The ecotropic viral integration site-1 (Evi1) locus was initially identified as a common site of retroviral integration in myeloid tumors of the AKXD-23 recombinant inbred mouse strain. The full-length Evi1 transcript encodes a putative transcription factor, containing ten zinc finger motifs found within two domains of the protein. To determine the biological function of the Evi1 proto-oncogene, the full-length, but not an alternately spliced, transcript was disrupted using targeted mutagenesis in embryonic stem cells. Evi1 homozygous mutant embryos die at approximately 10.5 days post coitum. Mutants were distinguished at 10.5 days post coitum by widespread hypocellularity, hemorrhaging, and disruption in the development of paraxial mesenchyme. In addition, defects in the heart, somites, and cranial ganglia were detected and the peripheral nervous system failed to develop. These results correlated with whole-mount in situ hybridization analyses of embryos which showed expression of the Evi1 proto-oncogene in embryonic mesoderm and neural crest-derived cells associated with the peripheral nervous system. These data suggest that Evi1 has important roles in general cell proliferation, vascularization, and cell-specific developmental signaling, at midgestation.
Ectopic production of the EVI1 transcriptional repressor zinc finger protein is seen in 4 -6% of human acute myeloid leukemias. Overexpression also transforms Rat1 fibroblasts by an unknown mechanism, which is likely to be related to its role in leukemia and which depends upon its repressor activity. We show here that mutant murine Evi-1 proteins, lacking either the N-terminal zinc finger DNA binding domain or both DNA binding zinc finger clusters, function as dominant negative mutants by reverting the transformed phenotype of Evi-1 transformed Rat1 fibroblasts. The dominant negative activity of the non-DNA binding mutants suggests sequestration of transformation-specific cofactors and that recruitment of these cellular factors might mediate Evi-1 transforming activity. C-terminal binding protein (CtBP) co-repressor family proteins bind PLDLS-like motifs. We show that the murine Evi-1 repressor domain has two such sites, PFDLT (site a, amino acids 553-559) and PLDLS (site b, amino acids 584 -590), which independently can bind CtBP family co-repressor proteins, with site b binding with higher affinity than site a. Functional analysis of specific CtBP binding mutants show site b is absolutely required to mediate both transformation of Rat1 fibroblasts and transcriptional repressor activity. This is the first demonstration that the biological activity of a mammalian cellular transcriptional repressor protein is mediated by CtBPs. Furthermore, it suggests that CtBP proteins are involved in the development of some acute leukemias and that blocking their ability to specifically interact with EVI1 might provide a target for the development of pharmacological therapeutic agents.A small number of transcription factors are frequently targets for de-regulation by recurring chromosome translocations in acute leukemias, and these events play a pivotal role in disease progression (1). The EVI-1 gene encodes one of these transcription factors, which is activated in 4 -6% of acute myeloid leukemia (AML) 1 patients with various karyotypic abnormalities of chromosome 3q26 (2), which result in the ectopic production of intact or, occasionally, C-terminal-truncated EVI1 proteins (3-6). In addition, novel EVI1 fusion proteins are sometimes produced. For example, patients with karyotypes t(3;21) (q26;q22) or t(3;12) (q26;p13) express AML1/EVI1 (7) and ETV6(TEL)/EVI1 (8) chimeras, respectively, and similar fusions with a naturally occurring MDS1/EVI1 isoform (9).The precise contribution of ectopic EVI1 and EVI1 fusion protein production in leukemia progression is unknown, but a combination of enforced transgene expression and intervention studies shows a causative role, affecting both cell differentiation and proliferation. Expression of AML1/MDS1/EVI1 induces AML in mice, resulting in the accumulation of myeloid blast cells and immature differentiated myelocytic and monocytic lineages (10). EVI1 or AML1/EVI1 expression in either 32Dcl3 cells or murine primary bone marrow cells abrogates granulocyte colony-stimulating factor and eryth...
The myeloid transforming gene Evi-1 encodes a protein with two zinc ®nger domains, designated ZF1 and ZF2, with distinct DNA binding speci®cities. For the ®rst time we demonstrate that Evi-1 has transcriptional repressor activity which is directly proportional to the amount of Evi-1 protein in cells. Repression has been observed with two distinct promoters: the minimal HSV-1 tk promoter and a VP16 inducible adenovirus E1b minimal promoter. Optimal repression is DNA binding dependent and is mediated by either ZF1 or a heterologous GAL4 DNA binding domain (GAL4DBD) but is signi®cantly less e cient through the ZF2 binding site. Both GAL4DBD/ Evi-1 fusion and non-fusion proteins have been used to map the repressor activity to a proline-rich region located within amino acids 514 ± 724 between the ZF1 and ZF2 domains. Constitutive expression of mutant proteins lacking the repressor domain are defective for transformation of Rat1 ®broblasts demonstrating that this region is required for the oncogenic activity of the Evi-1 protein. These studies show that the Evi-1 gene encodes a transcriptional repressor and has important implications for the mechanism of action of the Evi-1 protein both in development and in the progression of some myeloid leukaemias.
In this study, we investigated the impact of enhancing cholesterol delivery to mitochondrial sterol 27-hydroxylase, via steroidogenic acute regulatory protein (StAR), on the expression of genes involved in macrophage cholesterol homeostasis and efflux of cholesterol to apolipoprotein (apo) AI. Methods and results Stably transfected, murine (RAW 264.7) macrophages were used to investigate the role of StAR in cholesterol homeostasis. Cellular responses were analysed using quantitative PCR, immunoblotting, and an LXRE reporter plasmid; [ 3 H]cholesterol efflux was measured in the presence or absence of apoAI. Macrophage overexpression of mitochondrial cholesterol trafficking protein, StAR, activates and induces expression of liver X receptors (LXRs), and significantly alters expression of genes involved in cholesterol homeostasis, decreasing Fdps, Hmgcr, Mvk, Ldlr, and Scap, and markedly increasing Abca1 mRNA and protein. Overexpression of StAR, but not mutated 'loss-of-function' (R181L) StAR, enhanced efflux of [ 3 H]cholesterol to apoAI, and this effect was maintained in macrophages pretreated with LDL or acetylated LDL. The effect of StAR overexpression on apoAI-dependent [ 3 H]cholesterol efflux was mimicked by non-sterol agonist, T901317, and 27-hydroxycholesterol, and blocked by LXR inhibitor, geranylgeranyl pyrophosphate, sterol 27-hydroxylase inhibitor, GW273297x, and probucol, inhibitor of ATP binding cassette transporter A1 (ABCA1). Importantly, all observed effects of StAR overexpression were dependent upon cyclic AMP (cAMP analogue, dibutyryl cAMP), which is required for the full activity of the StAR protein to be manifested. Conclusion Macrophage overexpression of StAR significantly enhances LXR-dependent apoAI-and ABCA1-dependent cholesterol efflux, by which disposal of excess arterial cholesterol deposits and atheroma regression can be achieved.
Edited by Laszlo NagyKeywords: Atherosclerosis Macrophage 'Foam cell' Steroidogenic acute regulatory protein (StAR/STARD1) STARD3/metastatic lymph node 64 (MLN64) Liver X receptor Peroxisome proliferator activated receptor Retinoic acid X receptor Sterol regulatory element binding protein a b s t r a c tThe STARD1 subfamily of 'START' lipid trafficking proteins can reduce macrophage lipid content and inflammatory status (STARD1; StAR), and traffic cholesterol from endosomes (STARD3/MLN64). During macrophage differentiation, STARD1 mRNA and protein increase with sterol content, while the reverse is true for STARD3. Sterol depletion (methyl beta-cyclodextrin) enhances STARD3, and represses STARD1 expression. Agonists of Liver X receptors, peroxisome proliferator activated receptor-gamma and retinoic acid X receptors increase STARD1 expression, while hypocholesterolaemic agent, LY295427, reveals both STARD1 and STARD3 as putative SREBP-target genes. Pathophysiological 'foam cell' formation, induced by acetylated or oxidized LDL, significantly reduced both STARD1 and STARD3 gene expression. Differential regulation of STARD1 and D3 reflects their distinct roles in macrophage cholesterol metabolism, and may inform anti-atherogenic strategies.
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