Intermediate Filament Protein Vimentin Research Articles

The Roles and Molecular Mechanisms of Nestin Expression in Cancer with a Focus on Pancreatic Cancer

Nestin is a class VI intermediate filament protein that is expressed in a variety of stem and progenitor cells, including primitive neuroepithelial cells and pancreatic exocrine progenitor cells. Nestin expression is closely associated with rapidly proliferating progenitor cells during development and repair processes, and higher nestin expression levels correlate with poor prognosis in some cancers. Nestin regulates many molecules during cell processes in both normal and neoplastic tissues, and its transcription is regulated by enhancer regions in the second intron of the nestin gene that bind to SOX, Class III POU, and N-Myc. Nestin forms heterodimers with other intermediate filaments, with the class III intermediate filament protein vimentin being its main partner. Nestin/ vimentin copolymers provide an anchor for glucocorticoid receptors, regulate insulin-degrading enzyme, and promote the phosphorylation-dependent disassembly of vimentin during mitosis. Nestin regulates cell proliferation, the cell cycle, cell survival, and apoptosis by regulating cyclin-dependent kinase-5 (Cdk5), phosphoinositide 3-kinase, and AKT. We reported previously that nestin regulates cell motility, invasiveness, and cell morphology in a pancreatic cancer cell line via regulation of F-actin and E-cadherin. This review focuses on the roles of nestin in cancer stem cells and in tumor-initiating cells in the pancreas. In the pancreas, nestin plays important roles in the proliferation, differentiation, and survival of both progenitor cells and cancer cells.

Astrocytes Negatively Regulate Neurogenesis Through the Jagged1‐Mediated Notch Pathway

Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin.

The Structure of Vimentin Linker 1 and Rod 1B Domains Characterized by Site-directed Spin-labeling Electron Paramagnetic Resonance (SDSL-EPR) and X-ray Crystallography

Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.

Molecular and Biological Characterization of Streptococcal SpyA-mediated ADP-ribosylation of Intermediate Filament Protein Vimentin

The Gram-positive bacterial pathogen Streptococcus pyogenes produces a C3 family ADP-ribosyltransferase designated SpyA (S. pyogenes ADP-ribosyltransferase). Our laboratory has identified a number of eukaryotic protein targets for SpyA, prominent among which are the cytoskeletal proteins actin and vimentin. Because vimentin is an unusual target for modification by bacterial ADP-ribosyltransferases, we quantitatively compared the activity of SpyA on vimentin and actin. Vimentin was the preferred substrate for SpyA (k(cat), 58.5 ± 3.4 min(-1)) relative to actin (k(cat), 10.1 ± 0.6 min(-1)), and vimentin was modified at a rate 9.48 ± 1.95-fold greater than actin. We employed tandem mass spectrometry analysis to identify sites of ADP-ribosylation on vimentin. The primary sites of modification were Arg-44 and -49 in the head domain, with several additional secondary sites identified. Because the primary sites are located in a domain of vimentin known to be important for the regulation of polymerization by phosphorylation, we investigated the effects of SpyA activity on vimentin polymerization, utilizing an in vitro NaCl-induced filamentation assay. SpyA inhibited vimentin filamentation, whereas a catalytic site mutant of SpyA had no effect. Additionally, we demonstrated that expression of SpyA in HeLa cells resulted in collapse of the vimentin cytoskeleton, whereas expression in RAW 264.7 cells impeded vimentin reorganization upon stimulation of this macrophage-like cell line with LPS. We conclude that SpyA modification of vimentin occurs in an important regulatory region of the head domain and has significant functional effects on vimentin assembly.

Proteomics reveals drastic increase of extracellular matrix proteins collagen and dermatopontin in the aged mdx diaphragm model of Duchenne muscular dystrophy

Duchenne muscular dystrophy is a lethal genetic disease of childhood caused by primary abnormalities in the gene coding for the membrane cytoskeletal protein dystrophin. The mdx mouse is an established animal model of various aspects of X-linked muscular dystrophy and is widely used for studying fundamental mechanisms of dystrophinopathy and testing novel therapeutic approaches to treat one of the most frequent gender-specific diseases in humans. In order to determine global changes in the muscle proteome with the progressive deterioration of mdx tissue with age, we have characterized diaphragm muscle from mdx mice at three ages (8-weeks, 12-months and 22-months) using mass spectrometry-based proteomics. Altered expression levels in diaphragm of 8-week vs. 22-month mice were shown to occur in 11 muscle-associated proteins. Aging in the mdx diaphragm seems to be associated with a drastic increase in the extracellular matrix proteins, collagen and dermatopontin, the molecular chaperone αB-crystallin, and the intermediate filament protein vimentin, suggesting increased accumulation of connective tissue, an enhanced cellular stress response and compensatory stabilization of the weakened membrane cytoskeleton. These proteomic findings establish the aged mdx diaphragm as an excellent model system for studying secondary effects of dystrophin deficiency in skeletal muscle tissue.

Abstract 344: Mifepristone alters morphology, cytoskeletal architecture, and epithelial-mesenchymal transition-related proteins in human metastatic cancer cells

Abstract The burden of secondary metastatic spread accounts for nearly 90% of cancer-related deaths. The ability of cancer cells to metastasize is often accompanied by biochemical changes leading to transition from an epithelial to a mesenchymal phenotype (EMT), changes in cell morphology, loss of tight junctions, remodeling of the cytoskeleton, and acquisition of migratory and invasive capacities. Previous work in our laboratory demonstrated that the antiprogestin mifepristone (MF) inhibits growth of known metastatic ovarian, prostate, breast, and brain cancer cells. In response to MF the cells displayed changes in morphology, and had reduced adhesive, migratory, and invasive capabilities. In this study, we assessed if such anti-metastatic phenotype induced by MF is associated with changes in the expression of EMT biomarkers (vimentin, slug, E-cadherin, and high and low molecular weight keratins), abundance of actin and tubulin, and/or modifications in the composition or intracellular localization of focal adhesion proteins [vinculin, phospho-focal adhesion kinase (p-FAK), and total FAK]. We studied all these parameters in cancer cells of the ovary (SK-OV-3), breast (MDA-MB-231), prostate (LNCaP), and brain (U87MG) upon exposure to concentrations of MF that block growth, adhesion, migration and invasion. MF induced a striking change in cellular morphology that was similar in all cell lines, and that included shrinkage of the cell body, long, thin cellular extensions, and a loss of cell-to-cell connections. Morphology changes were dependent on time of drug exposure, not influenced by cell density, and not associated with changes in the abundance of actin and tubulin, but instead linked to a remarkable cytoplasmic redistribution of actin and tubulin filaments. Upon removal of MF, cell morphology and proliferation returned to normal in all cell lines expect for LNCaP in which MF appears to induce irreversibly quiescence. The epithelial marker E-cadherin was found expressed only in LNCaP cells, and was not modified by MF. The mesenchymal intermediate filament protein vimentin was found in SK-OV-3, MDA-MB-231, and U87MG cells, but not in LNCaP cells. MF had a slight tendency to increase vimentin expression in SK-OV-3 and U87MG cells, and altered the expression of cytokeratins in LNCaP, MDA-MB-231 and U87MG, but not in SK-OV-3 cells. The focal adhesion marker, vinculin, was highly expressed in all cell lines and was not modified by MF, whereas FAK and p-FAK were reduced by MF in LNCaP, MDA-MB-231, and U87MG cells, but not in SK-OV-3 cells. In summary the anti-cancer effect of MF manifested by inhibition of cell growth, adhesion, migration, and invasion, is also associated with profound changes in cell morphology, actin and tubulin distribution, and, to a lesser extent, dysregulation of FAK, p-FAK and EMT-related proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 344. doi:1538-7445.AM2012-344

Umbilical Cord Stromal Cell Differentiation: little correlation with known markers of the chondrocyte phenotype or cartilage extracellular matrix

The increasing prevalence of degenerative joint diseases, in particular osteoarthritis, has driven the quest for chondrocyte precursor cells and biomaterials suitable for autologous cartilage repair (ACR). As an alternative to mesenchymal stem cells (MSCs) generated in situ from the subchondral bone, the differentiation and expansion of autologous MSCs in the presence of pro-chondrogenic factors such as BMP-2 and/or TGF beta holds great promise as a therapeutic approach. However, a typical feature of these chondrogenesis systems, including MSCs derived from umbilical cord blood (1) and the stromal model used in the current report (2) is differentiation towards hypertrophy marked by the production of collagen X. To quote Hardingham et al., (3) “chondrocyte hypertrophy and ECM calcification would be an undesirable characteristic of any cartilage repair tissue.” Central to the study by De la Fuente et al., (4) is the claim that umbilical cord MSCs are “similar to chondrocytes and expressed proteins characteristic of the cartilage extracellular matrix”. Apart from the immunohistochemistry for collagen II published previously (2), there is scant evidence to support this claim, as none of the proteomic data provides specific evidence for biosynthesis of a cartilaginous matrix. The cartilage ECM contains a plethora of well-known proteoglycans, non- collagenous proteins and glycoproteins. None of the typical cartilage ECM components, including aggrecan, link protein and the matrilins are found in this study and neither are the sensitive markers for the chondrocyte differentiation status such as cartilage oligomeric matrix protein and collagen IX (5). The authors instead report a collection of proteins (PLOD2, PDI, GRP78, PPIA and HSP90) that are involved in protein biosynthesis and post- translational modification and are found in a diverse range of tissues, if not ubiquitous. The claim that these proteins are involved in “cartilage extracellular matrix metabolism” is misleading, as none of these proteins are cartilage-specific. A second set of differentially- abundant proteins (CALU, CALR3, VIM, PDIA3, ZYX and ANXA5 in Table III), was provided as evidence of chondrogenic differentiation: “Results demonstrate that MSCs cultured in chondrogenic media become chondrocyte- like, as the cells express proteins observed in differentiated chondrocytes or in cartilage”. Again, these proteins are expressed in bone cells (eg osteoblasts), fibroblasts etc, and so their altered expression reveals nothing about the differentiation of the MSCs into “chondrocyte-like” cells. None are chondrocyte-specific. Finally, these results are drawn together in a protein network diagram summarizing the relationships between the proteins based on “linked bibliographic information”. It is difficult to extract any biological significance from this figure without all the citations on which these connections are based, but many of the connections are tenuous and some are contradictory. For example, the intermediate filament protein vimentin, linked to “extracellular matrix proteins” and “cartilage”, is down regulated in the current report. However this contradicts the findings of Bobick et al., (6) showing a positive role for vimentin in chondrogenesis. The field of autogolous cartilage repair has been plagued by the well documented phenotypic instability of chondrocytes: de-differentiation towards fibroblastic cells and/or the uncontrolled hypertrophy and matrix calcification analogous to differentiation of growth plate chondrocytes. The holy grail of ACR is the suppression these processes (marked by expression of collagen I and collagen X, respectively) during the expansion of phenotypically stable MSC precursors. Given the critical role of the ECM in cartilage function, proteomics is a promising method for determining the optimal source of MSCs, as the matrix proteins accumulated at the time of sampling may not be reflected by their respective mRNA levels. I anticipate that further studies using proteomics to compare engineered constructs and authentic cartilage will lead to a greater understanding of MSC-based chondrogenesis and improved materials for tissue engineering.

The Function of Ocular Lens Intermediate Filaments in Normal and Pathogenic Biology

The ocular lens sequentially expresses two different classes of Intermediate Filament (IF) proteins in the course of terminal differentiation. The less differentiated lens epithelial cells express the common IF protein vimentin. However, as lens cells enter into terminal differentiation they expresses two lens‐specific IF proteins, CP49 and filensin. These two IF proteins are unique among IF proteins in that they are found not in canonical 8–11 nm IFs, but in a structure termed the Beaded Filament (BF). To assign function to the Beaded Filament (BF), we used a knockout approach to delete both CP49 and filensin. The data suggest that the Beaded Filament is not required for the remarkable structural differentiation of either lens fiber cells, or the lens as a tissue. However, the BF is required to maintain these structural phenotypes with age. The knock‐in mouse suggests that the human disease‐causing mutation identified in CP49 results in a loss‐of‐function phenotype due to the absence of BF, compounded by a “gain‐of‐toxic function phenotype: the mutation in CP49 prevents the efficient removal of CP49, resulting in accumulation of protein aggregates.

Chronic treatment with long acting phosphodiesterase-5 inhibitor tadalafil alters proteomic changes associated with cytoskeletal rearrangement and redox regulation in Type 2 diabetic hearts

Diabetic patients are prone to metabolic perturbations that progressively contribute to structural, functional and proteomic alterations in the myocardium. Phosphodiesterase-5 (PDE-5) inhibitors exhibit cardioprotective effects against ischemic/reperfusion injury, however the effects of chronic administration of PDE-5 inhibitors, particularly under diabetic conditions, remain unknown. Hence, the present study was designed to identify novel protein targets related to long-acting PDE-5 inhibitor tadalafil-induced cardioprotection in diabetes. Using two-dimensional differential in-gel electrophoresis with 3 CyDye labeling and MALDI-TOF/TOF tandem mass spectrometry we identified alterations in the expressions of cardiac proteins in diabetic db/db mice treated with tadalafil. Tadalafil reversed the coordinated alterations of cytoskeletal/contractile proteins such as myosin light chain (MLY) 2 and 4, myosin heavy chain α and myosin-binding protein C which contributes to contractile dysfunction. The expression of intermediate filament protein vimentin and extra-cellular matrix proteins like cysteine and glycine rich protein-3 and collagen type VI α were upregulated in db/db mice indicating cardiac remodeling in diabetes. These detrimental proteomic alterations were reflected in cardiac function which were reversed in tadalafil treated mice. Tadalafil also enhanced antioxidant enzyme glutathione S-transferase Kappa-1 (GSKT-1) and downregulated redox regulatory chaperones like heat shock protein 8 (HSPA8), and 75kD glucose regulatory protein (75GRP). Furthermore, tadalafil treatment significantly attenuated GSSG/GSH ratio and improved the metabolic status of db/db mice. Chronic treatment with tadalafil in db/db mice modulates proteins involved in cytoskeletal rearrangement and redox signaling of the heart, which may explain the beneficial effects of PDE-5 inhibition in diabetes.

Soluble CD44 Interacts with Intermediate Filament Protein Vimentin on Endothelial Cell Surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12–37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.

FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant hereditary neuromuscular di- sorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been sho- wn previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to nor- mal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myo- blasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vi- mentin could be an easily detectable marker of FSHD myotubes.

Vimentin binding is critical for infection by the virulent strain of Japanese encephalitis virus

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis with high mortality in humans. We used a pair of virulent (RP-9) and attenuated (RP-2ms) variants of JEV to pull down the cell surface molecules bound with JEV particle; their identities were revealed by LC-MS/MS analysis. One major protein bound with RP-9 and weakly with RP-2ms was identified as the intermediate filament protein vimentin. Infection of RP-9 but not that of RP-2ms was blocked by anti-vimentin antibodies and by recombinant-expressed vimentin proteins. Knockdown of vimentin expression reduced the levels of viral binding and viral production of RP-9, but not that of RP-2ms. The different vimentin dependency for JEV infection could be attributed to the major structural envelope protein, as the recombinant RP-9 with an E-E138K mutation became resistant to anti-vimentin blockage. Furthermore, RP-2ms mainly depended on cell surface glycosaminoglycans for viral binding and it became vimentin-dependent only when binding to glycosaminoglycans was blocked. Thus, we suggest that vimentin contributes to virulent JEV infection and might be a new target to intervene in this deadly infection.

The vimentin-tubulin binding site peptide (Vim-TBS.58-81) crosses the plasma membrane and enters the nuclei of human glioma cells

Cell-penetrating peptides (CPPs) can translocate through the plasma membrane and localize in different cell compartments providing a promising delivery system for peptides, proteins, nucleic acids, and other products. Here we describe features of a novel cell-penetrating peptide derived from the intermediate filament protein vimentin, called Vim-TBS.58-81. We show that it enters cells from a glioblastoma line via endocytosis where it distributes throughout the cytoplasm and nucleus. Moreover, when coupled to the pro-apoptogenic peptide P10, it localizes to the nucleus inhibiting cell proliferation. Thus, the Vim-TBS.58-81 peptide represents an effective vector for delivery of peptides and potentially a broad range of cargos to the nucleus.

Abstract 5127: G6-mediated inhibition of Jak2 tyrosine kinase correlates with the cleavage and cellular reorganization of vimentin

Abstract Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including various leukemias, lymphomas, myelomas and the myeloproliferative neoplasms. Using structure-based virtual screening, our lab previously identified a novel Jak2 inhibitor named G6. We showed that G6 has a specific inhibitory effect on Jak2 tyrosine kinase activity and potently suppresses Jak2-mediated cellular proliferation. To elucidate the biochemical and cellular mechanisms by which G6 inhibits Jak2-mediated cell proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 hours with either vehicle control (DMSO) or 25 µM of the drug and then compared their protein expression profiles using two-dimensional gel electrophoresis. One protein that was found to be differentially expressed was the intermediate filament protein vimentin. Specifically, we found that full length vimentin was robustly expressed in untreated cells and absent in G6 treated cells. We hypothesized that inhibition of Jak2-V617F dependent pathogenic cell growth by G6 correlates with the cleavage and cellular reorganization of intermediate vimentin filaments. To this end, we found that the treatment of HEL cells with G6 induced a time- and dose-dependent cleavage of vimentin. This G6-induced cleavage of vimentin was both Jak2-dependent and calpain-mediated. Using indirect immunofluorescence, we found that G6 treatment resulted in a marked reorganization of vimentin filaments within cells. Finally, using a mouse model of Jak2-V617F mediated neoplasia, we found that G6 decreased the levels of vimentin protein within the bone marrow of G6 treated mice when compared to animals that received vehicle control. Overall, these results demonstrate that G6-mediated inhibition of Jak2-dependent pathogenic cell growth correlates with the proteolytic cleavage of full length vimentin and the reorganization of intermediate vimentin filaments within cells. As such, we may have identified a biochemical and cellular mechanism whereby G6 inhibits Jak2-V617F mediated cellular proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5127. doi:10.1158/1538-7445.AM2011-5127

RIG-LIKE receptor signaling modulates the innate immune response and astrocyte activation after injury to the spinal cord (157.6)

Abstract Injury to the central nervous system (CNS) induces a glial response, in which astrocytes become activated and produce inflammatory mediators. However, the molecular basis for glial activation and the signaling pathways regulating glial/innate immune responses remains poorly understood. Here, we examine the activation of retinoic acid inducible gene-like (RIG) receptors (RLRs) and the involvement of RLR signaling in the regulation of type I IFNs following spinal cord injury (SCI). Immunohistochemical analysis followed by confocal microscopy procedures reveals that astrocytes express two intracellular RLRs, RIG-I and melanoma-associated gene 5 (MDA5). Immunoblotting of spinal cord protein lysates after SCI and stretch injury of culture astrocytes indicates that injury to the CNS activates RLR signaling as determined by phosphorylation of IRF3 leading to production of type I IFNs. Stimulation of RIG-I and MDA5 with synthetic RNA (poly(I:C)) results in RLR signaling activation, phosphorylation of IRF3, and increased expression of glial fibrillary acidic protein and the intermediate filament protein vimentin, two hallmarks of reactive astrocytes. To the best of our knowledge this is the first time that the RLR signaling pathway is described as being involved in regulating astrocyte activation and modulation of the innate immune response after SCI.

A Knock-In Mouse Model for the R120G Mutation of αB-Crystallin Recapitulates Human Hereditary Myopathy and Cataracts

An autosomal dominant missense mutation in αB-crystallin (αB-R120G) causes cataracts and desmin-related myopathy, but the underlying mechanisms are unknown. Here, we report the development of an αB-R120G crystallin knock-in mouse model of these disorders. Knock-in αB-R120G mice were generated and analyzed with slit lamp imaging, gel permeation chromatography, immunofluorescence, immunoprecipitation, histology, and muscle strength assays. Wild-type, age-matched mice were used as controls for all studies. Both heterozygous and homozygous mutant mice developed myopathy. Moreover, homozygous mutant mice were significantly weaker than wild-type control littermates at 6 months of age. Cataract severity increased with age and mutant gene dosage. The total mass, precipitation, and interaction with the intermediate filament protein vimentin, as well as light scattering of αB-crystallin, also increased in mutant lenses. In skeletal muscle, αB-R120G co-aggregated with desmin, became detergent insoluble, and was ubiquitinated in heterozygous and homozygous mutant mice. These data suggest that the cataract and myopathy pathologies in αB-R120G knock-in mice share common mechanisms, including increased insolubility of αB-crystallin and co-aggregation of αB-crystallin with intermediate filament proteins. These knock-in αB-R120G mice are a valuable model of the developmental and molecular biological mechanisms that underlie the pathophysiology of human hereditary cataracts and myopathy.

Plectin interacts with the rod domain of type III intermediate filament proteins desmin and vimentin

Plectin is a versatile cytolinker protein critically involved in the organization of the cytoskeletal filamentous system. The muscle-specific intermediate filament (IF) protein desmin, which progressively replaces vimentin during differentiation of myoblasts, is one of the important binding partners of plectin in mature muscle. Defects of either plectin or desmin cause muscular dystrophies. By cell transfection studies, yeast two-hybrid, overlay and pull-down assays for binding analysis, we have characterized the functionally important sequences for the interaction of plectin with desmin and vimentin. The association of plectin with both desmin and vimentin predominantly depended on its fifth plakin repeat domain and downstream linker region. Conversely, the interaction of desmin and vimentin with plectin required sequences contained within the segments 1A–2A of their central coiled-coil rod domain. This study furthers our knowledge of the interaction between plectin and IF proteins important for maintenance of cytoarchitecture in skeletal muscle. Moreover, binding of plectin to the conserved rod domain of IF proteins could well explain its broad interaction with most types of IFs.

Degenerative disc disease of herniated intervertebral discs is associated with extracellular matrix remodeling, vimentin-positive cells and cell death

We studied patients with degenerative disc disease (DDD) to demonstrate that i) remodeling of the extracellular matrix (ECM) in the intervertebral disc (IVD), particularly the elastic fiber system, of subjects with herniated discs is dysregulated and that ii) it is accompanied by accelerated elastin degradation due to increased expression of matrix metalloprotease-9 (MMP-9). Moreover we wanted to obtain a deeper insight into the pathogenesis of DDD through the study of ECM calcification, DNA fragmentation using TUNEL analysis, BAX, bcl-2 and vimentin immunopositive cells. We studied herniated discs from patients of three age groups (group 1=30-40 years; group 2=40-50 years; and group 3=50-65 years) to evaluate the oxytalan fiber systemMMP-9, apoptosis and vimentin immunopositive cells. The results demonstrated the presence of oxytalan fibers in the annulus fibrosus (AF) and the nucleus pulposus (NP) of herniated discs. In the AF oxytalan fibers replaced disrupted mature elastic fibers in calcified areas, while in the NP they were mostly found in nests at the periphery of chondrocytes. MMP-9 was prevalently observed in NP nests above all in group 1 and group 3 discs while group 2 exhibited a lower MMP-9 immunostaining. Activation of the apoptotic process was demonstrated by upregulated BAX expression in group 3. BAX immunopositivity was inversely mirrored by a significant decrease in bcl-2 expression. Intermediate filament protein vimentin was strongly expressed only in group 1 samples. A large number of apoptotic TUNEL+ cells was observed in group 3 specimens. The presence of oxytalan fibers may be the result of a process of incomplete elastogenesis, or a response to mechanical stress trying to functionally replace the lack of elastic fibers. MMP-9 expression seems to relate to disc damage, while chondrocyte BAX upregulation and TUNEL+ cell staining revealed apoptosis activation regardless of patient age. Vimentin immunopositivity was clearly detected in group 1 annulus fibrosus and nucleus pulposus cells. In conclusion, as demonstrated by the vimentin-positive cells, the injured IVD has endogenous resources that can stem the DDD damage, including substitution of damaged elastic fibers by oxytalan fibers. In addition, induction of apoptosis suggests an increased cell turnover in response to repair needs.

Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer

Epithelial-to-mesenchymal transition (EMT) is a critical event in the progression toward cancer metastasis. The intermediate filament protein vimentin is an important marker of EMT and a requisite regulator of mesenchymal cell migration. However, it is not known how vimentin functionally contributes to cancer cell invasion. Here, we report that ectopic expression of oncogenic H-Ras-V12G and Slug induces vimentin expression and migration in pre-malignant breast epithelial cells. Conversely, vimentin expression is necessary for Slug- or H-Ras-V12G-induced EMT-associated migration. Furthermore, silencing of vimentin in breast epithelial cells results in specific changes in invasiveness-related gene expression including upregulation of RAB25 (small GTPase Rab25) and downregulation of AXL (receptor tyrosine kinase Axl), PLAU (plasminogen activator, urokinase) and ITGB4 (integrin β4-subunit). Importantly, gene expression profiling analyses reveal that vimentin expression correlates positively/negatively with these genes also in multiple breast cancer cell lines and breast cancer patient samples. Focusing on the tyrosine kinase Axl, we show that induction of vimentin by EMT is associated with upregulation of Axl expression and that Axl enhances the migratory activity of pre-malignant breast epithelial cells. Using null and knock-down cells and overexpression models, we also show that regulation of breast cancer cell migration in two- and three-dimensional matrices by vimentin is Axl- dependent and that Axl functionally contributes to lung extravasation of breast cancer cells in mice. In conclusion, our data show that vimentin functionally contributes to EMT and is required for induction of Axl expression. Moreover, these results provide a molecular explanation for vimentin-dependent cancer cell migration during EMT by identifying Axl as a key proximal component in this process.

A Nuclear Function for Plakophilin-1 in the DNA Damage Response?

Plakophilins are proteins of the desmosomal plaque. Based on the observation that plakophilins localize not only to desmosomes but also to the cytoplasm and nucleus, additional functions in cell signaling have been proposed. In this issue, Sobolik-Delmaire et al. address the nuclear function of Plakophilin-1. The authors show that Plakophilin-1 interacts with ssDNA in vitro and may have a function in protecting cells from DNA damage.