Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Summary Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.     

Microinjection of monoclonal antibodies to vimentin, desmin, and GFA in cells which contain more than one IF type

Microinjection of antibodies to vimentin into fibroblast cell lines causes intermediate filaments (IFs) to build perinuclear caps. We have extended these findings by microinjection of monoclonal antibodies specific for different IF types to non-epithelial cell lines of human origin, which co-express two different IF proteins. Thus GFA and vimentin IgGs have been microinjected in separate experiments into a glioma cell line, desmin and vimentin IgGs into RD cells, and vimentin IgGs into a cell line which co-expresses neurofilaments and vimentin. In all instances, microinjection of a single antibody causes the formation of perinuclear caps in which the two different IF proteins co-localize, suggesting that vimentin and the second IF type present in each cell line localize to the same 10-nm filaments. Immunoelectron microscopy using desmin and vimentin antibodies made in different species and appropriate second antibodies labelled with 5 and 20 nm gold particles confirm this result for RD cells. When Fab' fragments of the vimentin IgGs are microinjected into different cell types, formation of perinuclear caps is observed in immunofluorescence microscopy. In RD cells immunoelectron microscopy shows that the Fab' fragments induce caps which appear less dense than the caps seen after microinjection of IgGs.     

Occurrence of two different intermediate filament proteins in the same filament in situ within a human glioma cell line : An immunoelectron microscopical study

Intermediate filament systems of an established glioma cell line have been characterized by double immunofluorescence microscopy and by immunoelectron microscopy using two antibodies, one of which recognizes glial fibrillary acid protein (GFA) but not vimentin, and the second which recognizes vimentin but not GFA. The results show that glioma cells express two immunologically distinct IF polypeptides which are found in the same 10-nm filaments. Juxtanuclear caps formed after exposure of the cells to colcemid consisted of intermediate filaments composed of both GFA and vimentin. In immunoelectron microscopy both untreated cells and cells treated with colcemid show discontinuous labelling when only a single antibody is used, but continuous labelling when both antibodies are used simultaneously.     

Nordy, a synthetic lipoxygenase inhibitor, inhibits the expression of formylpeptide receptor and induces differentiation of malignant glioma cells

We recently found that formylpeptide receptor (FPR), a G-protein-coupled receptor that mediates chemotaxis of phagocytic leukocytes induced by bacterial peptide N-formyl-methionyl-leucyl-phenylalanine, is expressed by malignant human glioma cells and promotes tumor growth and angiogenesis. In this study, we examined the effect of Nordy, a novel chiral lipoxygenase inhibitor which was synthesized based on the structure of a natural nordihydroguaiaretic acid, on the expression of FPR by human glioblastoma cells. We found that FPR was expressed at the protein level by highly malignant human glioma cell lines U87 and BT325, and a rat glioma cell line C6. The expression level of FPR was correlated with the degree of the malignancy of tumor cells. The poorly differentiated glioma cell line U87 expressed the highest level of FPR. In U87 glioma cells, the expression of FPR was attenuated at the protein level by Nordy treatment for 48 (P<0.05). Nordy did not affect FPR mRNA expression in U87 cells. In addition, Nordy treatment seemed to promote glioma cell differentiation, as evidenced by their reduced expression of vimentin and increased expression of GFAP. Our results suggest that Nordy was capable of reducing the level of malignancy of glioma cells.     

Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co- expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.     

Intermediate filament heterogeneity in normal and hypercholesterolemic rabbit vascular smooth muscle cells

We have examined the intermediate filament (IF) protein content of vascular smooth muscle (SM) cells from several arteries and veins in rabbits and quantitated the changes which occur in SM cell expression of these proteins in response to cholesterol feeding. Cells from control rabbit arteries expressed 30% of their IF protein as desmin, while veins expressed 50% as desmin. During development of diet-induced atherosclerosis, morphological changes in arterial SM cells in the intima correlate with changes in IF expression. There is a significant increase in total IF protein content, vimentin increased differentially in thoracic aorta and desmin in pulmonary artery. In abdominal aorta both increase equally. Cholesterol feeding also resulted in changes in the expression of subspecies of desmin, vimentin, and actin in the thoracic arch. Although cholesterol feeding did not produce obvious morphological changes in the veins examined, venous SM IF protein expression was also altered. In the vena cava of cholesterol-fed rabbits there was an increase in vimentin expression without the parallel increase in desmin that occurred in the arterial system. These studies show that cholesterol feeding of rabbits induces measurable changes in the amounts of IF proteins in both arterial atherosclerotic lesions and venous SM cells.     

Vimentin intermediate filament and plectin provide a scaffold for invadopodia,facilitating cancer cell invasion and extravasation for metastasis

To investigate the molecular mechanisms of cancer metastasis, we have isolated a high-metastatic bladder cancer cell subpopulation from a low-metastatic cell line by using an in vivo selection system. Cells in the subpopulation showed a high ability to form invadopodia, the filamentous actin (F-actin)-based membrane protrusions that play an essential role in cancer cell invasion. Analysis of the gene expression profile revealed that the expression of an intermediate filament (IF) protein, vimentin and a cytoskeletal linker protein, plectin was up-regulated in the high-metastatic subpopulation compared with the low metastatic cell line. Here we report a novel role of vimentin IF and plectin in metastasis. In invasive bladder cancer cells, the vimentin IF-plectin-invadopodia F-actin link was formed. Disruption of this link severely impaired invadopodia formation, reducing the capacities of extracellular matrix degradation, transendothelial migration and metastasis. In addition, the vimentin assembly into the filaments was required for invadopodia formation. Our results suggest that plectin anchoring invadopodia to vimentin IF scaffolds and stabilizes invadopodia, which is a critical molecular process for cancer cell invasion and extravasation for metastasis.     

Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation

We recently demonstrated that inherited disease-causing mutations clustered in the alpha-helical coiled-coil "rod" domain of the muscle-specific intermediate filament (IF) protein desmin display a wide range of inhibitory effects on regular in vitro assembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area.     

Review on intermediate filaments of the nervous system and their pathological alterations

Intermediate filaments (IFs) of the nervous system, including neurofilaments, α-internexin, glial fibrillary acidic protein, synemin, nestin, peripherin and vimentin, are finely expressed following elaborated cell, tissue and developmental specific patterns. A common characteristic of several neurodegenerative diseases is the abnormal accumulation of neuronal IFs in cell bodies or along the axon, often associated with impairment of the axonal transport and degeneration of neurons. In this review, we also present several perturbations of IF metabolism and organization associated with neurodegenerative disorders. Such modifications could represent strong markers of neuronal damages. Moreover, recent data suggest that IFs represent potential biomarkers to determine the disease progression or the differential stages of a neuronal disorder. Finally, recent investigations on IF expression and function in cancer provide evidence that they may be useful as markers, or targets of brain tumours, especially high-grade glioma. A better knowledge of the molecular mechanisms of IF alterations, combined to neuroimaging, is essential to improve diagnosis and therapeutic strategies of such neurodegenerative diseases and glioma.     

Properties of the nonhelical end domains of vimentin suggest a role in maintaining intermediate filament network structure

To investigate the functional role of the nonhelical domains of the intermediate filament (IF) protein vimentin, we carried out transient transfection of constructs encoding fusion proteins of these domains with enhanced green fluorescent protein (EGFP). Expression of these fusion proteins did not have any effect on the endogenous IF networks of transfected cells. However, the head domain-EGFP fusion protein localized almost exclusively to the nucleus. This localization could be disrupted in a reversible fashion by chilling cells. Furthermore, the head domain was capable of targeting to the nucleus a strictly cytoplasmic protein, pyruvate kinase. Thus, the vimentin head domain contains information that specifically directs proteins into the nucleus. In contrast, the nonhelical tail domain of vimentin, when expressed as a fusion protein with EGFP, was retained in the cytoplasm. Cytoplasmic retention of tail domain-containing fusion proteins appeared to be dependent on the integrity of the microtubule network. Our results are consistent with a proposal that the nonhelical end domains of vimentin are involved in maintaining an extended IF network by exerting oppositely directed forces along the filaments. The head domains exert a nuclear-directed force while the tail domains extend the IF network toward the cell periphery via a microtubule-dependent mechanism.     

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.     

Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells

Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation- and tissue-specific, and is maintained during tumorigenesis. Vimentin IF is often co-expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over-expression of Akt1 increases K8/18 proteins. Akt2 up-regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.     

Interaction of surfactant protein A with the intermediate filaments desmin and vimentin

Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.     

Intermediate filament proteins in the developing chick spinal cord

The distribution of different intermediate filament (IF) proteins in the embryonic chick spinal cord was examined at several stages of development using immunohistochemical techniques, analytic gel electrophoresis, and electron microscopy. We have found that: (1) the fibroblast-type IF protein (vimentin) is present in virtually all of the replicating neuroepithelial cells of the early neural tube, as well as in radial glia, astrocytes, and Schwann cells in later stages of development; (2) the fibroblast-type IF protein is not detectable in definitive neurons; (3) the neurofilament proteins are first detectable in postmitotic neuroblasts at about the time of initial axon formation and they are restricted to neurons; (4) the astrocyte-type IF protein (glial fibrillary acidic protein) is in definitive astrocytes, but not in radial glia; (5) the prekeratin proteins are restricted to cells of the leptomeninges; and (6) the muscle-type IF protein (desmin) is restricted to vascular tissue in and around the developing spinal cord. These findings suggest that the fibroblast-type IF protein is the only IF protein in the early neuroepithelial cells and that the progeny of these cells will follow one of three different patterns of IF protein expression: (1) continued expression of only the fibroblast-type IF protein (radial glia); (2) expression of both the fibroblast-type IF protein and the astrocyte-type IF protein (astrocytes); or (3) expression of only the neurofilament proteins (neurons).     

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.     

Effect of sodium butyrate on S-100 protein levels and the cAMP response

Sodium butyrate (NaB), when added to cell cultures, produces a variety of morphological and biochemical changes. We examined its effects, in nM concentrations, on the expression of two glioma cell-associated proteins, glial fibrillary acidic protein (GFAP) and S-100 protein in human glioma-derived cell line (RF), and of S-100 protein in the C6 rat glioma cell line. GFAP levels decreased by about 50% in the RF cell line, and S-100 protein levels decreased protein levels decreased by about 40% after treatment with 1 mM NaB for 48 h. In the C6 rat glioma cell line, isoproterenol with theophylline was found to increase S-100 levels by two-fold over basal levels. NaB was found to inhibit the induction of S-100 protein but exhibited no effect on the basal levels of the protein. Other short chain fatty acids, including sodium propionate and sodium isobutyrate, exhibited partial inhibitory activity. NaB, at an EC50 of 1 mM, was also found to inhibit both the beta-adrenergic and the forskolin-mediated increase in cAMP levels in these cells. This suggests that NaB may inhibit cells from expressing S-100 protein by attenuating cAMP levels.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Vimentin and glial fibrillary acidic protein are codistributed in the same intermediate filament system of malignant glioma cells in vivo

Normal, reactive, and neoplastic astrocytes express two types of intermediate filament (IF) proteins, namely glial fibrillary acidic protein (GFAP) and vimentin. Their submicroscopical distribution in vivo is so far unknown. We therefore investigated four malignant gliomas by electron microscopy, applying postembedding double immunogold labeling. The IF proteins were randomly scattered over the same filament bundles, as in previous experiments on glioma cultures. No clustering or preferential intracytoplasmic location of either IF protein was visible. The demonstration of IF proteins within nuclei gives some support to the suggested intranuclear functions of IF proteins.