Effect of TNFα on activities of different promoters of human apolipoprotein A-I gene

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5′-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching.     

Epigenetic alterations of CDH1 and APC genes: relationship with activation of Wnt/beta-catenin pathway in invasive ductal carcinoma of breast

Activation of canonical Wnt/beta-catenin pathway in Invasive Ductal Carcinoma of Breast (IDCs) was recently reported from our laboratory. Herein, we analyzed promoter methylation status of CDH1 and Adenomatous polyposis coli (APC) genes in 50 IDCs and correlated with expression of E-cadherin (E-CD) and APC proteins and with activation of oncogenic Wnt/beta-catenin signaling pathway components, Dvl, beta-catenin and CyclinD1. Further, Wnt/beta-catenin driven epithelial mesenchymal transition (EMT) was investigated by correlating the expression of Dvl, beta-catenin and CyclinD1 with vimentin expression in these IDCs. Promoter hypermethylation was observed in 25/50 (50%) IDCs for CDH1 and in 11/50 (22%) tumors for APC, associated with loss of expression of E-CD and APC proteins; concordant hypermethylation of these genes was observed in paired patients' sera. Further, 57% of tumors harboring CDH1 methylation and 50% tumors harboring the methylated APC gene showed nuclear localization of beta-catenin, suggesting activation of the canonical Wnt/beta-catenin pathway. Our study demonstrates significant association between vimentin expression and nuclear beta-catenin (p=0.001; Odds ratio (OR)=25.6) and Dvl (p=0.023; OR=8.0), suggesting that EMT may be driven by Wnt/beta-catenin activation in IDCs. In conclusion, we demonstrate correlation of CDH1 and APC promoter methylation with loss of E-CD and APC proteins and with activation of Wnt/beta-catenin signaling pathway. Association of nuclear Dvl and beta-catenin with vimentin expression suggests the importance of Wnt/beta-catenin pathway driven EMT in IDCs. The concordance between CDH1 and APC methylation in IDCs and paired circulating DNA underscores the utility of serum DNA as a non-invasive tool for methylation analysis in IDC patients.     

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene.

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.     

Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus type I) and that responsiveness to this viral protein was mediated by the presence of an NF-kappa B binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-kappa B binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-kappa B binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a human 12/15-lipoxygenase promoter variant associated with atherosclerosis identifies vimentin as a promoter binding protein

Background

Sequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden.

Methodology/Principal Findings

We demonstrate specific in vitro and in vivo binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in in-vitro vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin.

Conclusions/Significance

This study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation.     

AP-1/jun binding sites mediate serum inducibility of the human vimentin promoter.

The vimentin gene is inducible by serum in quiescent Balb/c 3T3 cells, but the molecular mechanism of this induction is unknown. The results presented here represent a first step towards the elucidation of the pathway of events leading from growth factor-receptor interaction to this induction. A series of Bal 31 deletions of the human vimentin promoter are used to show that a sequence residing at -700 is responsible for the serum, and also TPA inducibility of this gene. This sequence is able to confer serum inducibility upon uninducible constructs regardless of its position and orientation, indicating that it is this element alone which is required for this induction. The isolated sequence is a strong enhancer as well. Further deletions and the use of synthetic oligonucleotides demonstrate that a 24-mer containing two AP-1/jun binding sites confer both serum and TPA inducibility upon the human vimentin gene. Gel retardation analysis confirms that this sequence binds an AP-1 -like protein.     

The vimentin promoter as a tool to analyze the early events of retinoic acid-induced differentiation of cultured embryonal carcinoma cells

The vimentin gene encodes an intermediate filament protein expressed in the parietal endoderm, mesodermal, and early neural cells in vivo but by most in vitro-cultured cells regardless of their embryonic origin. Here we show that the vimentin gene promoter is very active in F9 embryonal carcinoma cells and increases in activity during differentiation. Using a series of 5'-deletion mutants, we provide evidence that the regions of the promoter involved in F9 cell activity are different from those previously demonstrated to be active in differentiated cell lines. Furthermore, we show that in differentiating F9 cells the activities of two different regions of the promoter are significantly enhanced. A distal region (-1710/-957) appears to contain functional binding sites for the murine Hox-A5 homeoprotein as demonstrated by band shift and footprinting experiments. A proximal region (-140/-78) contains a 30-bp repetitive sequence found in other genes activated during differentiation of F9 cells. Using band shift assays and methylation interference, we present evidence that a sequence-specific single-stranded DNA-binding protein(s) specifically interacts with the minus strand of the 30-bp sequence.