas/farmacologia , Trofoblastos/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Gravidez , Primeiro Trimestre da Gravidez/genética , Primeiro Trimestre da Gravidez/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Trofoblastos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the conversion of 17beta-OH (-hydroxy)/17-oxo groups of steroids, and are essential in mammalian hormone physiology. At present, eleven 17beta-HSD isoforms have been defined in mammals, with different tissue-expression and substrate-conversion patterns. We analysed 17beta-HSD type 10 (17beta-HSD10) from humans and Drosophila, the latter known to be essential in development. In addition to the known hydroxyacyl-CoA dehydrogenase, and 3alpha-OH and 17beta-OH activities with sex steroids, we here demonstrate novel activities of 17beta-HSD10. Both species variants oxidize the 20beta-OH and 21-OH groups in C21 steroids, and act as 7beta-OH dehydrogenases of ursodeoxycholic or isoursodeoxycholic acid (also known as 7beta-hydroxylithocholic acid or 7beta-hydroxyisolithocholic acid respectively). Additionally, the human orthologue oxidizes the 7alpha-OH of chenodeoxycholic acid (5beta-cholanic acid, 3alpha,7alpha-diol) and cholic acid (5beta-cholanic acid). These novel substrate specificities are explained by homology models based on the orthologous rat crystal structure, showing a wide hydrophobic cleft, capable of accommodating steroids in different orientations. These properties suggest that the human enzyme is involved in glucocorticoid and gestagen catabolism, and participates in bile acid isomerization. Confocal microscopy and electron microscopy studies reveal that the human form is localized to mitochondria, whereas Drosophila 17beta-HSD10 shows a cytosolic localization pattern, possibly due to an N-terminal sequence difference that in human 17beta-HSD10 constitutes a mitochondrial targeting signal, extending into the Rossmann-fold motif.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases , Ácidos e Sais Biliares/metabolismo , Hormônios Esteroides Gonadais/metabolismo , 17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Drosophila melanogaster/enzimologia , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Cinética , Mitocôndrias/química , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Esteroides/metabolismo , Especificidade por SubstratoRESUMO
The processes of implantation and placentation involve the degradation and remodeling of extracellular matrix, cellular proliferation, apoptosis, and differentiation. Evidence indicates that members of the matrix metalloproteinase (MMP) family play crucial roles in these processes. In the present study, we identified the expression and localization of MMP26/endometase/ matrilysin-2 in human placentae at different stages of gestation using methods of reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. MMP26 was widely localized to villous cytotrophoblast cells, syncytiotrophoblast cells, and to column trophoblasts during early pregnancy. The mRNA and protein level of MMP26 in chorionic villi was highest at Weeks 6-7, and decreased thereafter, reaching its lowest level at the second trimester. The mRNA level was significantly up-regulated in term placenta, while the immunoreactivity remained undetectable. Notably, intense expression of MMP26 was found in fetal nucleated red cells inside the villous capillaries during gestational Weeks 6-9. Strong expression of MMP26 mRNA was also demonstrated in fetal red cells isolated from the whole blood of fetuses at midpregnancy. The expression patterns of MMP26 in human placenta suggests complicated roles for MMP26 during the processes of placentation and hematopoiesis, perhaps working in concert with other members of the MMP family, such as MMP9.