Vimentin Gene Expression Research Articles

A GC-box is required for expression 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 (-1416 to +73) was shown to contain two positive-acting regions, separated by a negative region, and at least eight GC-boxes as determined by sequence homology (Rittling, S.R., Baserga, R., 1987. Mol. Cell. Biol. 7, 3908-3915). We have analyzed the region -900 to +41 for protein binding by in vivo footprinting experiments using ligation-mediated PCR. For the various GC-boxes, we detect protein binding only to that GC-box (at position -64 and -55) closest to the transcriptional start site. Transient transfection assays of various vimentin 5'-end fragments and mutations thereof fused to the reporter gene cat indicate that this sequence is indispensable for promoter function regardless of the inclusion of upstream DNA sequences. In vitro binding studies confirm that this region binds protein specifically. We suggest that this GC-box and its binding factor are required for regulated expression of the human vimentin gene.

Differential Display of Reticulocalbin in the Highly Invasive Cell Line, MDA-MB-435, versus the Poorly Invasive Cell Line, MCF-7

Matrigel invasion assays were used to characterize the invasive abilities of five breast cancer cell lines. Reverse Transcription Polymerase Chain Reaction (RT-PCR) was used to detect the differential gene expression of estrogen receptor (ER), E-cadherin, vimentin and cathepsin D in these cell lines. Using mRNA differential display, we identified novel cDNA clones representing the partial sequences of genes overexpressed in the invasive MDA-MB-435 cells as compared to that of the less invasive MCF-7 cells. One of the cDNAs was homologous to reticulocalbin. The studies were repeated in all of the cell lines and the overexpression of this cDNA was confirmed by RT-PCR and Northern hybridization analysis. Reticulocalbin was expressed in the highly invasive breast cancer cell lines but was not expressed in poorly invasive ones. Although its function is still unknown, reticulocalbin is implicated in tumor cell invasiveness because of its differential expression in breast tumor cell lines.

Developmental pattern of GFAP and vimentin gene expression in rat brain and in radial glial cultures

In the present study we analyze the events which occur during the early stages of astrogliogenesis by examining the pattern of both GFAP and vimentin gene expression and their corresponding immunoreactive proteins during rat brain development. This study was carried out "in vivo" (whole brain) and "in vitro" (primary culture of radial glia) using immunofluorescence, immunoblotting, and Northern blot analysis. Our results demonstrate that although GFAP immunostaining appeared late in gestation and at day 5 in radial glia cultures, GFAP mRNA expression was first detected, at very low levels, on fetal (F) day 15 and increased to F21. During postnatal development a striking increase in GFAP and its encoding messenger occurs. In contrast, the levels of vimentin and its mRNA expression were very high during the fetal stage (F15 to F21). Thereafter vimentin expression declined during postnatal (P) development until P21 and then remained constant at adult levels. In contrast, an increase in vimentin expression was observed in glial cells throughout the entire culture period. The biological significance of the developmental patterns of GFAP and vimentin expression in astroglial cells. during brain development is discussed.

Multiple silencer elements are involved in regulating the chicken vimentin gene.

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

Developmental patterns of intermediate filament gene expression in the normal hamster brain

We have examined the patterns of expression of the major intermediate filament (IF) protein mRNAs during development of the hamster brain. Quantitative northern blotting was used to examine changes in the levels of mRNAs for the low, middle and high molecular weight neurofilament proteins (NF-L, NF-M, NF-H) as well as peripherin, vimentin and glial fibrillary acidic protein (GFAP). Total RNA was isolated from hamster brains at embryonic (E) days 12 and 14 and postnatal (P) days 1, 3, 5, 7, 9, 11, 13, 15, 20, 28 and 60–90 (adult), and probed with specific IF cDNAs. Northern blotting revealed that NF-L and NF-M mRNAs were present at very low levels in embryonic brain and that significant expression of these genes only occurred postnatally when the levels increased dramatically until P28 and then declined again in the adult. Increases in NF-H mRNA levels were somewhat delayed relative to those of NF-L and NF-M. NF-H mRNA was not seen at embryonic stages and was expressed at very low levels prior to P9; after that time the levels increased rapidly until P28 and then declined in the adult. Two of the type III IF genes, peripherin and vimentin, followed a pattern of expression opposite that of the NF genes. Both peripherin and vimentin mRNAs were present in embryonic brain and were expressed at higher levels during early postnatal stages than at later times. The magnitude and rate of reduction in vimentin gene expression in the postnatal interval was much greater than that of peripherin. GFAP mRNA levels were extremely low prior to P9 alter which a robust increase occurred, followed by a decline in the adult. We discuss the implication of the dramatic changes in IF isotype expression in brain to the pathways of both neuronal and glial development in vivo.

Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression.

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression.

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

Expression of histone H3 cell cycle-related gene, Vimentin and MYC genes in pediatric brain tumors. A preliminary analysis showing the different malignant cell growth potential

Eleven pediatric brain tumors were studied for the histone H3, Vimentin and MYC gene expression. H3, an S phase cell cycle-related gene (ccr), was found prevalently expressed in tumors with a high mitotic index (MI). Vimentin gene, which contributes to maintaining the cell structure but is also demonstrated to be an early responder gene to growth stimulation was found variously expressed. The different expression of Vimentin gene in the examined samples suggests the active proliferation of the tumor cells. Analysis of MYC gene expression was found increased only in a mesenchymal chondrosarcoma while in other samples MYC mRNA was undetectable. Medulloblastoma, chondrosarcoma, and choroid plexus carcinoma have high S phase H3 gene expression associated with a high MI. Differently an astrocytoma shows a low MI associated with high H3 gene expression. This first preliminary report of H3, Vimentin and MYC gene expression in brain tumors demonstrates that malignant cells are characterized by a different gene expression and different growth potentials.

Detection of translocations of 10p by non-radioactive in situ hybridization of VIM gene in SV40-transformed human cell lines

SV40-transformed human fibroblasts exhibit characteristic chromosome imbalances, fairly well correlated with the activity of enzymes encoded by genes located on chromosome segments either in deficiency or in excess. However, a major discrepancy existed for the expression of vimentin gene (VIM), which was high, even though the map location of the gene (10p) was missing in many cell lines. An in situ hybridization technique using a biotinylated probe for the human VIM was applied to detect eventual cryptic translocations, as chromosome 10p is difficult to identify. In two cell lines (WI 98 and HEL1 HBLT) in which a loss of copy number of 10p was assumed after karyotyping, a signal for VIM was detected in unidentified short arms of derivative chromosomes. This exemplifies that in situ hybridization is a powerful complement to classical cytogenetics to detect rearrangements in highly rearranged karyotypes from transformed or cancerous cells. These results also strengthen the interpretation of the correlation between karyotypic and metabolic imbalances in transformed cells.

Vimentin mRNA expression increases after corticospinal axotomy in the adult hamster

We examined changes in vimentin gene expression during Wallerian degeneration after corticospinal axotomy in the adult hamster. Vimentin, which is the product of a type III intermediate filament (IF) gene, is expressed in various cells of mesenchymal origin, including microvascular endothelial cells, microglia and developing astrocytes. While increases in vimentin protein have been observed after various types of central nervous system (CNS) injury, it is not known whether this increase is due to increased vimentin mRNA expression. There is also conflicting evidence as to which cells are expressing increased levels of vimentin. In the present study we used in situ hybridization and double-label immunofluorescence techniques to address these issues. A 35S-labeled vimentin cDNA probe was used for in situ hybridizations of brain stem sections obtained 2, 7 and 14 days after unilateral transection of the corticospinal tract in the caudal medulla of adult hamsters. Autoradiography showed that an increase in vimentin mRNA associated with the degenerating corticospinal tract occurred by 2 days after axotomy and that the levels remained elevated for at least 14 days. Immunoblotting and immunocytochemical studies indicated that vimentin protein levels were increased in the degenerating corticospinal tract. Double-label immunofluorescence revealed many vimentin-positive cells and processes that were also labeled with GFAP antibody. In addition, cells and processes that were vimentin-negative but GFAP-positive were also found in the degenerating tract. We suggest that the reactive cells which possessed both vimentin and GFAP were reactive astrocytes of astroblastic origin while those that expressed only GFAP were derived from mature astrocytes. Other vimentin-positive cells/processes did not label with anti-GFAP and thus were either microglial, endothelial or inflammatory cells. These results demonstrate that an increase in vimentin mRNA occurs during Wallerian degeneration after corticospinal axotomy and that this increase is likely to be due to contributions from more than one cell type.

Vimentin downregulation is an inherent feature of murine erythropoiesis and occurs independently of lineage

In mammalian erythropoiesis, the mature cells of the primitive lineage remain nucleated while those of the definitive lineage are anuclear. One of the molecular and structural changes that precedes enucleation in cells of the definitive lineage is the cessation in the expression of the gene for the intermediate filament (IF) protein vimentin and the removal of all vimentin filaments from the cytoplasm. We show here that in immature primitive cells vimentin is synthesized and forms a cytoplasmic network of IFs. As differentiation proceeds in vivo, vimentin gene expression is downregulated in these cells; this is accompanied by the loss of vimentin filaments from the cytoplasm. This loss temporally coincides with the nucleus becoming freely mobile within the cytoplasm, suggesting that, while IF removal is not directly linked to the physical process of enucleation, it may be a prerequisite for the initiation of nuclear mobility in both lineages. These changes are also observed in early primitive cells cultured in vitro, suggesting that they constitute an intrinsic part of the murine erythroid differentiation program independent of lineage and hematopoietic microenvironment.

Regulation of vimentin gene expression in the ocular lens

Vimentin expression in the lens is striking due to the reported mesenchymal preference of vimentin and the epithelial origin of the lens. The amount of chicken vimentin mRNA levels determined by Northern blot analysis increased 3-fold from 7 to 14 days of embryonic lens development and then decreased 10-fold at 16 days of development, suggesting that post-transcriptional processes may contribute to the level of cytoplasmic vimentin mRNA during lens development. To analyze the mechanisms governing vimentin gene expression in the lens at the level of transcription, a series of chicken vimentin 5′-flanking region deletions were fused to the bacterial CAT gene and transfected into fibroblasts and lens cultures derived from three species. The −160 to +1 sequence conferred equal promoter activity in cultured chicken lens epithelial cells and fibroblasts. The −321 to −160 sequences increased promoter activity in all cultures, but more strongly in fibroblasts than in lens cells. Sequence elements in the region −608 to −321 repressed promoter activity in lens cells and fibroblasts. Promoter activity was partially restored in fibroblasts but not in lens cells by −767 to −608 sequences. Vimentin gene expression in the lens thus appears to be controlled by multiple positive- and negative-acting elements in its 5′-flanking sequence.

Regulation of the expression of vimentin gene during the differentiation of mouse myeloid leukemia cells.

We have examined the expression of vimentin during the differentiation of mouse myeloid leukemia cells (M1), which were induced to differentiate into macrophages by exposure to conditioned medium (CM) obtained from rat embryo fibroblasts. The synthesis of vimentin, which was examined by two-dimensional gel electrophoresis, increased after 12-24 h of incubation of M1 cells in CM and the elevated level of synthesis continued up to 96 h. A macrophage cell line (Mm1) that was derived from spontaneously differentiated M1 cells constantly synthesized much higher levels of vimentin. The amount of vimentin, which was revealed by immunoblot analysis using an mAb against human vimentin, also increased after differentiation by a factor of 7 when compared on the basis of constant protein and by a factor of 17 on the basis of constant cell numbers. Mm1 cells contained greater than 12- and 45-fold more vimentin compared with undifferentiated M1 cells on the bases of constant protein and constant cell numbers, respectively. Northern blot analysis using vimentin cDNA as a probe revealed increases in vimentin mRNA in the differentiated M1 cells and Mm1 cells. Nuclear run-on assay showed that the expression of vimentin gene during the differentiation of M1 cells was transcriptionally regulated. Observations in indirect immunofluorescence microscopy and EM clearly showed that vimentin bundles were rarely observed in undifferentiated M1 cells, and increased amounts of and large-size vimentin bundles were easily observed in differentiated M1 and Mm1 cells. These results suggest the participation of increased amounts of vimentin filaments in the maldistribution of nuclei in M1 cells during differentiation.

A Negative Element Involved in Vimentin Gene Expression

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

A negative element involved in vimentin gene expression.

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

The induction of vimentin gene expression by sodium butyrate in human promonocytic leukemia U937 cells

The administration of 1 m M sodium butyrate induced the phenotypic differentiation of human promonocytic leukemia U937 cells, as judged by the expression of cD11b and cD11c antigens, two differentiation-specific surface markers. At the same time, butyrate greatly induced the expression at the mRNA level of the vimentin gene. The increase in the level of this RNA started at 6 h of treatment and reached the maximum at Hour 24. Such an increase was caused at least in part by a stimulation in the rate of gene transcription, as suggested by transcription assays in isolated nuclei. Experiments in the presence of cycloheximide suggested that vimentin induction is probably a direct response to the action of butyrate, not mediated by the prior induction of other gene products. Unlike the case of vimentin, the levels of other RNAs, namely β-actin, ornithine decarboxylase, and c-myc, were not enhanced, but they decreased at different times of treatment with butyrate. Finally, we observed that butyrate induced also the differentiation of HL60 cells, another human myeloid cell type. Nevertheless, the drug failed to stimulate the expression of vimentin in this cell line.

Expression of the chicken vimentin gene in transgenic mice: efficient assembly of the avian protein into the cytoskeleton.

To study expression and function of the vimentin gene, transgenic mice were generated by microinjecting the entire chicken gene plus 2.4 kilobases of 5' and 2.6 kilobases of 3' flanking sequences. All the transgenic mice obtained had incorporated multiple copies of the gene. RNA analyses demonstrated that the chicken vimentin gene was efficiently expressed in an appropriate tissue-specific pattern and that the transcripts were properly processed, as in chicken, giving rise to two RNAs. The vimentin transgene was predominantly expressed in lens at levels of up to 10-fold the endogenous level in every transgenic line studied. The chicken vimentin transcripts were efficiently translated into polypeptides that were modified posttranslationally and could assemble into the mouse cytoskeleton. Overexpression of the chicken vimentin gene did not obviously affect the expression of the endogenous gene at the RNA or the protein level. Immunofluorescence microscopy further demonstrated that the chicken protein was properly expressed spatially in lens. However, the levels were much higher in the transgenic animals.

Down-regulation of vimentin gene expression during myogenesis is controlled by a 5′-flanking sequence

During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes ( Vim) are expressed in fibroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5 ' -end Vim ::cat constructs demonstrate that this repression is controlled by a 5′-element (nt −321 to −160). This region is distinct from Vim promoter elements (nt −160 to + 71) which do not contribute to vimentin's down-regulation during myogenesis.

Multiple elements are required for expression of an intermediate filament gene.

The expression of vimentin is unique within the intermediate filament multigene family. It is the only member which deviates from its usual tissue-specific expression pattern and whose 5'-flanking region contains multiple GC boxes, the binding site for Sp1. The activity of vimentin 5'-end:CAT fusions has been compared in cells where vimentin is highly expressed (mouse L cells) or not expressed at all (MH1C1). In addition, CAT activity has been examined by microinjection into Xenopus oocytes. Both in vivo expression and in vitro binding studies implicate Sp1 as a general regulatory factor in vimentin gene expression. Increased expression of 5'-end:CAT fusions in mouse L cells suggests that a fibroblast-specific enhancer element resides in the region -321 to -160. Low transcriptional activity in MH1C1 cells may be due to either the lack of this positive transcription factor(s) or the presence of a repressor element. Here, we demonstrate that the unique and complex pattern of vimentin gene expression is controlled by multiple cis-acting elements.

Expression of transfected vimentin genes in differentiating murine erythroleukemia cells reveals divergent cis-acting regulation of avian and mammalian vimentin sequences.

We studied the expression of transfected chicken and hamster vimentin genes in murine erythroleukemia (MEL) cells. MEL cells normally repress the levels of endogenous mouse vimentin mRNA during inducermediated differentiation, resulting in a subsequent loss of vimentin filaments. Expression of vimentin in differentiating MEL cells reflects the disappearance of vimentin filaments during mammalian erythropoiesis in vivo. In contrast, chicken erythroid cells express high levels of vimentin mRNA and vimentin filaments during terminal differentiation. We demonstrate here that chicken vimentin mRNA levels increase significantly in differentiating transfected MEL cells, whereas similarly transfected hamster vimentin genes are negatively regulated. In conjunction with in vitro nuclear run-on transcription experiments, these results suggest that the difference in vimentin expression in avian and mammalian erythropoiesis is due to a divergence of cis-linked vimentin sequences that are responsible for transcriptional and posttranscriptional regulation of vimentin gene expression. Transfected chicken vimentin genes produce functional vimentin protein and stable vimentin filaments during MEL cell differentiation, further demonstrating that the accumulation of vimentin filaments is determined by the abundance of newly synthesized vimentin.