Abstract-A potent transcriptional enhancer was previously identified upstream of the mouse renin gene. Within the enhancer is a TGACCT direct-repeat motif, required for enhancer activity, that is the consensus sequence recognized by members of the thyroid hormone subfamily of steroid hormone receptors. We previously reported that RAR/RXR bind to this sequence and mediate the induction of renin promoter activity by retinoids. However, gel mobility shift assays clearly show that other as yet unidentified factors also bind to this motif. In order to identify some of these TGACCT binding factors, we screened a yeast one-hybrid cDNA library derived from mouse As4.1 cells. One of these encoded the orphan nuclear receptor Ear2. Recombinant Ear2 was purified from Escherichia coli and an antipeptide antisera was generated. EMSA showed that purified recombinant Ear2 specifically binds the TGACCT direct-repeat motif. Transfection assays showed that Ear2 potently decreases both baseline and retinoid-induced mouse renin promoter activity in a dose-dependent, enhancer-dependent, and sequence-specific manner. Mutations in Ear2, which abolish its binding to the TGACCT motif, also abolish transcriptional repression. Ear2 was identified as a nuclear protein in As4.

The T to C substitution at position ؊175 of the ␥-globin gene has been identified in some individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH). In this study, the HPFH phenotype was reestablished in transgenic mice carrying the LCRA␥ ؊175 ␤␦␤ construct, which contained a 3.1-kb LCR cassette linked to a 29-kb fragment from the A␥-to ␤-globin gene with the natural chromosome arrangement but with the ؊175 mutation, which provided evidence for this single mutation as the cause of this form of HPFH. The HPFH phenotype was also reproduced in transgenic mice carrying the LCRA␥ ؊173 ␤␦␤ construct, in which the ؊175 T to C A␥ gene was substituted with the ؊173 T to C A␥ gene. In vitro experiments proved that the ؊175 mutation significantly reduced binding of Oct-1 but not GATA-1, whereas the ؊173 mutation dramatically decreased binding of GATA-1 but not Oct-1. These results suggest that abrogation of either GATA-1 or Oct-1 binding to this promoter region may result in the HPFH phenotype. An in vivo footprinting assay revealed that either the ؊175 mutation or the ؊173 mutation significantly decreased overall protein binding to this promoter region in adult erythrocytes of transgenic mice. We hypothesize that a multiprotein complex containing GATA-1, Oct-1, and other protein factors may contribute to the formation of a repressive chromatin structure that silences ␥-globin gene expression in normal adult erythrocytes. Both the ؊173 and ؊175 T to C substitutions may disrupt the complex assembly and result in the reactivation of the ␥-globin gene in adult erythrocytes.The human fetal globin genes (G␥ and A␥) are mainly expressed in fetal liver. The switch from fetal to adult (␦ and ␤) globin gene expression occurs in the perinatal period concomitantly with the establishment of the bone marrow as the main site of erythropoiesis. In some individuals, however, this switching is incomplete, and ␥-globin expression persists in adult erythrocytes. Such a condition, found in syndromes of hereditary persistence of fetal hemoglobin (HPFH) 1 and (␦␤) 0 -thalassemias, can result from deletions within the ␤-globin gene cluster (1). However, in some syndromes known as nondeletion HPFH, point mutations in the ␥-globin gene promoter seem to be responsible for the continuance of ␥-globin expression (1). It has been revealed that most of the point mutations lie in or near the trans-acting factor binding motifs within the promoter and that the mutations usually affect the binding of some trans-acting factors in vitro (2).Studies in transgenic mice carrying either Ϫ117 A␥ HPFH cosmid or Ϫ117 A␥ HPFH yeast artificial chromosome showed that transgenic expression could mimic the developmental expression program observed in HPFH, providing powerful proof that the single point mutation was the cause of the Greek form of HPFH (3, 4). Berry et al. (3) speculated that this change of ␥-globin expression during development could be correlated with the loss of GATA-1 binding to the ␥-promoter region around Ϫ117 and deduced that ...

BackgroundMelatonin, a well-known antioxidant, has been shown to possess anti-invasive properties for glioma. However, little is known about the effect of melatonin on glioma cell migration and invasion under hypoxia, which is a crucial microenvironment for tumor progress. In addition, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration and invasion. Therefore, we investigated the possible role of these kinases and its related signaling in the regulation of human U251 glioma cells behavior by melatonin under hypoxia.MethodsThe abilities of migration and invasion of U251 glioma cells were determined by wound healing and transwell assay in vitro. The intracellular production of reactive oxygen species (ROS) was measured by using the fluorescent probe 6-carboxy-2′, 7′-dichorodihydrofluorescein diacetate (DCFH-DA). Immunofluorescence experiments and western blotting analysis were used to detect the expression level of protein. Small interfering RNAs (siRNA) was used to silence specific gene expression.ResultsThe pharmacologic concentration (1 mM) of melatonin significantly inhibited the migration and invasion of human U251 glioma cells under hypoxia. The inhibitory effect of melatonin was accompanied with the reduced phosphorylation of FAK and Pyk2, and decreased expression of alpha v beta 3 (αvβ3) integrin. Additionally, inhibition of αvβ3 integrin by siRNA reduced the phosphorylation of FAK/Pyk2 and demonstrated the similar anti-tumor effects as melatonin, suggesting the involvement of αvβ3 integrin- FAK/Pyk2 pathway in the anti-migratory and anti-invasive effect of melatonin. It was also found that melatonin treatment decreased the ROS levels in U251 glioma cells cultured under hypoxia. ROS inhibitor apocynin not only inhibited αvβ3 integrin expression and the phosphorylation levels of FAK and Pyk2, but also suppressed the migratory and invasive capacity of U251 glioma cells under hypoxia.ConclusionsThese results suggest that melatonin exerts anti-migratory and anti-invasive effects on glioma cells in response to hypoxia via ROS-αvβ3 integrin-FAK/Pyk2 signaling pathways. This provides evidence that melatonin may be a potential therapeutic molecule targeting the hypoxic microenvironment of glioma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0454-8) contains supplementary material, which is available to authorized users.

Wnt ligand-driven tumor growth is inhibited by the soluble Wnt inhibitor Fzd8CRD, but the mechanism through which this effect is mediated is unknown. In the MMTV-Wnt1 mouse model, regression of mammary tumors by Fzd8CRD treatment coincides with an acute and strong induction of insulin-like growth factor (IGF)-binding protein IGFBP5, an antagonist of IGF signaling that mediates involution of mammary gland in females after offspring are weaned. In this study, we show that repression of this IGF inhibitory pathway is crucial for Wntdriven growth of mammary tumors. We found that IGFBP5 regulation was mediated by the b-catenin-dependent Wnt pathway. Wnt, in addition to IGF ligands, facilitated tumor growth by paracrine communication among tumor cells. In addition, Fzd8CRD caused precocious induction of IGFBP5 in normal mammary glands undergoing involution, implying an acceleration of the involution process by inhibition of Wnt signaling. The molecular and phenotypic parallel between tumor regression and mammary gland involution suggests that Wntdriven mammary tumors use the same growth mechanism as proliferating normal mammary glands. Cancer Res; 72(6); 1568-78. Ó2012 AACR.