Vimentin Promoter Research Articles

T-antigen regulated expression reduces apoptosis of tag-transformed human myoblasts.

The generation of human myogenic cell lines could potentially provide a valuable source for cell transplantation in myopathies. The dysregulation of proliferative-differentiative signals by viral oncogenes can result in the induction of apoptosis. Whether apoptosis occurred in myogenic cells expressing large T antigen (Tag) from SV40 upon differentiation was unknown. Human muscle satellite cells were transfected with two different constructs, containing either an origin-defective SV40 genome or Tag under vimentin promoter control. When differentiation was triggered, Tag expression reduced the formation of myotubes and dead cells showing apoptotic features were present. However, the cells expressing SV40 Tag under vimentin promoter control retained their capacity to form myotubes and expressed the myofibrillar proteins as myosin heavy chain and dystrophin when Tag expression was silent. Their apoptotic rate was similar to that of untransfected cells. The observation that apoptosis can be prevented by the down-regulation of Tag suggests that the programmed cell death induced in transformed cells can be reversed, and confirms the regulatory efficiency of the human vimentin promoter.

The Zinc Finger Repressor, ZBP-89, Binds to the Silencer Element of the Human Vimentin Gene and Complexes with the Transcriptional Activator, Sp1

Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.

Vimentin contributes to human mammary epithelial cell migration

Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

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.

An antisilencer element is involved in the transcriptional regulation of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene was previously reported to contain two positive-acting regions, separated by a negative region (Rittling, S.R., Baserga, R., 1987. Functional analysis and growth factor regulation of the human vimentin promoter. Mol. Cell. Biol. 7, 3908-3915). Here, detailed studies reveal two additional regulatory elements, a new positive transcriptional element located between -717 and -757, and a new repressor element at -780 to -821. In transient transfections, the positive-acting element is able to completely override the effect of different silencer elements when fused to a heterologous promoter. However, this element does not enhance gene activity when the silencer element is absent and thus cannot be viewed as a true enhancer. Since it appears to overcome the effect of a silencer element, we refer to it as an antisilencer element. Gel mobility shift assays, UV-cross-linking experiments, and Southwestern blots reveal that a 105-kDa protein specifically binds to this region.

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control.

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells.

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 437–445, 1997

SV40 Large T Antigen Interferes with Adult Myosin Heavy Chain Expression, but Not with Differentiation of Human Satellite Cells

The growth of muscle fibers during late development as well as in regeneration following muscle injury is the result of the proliferation and differentiation of satellite cells. However, all human cells, including satellite cells, show a limit in their proliferation. In order to define a cellular system with enhanced proliferative capacity, human satellite cells were transfected with a construct containing large T antigen from SV40 under the control of the human vimentin promoter. Vimentin is normally expressed during proliferation, and its expression is down-regulated as differentiation proceeds. In transfected cells, the construct is regulated like the endogenous vimentin gene. The effect of exogenous T antigen expression on both the proliferation and differentiation of human satellite cells was investigated. T antigen expression reduced the doubling time of human satellite cells from 36 to 20 h and increased the final proliferative capacity from 46 to 69 mean population doublings. When differentiation was triggered, although T antigen did not prevent the formation of myotubes, fusion was delayed. A similar delay was observed in the appearance of myogenin protein, one of the HLH regulatory factors, but not in the corresponding mRNA. Finally, T antigen has an effect on adult myosin isoform expression, since both adult slow and fast isoforms were only detected in myotubes negative for T antigen. These results led us to propose a model of the possible interactions between T antigen and muscle-specific factors.

Relationships between intermediate filaments and cell-specific functions in renal cell lines derived from transgenic mice harboring the temperature-sensitive T antigen

Four renal cell lines were derived from glomeruli, proximal, distal, and cortical collecting tubules microdissected from the kidneys of transgenic mice carrying the temperature-sensitive mutant of the simian virus 40 large T antigen under the control of the vimentin promoter. All four cell lines contained large T antigen in their nuclei, grew rapidly, and contained vimentin filaments when grown in serum-enriched medium at the permissive temperature of 33 degrees C. The glomerular cell line formed multiple layers of cells and contained smooth muscle actin and desmin filaments, features of mesangial cells. The three tubule cell lines formed monolayers of polarized cuboid cells separated by tight junctions and having a patchy distribution of cytokeratins K8-K18. A shift from 33 degrees C to the restrictive temperature (39.5 degrees C) stopped cell growth in all cell lines and caused profound changes in the content of intermediate filaments. Vimentin was still present in mesangial-like cells, but the proximal, distal, and collecting tubule cells contained uniform networks of cytokeratins K8-K18 and desmoplakin I and II around the cell peripheries. Potassium transport, mediated by Na+-K+ ATPase pumps and specific cAMP hormonal sensitivities, significantly increased in proximal, distal, and collecting tubule cells when shifted from 33 degrees C to 39.5 degrees C. Thus, the temperature-dependent inactivation of large T antigen, responsible for the arrest of cell growth, did not affect the phenotype of mesangial-like glomerular cells but induced some changes in the expression of intermediate filaments and restored, at least partially, the main parental cell-specific functions in proximal, distal, and collecting tubule cultured cells.

Human multidrug resistance 3-P-glycoprotein expression in transgenic mice induces lens membrane alterations leading to cataract.

We have generated mice transgenic for a human multidrug resistance (MDR)3 mini-gene driven by a hamster vimentin promoter. The MDR3 gene encodes a P-Glycoprotein that resembles the mouse multidrug resistance 2 P-Glycoprotein shown to be involved in the translocation of the phospholipid phosphatidylcholine through the hepatocyte canalicular membrane (Smit et al., 1993. Cell. 75:451-462). The vimentin promoter drives expression of the MDR3 transgene in mesenchymal tissues and in the eye lens. We show here that the presence of human multidrug resistance 3 P-Glycoprotein in the lens results in a severe lenticular pathology. Lens structural abnormalities initiate at a late embryonic stage and increase during postnatal lens development. Differentiation of the primary fibers is affected, and the terminal differentiation of the lens epithelium into secondary fibers is also perturbed. The ultrastructural alterations, particularly of the lens plasma membranes, resemble those identified in congenital mouse osmotic cataract.

Induction of Blood-Brain Barrier Differentiation in a Rat Brain-Derived Endothelial Cell Line

An immortalized brain capillary endothelial cell line displaying blood-brain barrier characteristics may represent a useful tool for studying blood-brain barrier endothelial cell differentiation and for the in vitro prediction of drug brain penetration. In the present study, we have established a rat cerebral capillary endothelial cell line (CR3) by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter. The CR3 cell line displayed endothelial morphological and biochemical characteristics for up to 30 passages. However, the CR3 cell line did not spontaneously express the specific blood-brain barrier markers γ-glutamyl transpeptidase and mdr P-glycoprotein. However, when the cells were treated with the cell differentiating agent all-trans-retinoic acid, the blood-brain barrier markers were induced. Retinoic acid-treated CR3 cells may thus represent a useful tool for biological and pharmacological research related to the blood-brain barrier.

Negative regulation of a special, double AP-1 consensus element in the vimentin promoter: interference by the retinoic acid receptor.

The growth-regulated vimentin gene contains a functional double AP-1 binding site formed by two nearly perfect inverted repeats. We present evidence for down-regulation of vimentin expression by the retinoic acid receptor (RAR) in two mesodermally derived cell types. By mutation analysis we prove that the double consensus element is responsible for this negative regulation. From in vitro protein-DNA interaction studies we conclude that AP-1 binding is inhibited at RAR amounts required for occupation of the cognate RAR binding site in nuclear extracts from 3T3 cells and differentiated embryonal carcinoma cells. Furthermore, we show that, unlike in other cases, trans-activation of the vimentin AP-1 enhancer element can occur in undifferentiated embryonal carcinoma cells, despite the low amount of Jun and Fos proteins present in these cells. Here, however, down-regulation by retinoic acid cannot be detected.

The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice

The mouse mdr2 P-glycoprotein (P-gp) and its human MDR3 homologue are present in high concentrations in the canalicular membrane of hepatocytes. Mice lacking this protein are unable to secrete phosphatidylcholine (PC) into bile, suggesting that this P-gp is a PC translocator. We have tested this in fibroblasts from transgenic mice expressing the MDR3 gene under a vimentin promoter. Transgenic and control fibroblasts were incubated with [ 14C]choline to label PC. When the labeled cells were incubated with a PC transfer protein and acceptor liposomes, transfer of radioactive PC was enhanced in transgenic cells relative to the wild type controls. We conclude that the MDR3 P-glycoprotein is able to promote the transfer of PC from the inner to the outer leaflet of the plasma membrane, supporting the idea that this protein functions as a PC flippase.

Immortalization of Multiple Cell Types from Transgenic Mice Using a Transgene Containing the Vimentin Promoter and a Conditional Oncogene

Differentiated clonal cell lines were obtained from transgenic mice carrying a recombinant gene composed of DNA coding for a temperature-sensitive mutant of the simian virus large T antigen under the control of regulatory elements of the human vimentin gene. In response to mitogenic factors the vimentin promoter is activated in the presence of serum in almost all cultured cells independently of their origin. The expression of the T antigen could be controlled both at the level of transcription since the vimentin promoter is growth-regulated and at the level of the protein structure through the temperature stability of the T antigen. Indeed, the switch-off of the oncogene protein is obtained by serum deprivation of the culture and achieved with enhancement of the growth temperature. From transgenic mice several types of clonal differentiated cell lines were established and characterized including melanocytes, macrophages, mesangial, muscle, and endothelial cells. Melanocytes displayed melanin while endothelial cells from brain and heart expressed the related factor VIII and low density lipoprotein absorption capacities. Mesangial cells from kidney exhibited numerous desmosomes. Typical markers of macrophages from bone marrow were observed while skeletal muscle cells fused and contracted.

Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control.

Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1 beta (IL-1 beta) at 10 U.ml-1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 microM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

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)

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)

Activation of the human vimentin gene by the Tax human T-cell leukemia virus. I. Mechanisms of regulation by the NF-kappa B transcription factor.

The molecular basis for transactivation of the human vimentin gene by the Tax protein from the human T-cell leukemia virus (HTLV-1) is analyzed in this report. We first demonstrate that the factor NF-kappa B binds to the vimentin promoter. Using gel retardation assays, we found the putative NF-kappa B protein. Specific antibodies and competition experiments between the NF-kappa B-binding site in the interleukin-2R alpha and HIV-1 promoters and the vimentin promoter show that the three sites have identical affinity for the factor. We further show that the mechanisms of activation of NF-kappa B by the Tax protein involve a cellular inducer. Nuclear extract from lymphoid cells expressing Tax can induce in vitro a NF-kappa B binding activity in nonlymphoid cytosolic extract. This inducer, if preexisting in an inactivate state in T-cells which are not expressing Tax, cannot be switched to an active state by addition of partially purified Tax protein. While found in the nucleus of Tax-expressing cells in our experiments, this inducer might be cytoplasmic as well. In a first attempt to identify and characterize the inducer, we present the results of fractionation assays of nuclear extract.

Regulation of vimentin expression in cultured epithelial cells.

Most cell types start expressing vimentin when brought into tissue culture. Using both vimentin-expressing (HeLa) and vimentin-negative (MCF-7) epithelial cell lines, we have identified the cis-regulatory DNA elements involved in this process. Sequences located 1.1-0.6 kb upstream of the vimentin transcription-initiation site strongly enhance expression in HeLa cells, but are silenced in MCF-7 cells. Other regulatory elements in the vimentin promoter (an enhancer 3.2-2.6 kb upstream and a minimal promoter region including the CAAT-box) are potentially active in both cell types, but are silenced by the 0.5-kb fragment in MCF-7 cells. Deletion of this fragment restores transcriptional activity of a transfected vimentin promoter. Our data indicate that a double AP 1/jun-binding site present in the 0.5-kb fragment mediates the induction of vimentin expression in cultured epithelial cells, while silencing sequences located within the same fragment are responsible for the absence of vimentin expression in MCF-7 cells. In contrast to MCF-7 cells, a transfected vimentin promoter and gene are transcriptionally active in the vimentin-negative epithelial cell line T24. Transfection studies show that type-III-intermediate-filament expression is not impaired at any level in these cells. Upon transfection and expression of a desmin construct in T24 cells not only desmin, but also vimentin was detected. Both proteins assembled into intermediate filaments. This induction of vimentin expression appeared to be regulated at the post-transcriptional level.

Identification of two silencers flanking an AP-1 enhancer in the vimentin promoter

We have studied the 5′ upstream sequences required for the transcriptional regulation of the hamster gene encoding the intermediate filament protein, vimentin. Although vimentin is regarded as the intermediate filament protein of mesothelial tissue, it is also produced in most cultured cells. The human mammary carcinoma cell line, MCF-7, belongs to the exceptions. It contains no vimentin, and the complete upstream promoter region is inactive in this particular cell line. By using transient transfection of chimeric constructs into MCF-7 and HeLa cells, and subsequent chloramphenicol acetyltransferase assays, we were able to show the presence of two negative control regions flanking a double AP1 enhancer element. Our data indicate that these elements exert their effect irrespective of orientation and position, suggesting that they are silencers. In vitro footprinting assays, gel mobility assays and Southwestern (protein-DNA) blotting revealed the presence of trans-acting factors interacting with both silencer elements. The silencing effect was particularly pronounced in MCF-7 cells, although DNA-binding proteins are present in HeLa cells as well.