EF-hand Family Of Calcium-binding Proteins Research Articles

S100A12 in Digestive Diseases and Health: A Scoping Review.

Calgranulin proteins are an important class of molecules involved in innate immunity. These members of the S100 class of the EF-hand family of calcium-binding proteins have numerous cellular and antimicrobial functions. One protein in particular, S100A12 (also called EN-RAGE or calgranulin C), is highly abundant in neutrophils during acute inflammation and has been implicated in immune regulation. Structure-function analyses reveal that S100A12 has the capacity to bind calcium, zinc, and copper, processes that contribute to nutritional immunity against invading microbial pathogens. S100A12 is a ligand for the receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and CD36, which promote cellular and immunological pathways to alter inflammation. We conducted a scoping review of the existing literature to define what is known about the association of S100A12 with digestive disease and health. Results suggest that S100A12 is implicated in gastroenteritis, necrotizing enterocolitis, gastritis, gastric cancer, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, and digestive tract cancers. Together, these results reveal S100A12 is an important molecule broadly associated with the pathogenesis of digestive diseases.

Comprehensive analysis of the roles of ‘black’ and ‘gray’ clusters in structure and function of rat β-parvalbumin

Recently we found two highly conserved structural motifs in the proteins of the EF-hand calcium binding protein family. These motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif forms a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is much more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II includes a mix of aromatic, hydrophobic, and polar amino acids. The ‘black’ and ‘gray’ clusters in rat β-parvalbumin consist of F48, A100, F103 and G61, L64, M87, respectively. In the present work, we sequentially substituted these amino acids residues by Ala, except Ala100, which was substituted by Val. Physical properties of the mutants were studied by circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probe methods. The Ca2+ and Mg2+ binding affinities of these mutants were evaluated by intrinsic fluorescence and equilibrium dialysis methods. In spite of a rather complicated pattern of contributions of separate amino acid residues of the ‘black’ and ‘gray’ clusters into maintenance of rat β-parvalbumin structural and functional status, the alanine substitutions in the cluster I cause noticeably more pronounced changes in various structural parameters of proteins, such as hydrodynamic radius of apo-form, thermal stability of Ca2+/Mg2+-loaded forms, and total energy of Ca2+ binding in comparison with the changes caused by amino acid substitutions in the cluster II. These findings were further supported by the outputs of computational analysis of the effects of these mutations on the intrinsic disorder predisposition of rat β-parvalbumin, which also indicated that local intrinsic disorder propensities and the overall levels of predicted disorder were strongly affected by mutations in the cluster I, whereas mutations in cluster II had less pronounced effects. These results demonstrate that amino acids of the cluster I provide more essential contribution to the maintenance of structuraland functional properties of the protein in comparison with the residues of the cluster II.

S100A12-CD36 axis: A novel player in the pathogenesis of atherosclerosis?

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins and have a variety of intracellular and extracellular activities. It exerts its proinflammatory effects by binding to the receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4). CD36 is a class B scavenger receptor that acts as a fatty acid transporter. Both S100A12 and CD36 are implicated in vascular inflammation and atherosclerosis. It has recently been demonstrated that S100A12 binds with high affinity to CD36. On the other hand, RAGE and TLR4 play a key role in the regulation of CD36 expression. These observations point to the fact that S100A12 is an interesting molecular target for the development of therapeutics. This Cytokine stimulus will focus on the possible mechanisms of S100A12-CD36 axis in the pathogenesis of atherosclerosis.

Development of Calbindin- and Calretinin-Immunopositive Neurons in the Enteric Ganglia of Rats.

Calbindin D28K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.

Value of Calretinin Immunostaining in Diagnostic Pathology

Calretinin is a member of the EF-hand family of calcium-binding proteins. Because its expression is highly restricted to mesotheliomas, calretinin is, at present, the most commonly used positive mesothelioma marker that is most often recommended to be included in the various immunohistochemical panels used to assist in the differential diagnosis of these tumors. Calretinin expression has also been reported to be commonly expressed in a wide variety of other neoplasms, including sex cord-stromal tumors, adrenal cortical neoplasms, olfactory neuroblastomas, Schwann cell tumors, cardiac myxomas, and ameloblastomas. This article reviews the information that is currently available on calretinin expression in tumors and on its application as an immunohistochemical marker in diagnostic pathology.

The E144 residue of Scherffelia dubia centrin discriminates between the DNA repair protein XPC and the centrosomal protein Sfi1

Centrins are members of the EF-hand family of calcium-binding proteins, which are highly conserved among eukaryotes. Centrins bind to several cellular targets, through a hydrophobic triad. However, the W1xxL4xxxL8 triad in XPC (Xeroderma Pigmentosum Group C protein) is found in the reverse orientation, as in the L8xxxL4xxW1 triad in Sfi1 (Suppressor of Fermentation-Induced loss of stress resistance protein 1). As shown by previous NMR studies of human centrin 2 in complex with XPC or Sfi1, the E148 residue of human centrin 2 is in contact with XPC but is pushed away from the triad of Sfi1. We corroborated these findings using site-directed mutagenesis to generate mutations in Scherffelia dubia centrin (SdCen) and by using isothermal titration calorimetry to analyze the binding affinity of these mutants to XPC and Sfi1. We mutated the F109 residue, which is the main residue involved in target binding regardless of triad orientation, and the E144 residue, which was thought to be involved only in XPC binding. The F109L mutation reduced the binding of SdCen to XPC and Sfi1 and the negative effect was greater upon temperature increase. By contrast, the E144A mutation reduced the binding to XPC but had no effect on Sfi1 binding. The F109L-E144A mutation enhanced the negative effect of the two single mutations on XPC binding. Sfi1 proteins from Ostreococcus lucimarinus and Ostreococcus tauri, which belong to the same clade as S. dubia, were also investigated. A comparative analysis shows that the triad residues are more conserved than those in human Sfi1.

QM/MM Study on the Light Emitters of Aequorin Chemiluminescence, Bioluminescence, and Fluorescence: A General Understanding of the Bioluminescence of Several Marine Organisms

Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF-hand calcium-binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H-bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine-type luciferases).

Solution structure and dynamics of human S100A14.

Human S100A14 is a member of the EF-hand calcium-binding protein family that has only recently been described in terms of its functional and pathological properties. The protein is overexpressed in a variety of tumor cells and it has been shown to trigger receptor for advanced glycation end products (RAGE)-dependent signaling in cell cultures. The solution structure of homodimeric S100A14 in the apo state has been solved at physiological temperature. It is shown that the protein does not bind calcium(II) ions and exhibits a "semi-open" conformation that thus represents the physiological structure of the S100A14. The lack of two ligands in the canonical EF-hand calcium(II)-binding site explains the negligible affinity for calcium(II) in solution, and the exposed cysteines and histidine account for the observed precipitation in the presence of zinc(II) or copper(II) ions.

Calretinin interacts with huntingtin and reduces mutant huntingtin‐caused cytotoxicity

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expansion of CAG trinucleotide repeats encoding for polyglutamine (polyQ) in the huntingtin (Htt) gene. Despite considerable effort, the mechanisms underlying the toxicity of the mutated Htt protein remains largely uncertain. To identify novel therapeutic targets, we recently employed the approach of tandem affinity purification and discovered that calretinin (Cr), a member of the EF-hand family of calcium-binding proteins, is preferentially associated with mHtt, although it also interacts with wild-type Htt. These observations were supported by coimmunoprecipitation and by colocalization of Cr with mHtt in neuronal cultures. Over- expression of Cr reduced mHtt-caused cytotoxicity in both non-neuronal and neuronal cell models of HD, whereas knockdown of Cr expression in the cells enhanced mHtt-caused neuronal cell death. In addition, over-expression of Cr was also associated with reduction of intracellular free calcium and activation of Akt. These results suggest that Cr may be a potential therapeutic target for treatment of HD.

Copper-mediated cross-linking of S100A4, but not of S100A2, results in proinflammatory effects in melanoma cells

The aim of this study was to investigate the response to and the physiological consequences of copper-mediated cross-linking of S100A2 and S100A4, two members of the S100 family of EF-hand calcium-binding proteins. As demonstrated by electrophoresis and mass spectrometry techniques S100A2 and S100A4 show formation of cross-links due to copper-mediated oxidation of cysteine residues. For S100A4, but not for S100A2, this results in both increased activation of NFκB and secretion of TNF-α in human A375 and, to a higher extent, in RAGE-transfected melanoma cells. The data suggest that a prooxidative tumor microenvironment enhances proinflammatory and prometastatic action of S100A4.

S100A4 expression is increased in stricture fibroblasts from patients with fibrostenosing Crohn's disease and promotes intestinal fibroblast migration

Fibroblasts represent the key cell type in fibrostenosing Crohn's disease (FCD) pathogenesis. S100A4 is an EF-hand calcium-binding protein family member, implicated in epithelial-mesenchymal transition and as a marker of activated T lymphocytes and fibroblasts in chronic tissue remodeling. The aim of this study was to examine the expression profile of S100A4 in the resected ileum of patients with FCD. Mucosa, seromuscular explants, and transmural biopsies were harvested from diseased and proximal, macroscopically normal margins of ileocecal resections from patients with FCD. Samples were processed for histochemistry, immunohistochemistry, real-time RT-PCR, Western blotting, and transmission electron microscopy. Primary explant cultures of seromuscular fibroblasts were exposed to transforming growth factor (TGF)-beta1 (1 ng/ml), and S100A4 expression and scratch wound-healing activity were assessed at 24 h. CCD-18Co fibroblasts were transfected with S100A4 small interfering RNA, treated with TGF-beta1 (1 ng/ml) for 30 min or 24 h, and then assessed for S100A4 and Smad3 expression and scratch wound-healing activity. S100A4 expression was increased in stricture mucosa, in the lamina propria, and in CD3-positive intraepithelial CD3-positive T lymphocytes. Fibroblastic S100A4 staining was observed in seromuscular scar tissue. Stricture fibroblast explant culture showed significant upregulation of S100A4 expression. TGF-beta1 increased S100A4 expression in cultured ileal fibroblasts. In CCD-18Co fibroblasts, S100A4 small interfering RNA inhibited scratch wound healing and modestly inhibited Smad3 activation. S100A4 expression is increased in fibroblasts, as well as immune cells, in Crohn's disease stricture and induced by TGF-beta1. Results from knockdown experiments indicate a potential role for S100A4 in mediating intestinal fibroblast migration.

Changes in transcript and protein levels of calbindin D28k, calretinin and parvalbumin, and numbers of neuronal populations expressing these proteins in an ischemia model of rat retina

Excessive calcium is thought to be a critical step in various neurodegenerative processes including ischemia. Calbindin D28k (CB), calretinin (CR), and parvalbumin (PV), members of the EF-hand calcium-binding protein family, are thought to play a neuroprotective role in various pathologic conditions by serving as a buffer against excessive calcium. The expression of CB, PV and CR in the ischemic rat retina induced by increasing intraocular pressure was investigated at the transcript and protein levels, by means of the quantitative real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The transcript and protein levels of CB, which is strongly expressed in the horizontal cells in both normal and affected retinas, were not changed significantly and the number of CB-expressing horizontal cells remained unchanged throughout the experimental period 8 weeks after ischemia/reperfusion injury. At both the transcript and protein levels, however, CR, which is strongly expressed in several types of amacrine, ganglion, and displaced amacrine cells in both normal and affected retinas, was decreased. CR-expressing ganglion cell number was particularly decreased in ischemic retinas. Similar to the CR, PV transcript and protein levels, and PV-expressing AII amacrine cell number were decreased. Interestingly, in ischemic retinas PV was transiently expressed in putative cone bipolar cell types possibly those that connect with AII amacrine cells via gap junctions. These results suggest that these three calcium binding proteins may play different neuroprotective roles in ischemic insult by their ability to buffer calcium in the rat retina.

Human S100A8 and S100A9 activate phagocytes via Toll-like receptor 4 independent of RAGE

Endogenous Damage Associated Molecular Pattern (DAMP) proteins are known as important pro-inflammatory factors of the immune system, which are released during cellular stress. Recognition of DAMPs involves the multiligand Receptor for Advanced Glycation End products (RAGE) and Toll-like receptors (TLRs) in sensing not only Pathogen Associated Molecular Patterns (PAMPs) but also endogenous proteins. Members of the fast growing family of DAMP proteins are besides heat shock proteins, HMGB1 or defensins also some members of the family of S100 proteins, which promote inflammatory processes. It was claimed that RAGE is involved in almost all S100 protein activities. We here investigated the capacity of human S100A8 (MRP8, myeloid related protein 8) and human S100A9 (MRP14) on activation of human phagocytes. S100A8 and S100A9 form homodimers as well as heterodimers and belong to the S100 family of EF-hand calcium-binding proteins. Both proteins are the major cytoplasmic proteins of phagocytes and are released at sites of inflammation by activated or necrotic phagocytes. While human S100A8/S100A9-complexes did not show any phagocyte activation, S100A8 homodimers as well as S100A9 homodimers induce strong pro-inflammatory mechanisms in these cells. Human S100A9 induces intracellular translocation of MyD88 and activation of IRAK-1 as shown recently already for murine S100A8. Finally NF-kB activation results in elevated expression of TNF-alpha as well as other pro-inflammatory genes. In blocking experiments with TLR4-specific monoclonal antibodies we demonstrate that both S100 proteins specifically signals via TLR4 receptor complex on human phagocytes. Using TLR4/MD2/CD14 transfected HEK293 cells and RAGE transfected HEK293 cells we clearly can exclude the involvement of RAGE for at least these two S100 proteins. Our in vitro results could be further confirmed in vivo. Mice lacking S100A8/S100A9 are protected against abdominal sepsis induced by E. coli. Our present data clearly demonstrate the importance of TLR4 by which phagocytes promote their own activation via expression and secretion of endogenous ligands of this receptor.

15N Relaxation Studies of Apo-Mts1: A Dynamic S100 Protein

Mts1 is a member of the S100 family of EF-hand calcium-binding proteins. Like most S100 proteins, Mts1 exists as a dimer in solution and contains one canonical and one pseudo-EF-hand motif per monomer, each of which consists of two alpha helices connected by a loop capable of coordinating a calcium ion. The backbone dynamics of murine apo-Mts1 homodimer have been examined by nuclear magnetic resonance spectroscopy. Longitudinal and transverse relaxation data and steady-state (1)H- (15)N nuclear Overhauser effects were analyzed using model-free formalism. The extracted global correlation time is 9.94 ns. Results indicate that the protein backbone is most rigid at the dimer interface, made up of helices 1 and 4 from each monomer with mean S (2) ( S avg (2)) values approximately 0.9, flanked by helices 2 and 3 with lower S avg (2) values of 0.84 and 0.77, respectively. Each calcium-binding site along with the hinge joining the two EF-hands and the N- and C-termini are considerably more flexible than the dimer interface on a range of time scales and more flexible than the corresponding regions of other S100 proteins studied to date. As the hinge and the C-terminal tail are believed to interact with target proteins, these dynamic characteristics may have implications for Mts1 activity.

Human S100A12: a novel key player in inflammation?

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Human S100A12 is predominantly expressed and secreted by neutrophil granulocytes and, therefore, has been assigned to the S100 protein subfamily of calgranulins or myeloid-related proteins. Intracellular S100A12 exists as an anti-parallel homodimer and upon calcium-dependent activation interacts with target proteins to regulate cellular functions. Extracellular S100A12 exists majorily as homodimer and hexamer, respectively, and shows cytokine-like characteristics. It is part of the innate immune response and linked to certain autoimmune reactions. Human S100A12 is markedly overexpressed in inflammatory compartments, and elevated serum levels of S100A12 are found in patients suffering from various inflammatory, neurodegenerative, metabolic, and neoplastic disorders. In this regard, interaction of calcium-activated S100A12 with the multiligand receptor for advanced glycation endproducts (RAGE) and its soluble form (sRAGE) plays a central pathogenetic role. Recent clinical evidence suggests a high potential of S100A12 as a sensitive and specific diagnostic marker of localized inflammatory processes.

Metastasis-Associated Protein S100A4: Spotlight on its Role in Cell Migration

S100A4 (also known as Mts1, metastasin, p9Ka, pEL98, CAPL, calvasculin, Fsp-1, placental calcium-binding protein) belongs to the family of EF-hand calcium-binding proteins, whose expression is elevated in a number of pathological conditions. Although it is well documented that S100A4 is expressed in cancer cells and contributes to tumor cell motility and metastatic progression, the exact underlying mechanisms remain elusive. An important characteristic feature of S100 proteins is their dual function, inside and outside the cell. In this review, we focus on the intracellular function of S100A4. The review contains structural analysis of S1004 in comparison with other members of S100 proteins. Possible modes of the interaction of S100 proteins with targets are described. Several examples of best-studied molecular interactions involving S100A4 with heavy chain of nonmuscle myosin IIA, LAR-interacting protein liprin beta1 and tumor suppressor protein p53 are provided. We suggest that the binding of S100A4 to these molecules is critical for the S100A4 function. Further studies of the implications of these interactions in different molecular pathways may shed additional light on the role of S100A4 protein in the control of tumor cell motility and migration. We discuss the approaches for down-regulation of S100A4 expression and their potential for application in the clinics.

Purification, crystallization and preliminary X-ray diffraction of human S100A15.

Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

Mapping the Ca 2+-dependent binding of an invertebrate homolog of protein phosphatase 4 regulatory subunit 2 to the small EF-hand protein, calsensin

The EF-hand family of calcium-binding proteins regulates cellular signal transduction events via calcium-dependent interactions with target proteins. Here, we show that the COOH-terminal tail of the leech homolog of protein phosphatase 4 regulatory subunit 2 (PP4-R2) interacts with the small neuronal EF-hand calcium-binding protein, Calsensin, in a calcium-dependent manner. Using two-dimensional NMR spectroscopy and chemical shift perturbations we have identified and mapped the residues of Calsensin that form a binding surface for PP4-R2. We show that the binding groove is formed primarily of discontinuous hydrophobic residues located in helix 1, the hinge region, and helix 4 of the unicornate-type four helix structure of Calsensin. The findings suggest the possibility that calcium-dependent modulation of phosphatase complexes through interactions with small calcium-binding proteins may be a general mechanism for regulation of signal transduction pathways.

Parvalbumin deficiency in fast‐twitch muscles leads to increased ‘slow‐twitch type’ mitochondria, but does not affect the expression of fiber specific proteins

Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a 'slow-onset' Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV-/- mice. Unexpectedly, PV-/- fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV-/- fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slow-twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit beta revealed the PV-/- tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fast-twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast- and slow-twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca2+ transients in PV-/- muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin- and Ca2+/calmodulin-dependent kinase II-dependent pathways. We propose that 'slow-twitch type' mitochondria in PV-/- fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca2+ removal.

Role of intracellular S100A4 for migration of rat astrocytes

S100A4 is a member of the EF-hand family of calcium-binding proteins, first identified in tumor cells, and implicated in tumor invasion and metastasis. Intracellular upregulation of S100A4 is associated with increased motility of tumor cells. Extracellular application of S100A4 increases the motility of glioma cells in vitro. We showed previously that astrocytes in spinal cord and brain white matter also express S100A4. This expression is markedly increased in reactive white matter astrocytes after injury. Here, we have explored how changes in intracellular S100A4 affect migration of astrocytes. We produced cultures of white matter, S100A4 expressing astrocytes, and developed a small interfering (si) RNA approach to specifically eliminate S100A4 expression in these cells, and compared the migration of astrocytes expressing S100A4 with astrocytes transfected with S100A4 siRNA. As a "positive control" we used S100A4 expressing C6 glioma cells. In contrast to malignant cells, S100A4 expressing astrocytes increased their migration capacity after S100A4 siRNA treatment. At the same time, and in parallel with increased migration, white matter astrocytes increased their expression of metalloproteinases MMP-9 and MT1-MMP. The addition of MMP-2/MMP-9 inhibitor resulted in a significant inhibition of migration in S100A4 siRNA-treated astrocytes. These findings indicate that S100A4 has a stabilizing function in reactive white matter astrocytes, a function that may contribute to the development of a rigid, growth-inhibitory glial scar.