Actin Cytoskeleton-associated Proteins Research Articles

Specc1l deficiency leads to abnormal oocyte meiosis and reduced blastocyst development in mouse.

This study aimed to investigate the potential role of the mouse Specc1l (Sperm antigen with calponin homology and coiled-coil domains 1 like), a microtubule and actin cytoskeleton-associated protein during oocyte meiotic maturation and its potential effects on preimplantation development. This study shows that the transcriptional levels of Specc1l did not significantly change from the germinal vesicle (GV) stage to the metaphase II (MII) stage, but maternal transcripts rapidly and gradually degraded after fertilization. Specc1l was detected in both the cytoplasm and GV, but not in the nucleolus-like body in the GV intact oocyte. At the MII stages, Specc1l was widely distributed in the cytoplasm but did not co-localize with chromatin. Knockdown of Specc1l expression in oocytes resulted in abnormal spindle morphology and misaligned chromosomes, as well as a decrease in the rate of polar body extrusion and a reduced developmental competence of oocytes, leading to decreased blastocyst formation rate. In conclusion, this study provides evidence that Specc1l plays a critical role in mouse oocyte meiotic maturation and early embryo development, specifically in proper bipolar spindle assembly and extrusion of the first polar body.

A rapid degradation of calponin 2 is required for cytokinesis.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

Extracellular MIF, but not its homologue D-DT, promotes fibroblast motility independently of its receptor complex CD74/CD44.

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro, our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton.

Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging.

Modifications to cytoskeleton-associated proteins in dendritic spines underlie the adaptive plasticity involved in long term reference memory

Spatial learning and memory enables individuals to orientate themselves in an external environment. Synaptic stimulation of dendritic spines on hippocampal place cells underlies adaptive cognitive performance, inducing plastic changes such as spinogenesis, pruning and structural interconversion. Such plastic changes are driven by complex molecular machinery that relies on several actin cytoskeleton-associated proteins (ACAP's), these interacting with actin filaments in the postsynaptic density to guide the conformational changes to spines in accordance with the synaptic information they receive. However, the specific dynamics of the plastic changes in spines driven by ACAP's are poorly understood. Adult rats exhibit efficient allocentric reference memory 30days after training in a spatial learning paradigm in the Morris water maze. A Golgi study revealed this behavior to be associated with a reduction in both spine density and in mushroom spines, as well as a concomitant increase in thin spines. These changes were accompanied by the overexpression of mRNA encoding β-actin, Spinophilin and Cortactin, whilst the expression of Profilin, α-actinin, Drebrin, Synaptopodin and Myosin decreased. By contrast, no changes were evident in Cofilin, Gelsolin and Arp2/3 mRNA. From this analysis, it appears that neither spinogenesis nor new mushroom spines are necessary for long-term spatial information retrieval, while thin spines could be potentiated to retrieve pre-learned spatial information. Further studies that focus on the signaling pathways and their related molecules may shed further light on the molecular dynamics of the plastic changes to dendritic spines that underlie cognitive performance, both under normal and pathological conditions.

Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals-Illustrated with Four Actin Cytoskeleton Proteins.

The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: (1) yeast Hof1p/mammalian PSTPIP1, (2) yeast Rvs167p/mammalian BIN1, (3) yeast eEF1A/eEF1A1 and eEF1A2 and (4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.

Downregulation of calponin 2 contributes to the quiescence of lung macrophages.

Calponin 2 is an actin cytoskeleton-associated regulatory protein that inhibits the activity of myosin-ATPase and cytoskeleton dynamics. Recent studies have demonstrated that deletion of calponin 2 restricts the proinflammatory activation of macrophages in atherosclerosis and arthritis to attenuate the disease progression in mice. Here we demonstrate that the levels of calponin 2 vary among different macrophage populations, which may reflect their adaptation to specific tissue microenvironment corresponding to specific functional states. Interestingly, lung resident macrophages express significantly lower calponin 2 than peritoneal resident macrophages, which correlates with decreased substrate adhesion and reduced expression of proinflammatory cytokines and a proresolution phenotype. Deletion of calponin 2 in peritoneal macrophages also decreased substrate adhesion and downregulated the expression of proinflammatory cytokines. Providing the first line of defense against microbial invasion while receiving constant exposure to extrinsic antigens, lung macrophages need to maintain a necessary level of activity while limiting exaggerated inflammatory reaction. Therefore, their low level of calponin 2 may reflect an important physiological adaption. Downregulation of calponin 2 in macrophages may be targeted as a cytoskeleton-based novel mechanism, possibly via endoplasmic reticulum stress altering the processing and secretion of cytokines, to regulate immune response and promote quiescence for the treatment of inflammatory diseases.

ZNF185 is a p53 target gene following DNA damage.

The transcription factor p53 is a key player in the tumour suppressive DNA damage response and a growing number of target genes involved in these pathways has been identified. p53 has been shown to be implicated in controlling cell motility and its mutant form enhances metastasis by loss of cell directionality, but the p53 role in this context has not yet being investigated. Here, we report that ZNF185, an actin cytoskeleton-associated protein from LIM-family of Zn-finger proteins, is induced following DNA-damage. ChIP-seq analysis, chromatin crosslinking immune-precipitation experiments and luciferase assays demonstrate that ZNF185 is a bona fide p53 target gene. Upon genotoxic stress, caused by DNA-damaging drug etoposide and UVB irradiation, ZNF185 expression is up-regulated and in etoposide-treated cells, ZNF185 depletion does not affect cell proliferation and apoptosis, but interferes with actin cytoskeleton remodelling and cell polarization. Bioinformatic analysis of different types of epithelial cancers from both TCGA and GTEx databases showed a significant decrease in ZNF185 mRNA level compared to normal tissues. These findings are confirmed by tissue micro-array IHC staining. Our data highlight the involvement of ZNF185 and cytoskeleton changes in p53-mediated cellular response to genotoxic stress and indicate ZNF185 as potential biomarker for epithelial cancer diagnosis.

WIP-YAP/TAZ as A New Pro-Oncogenic Pathway in Glioma.

Wild-type p53 (wtp53) is described as a tumour suppressor gene, and mutations in p53 occur in many human cancers. Indeed, in high-grade malignant glioma, numerous molecular genetics studies have established central roles of RTK-PI3K-PTEN and ARF-MDM2-p53 INK4a-RB pathways in promoting oncogenic capacity. Deregulation of these signalling pathways, among others, drives changes in the glial/stem cell state and environment that permit autonomous growth. The initially transformed cell may undergo subsequent modifications, acquiring a more complete tumour-initiating phenotype responsible for disease advancement to stages that are more aggressive. We recently established that the oncogenic activity of mutant p53 (mtp53) is driven by the actin cytoskeleton-associated protein WIP (WASP-interacting protein), correlated with tumour growth, and more importantly that both proteins are responsible for the tumour-initiating cell phenotype. We reported that WIP knockdown in mtp53-expressing glioblastoma greatly reduced proliferation and growth capacity of cancer stem cell (CSC)-like cells and decreased CSC-like markers, such as hyaluronic acid receptor (CD44), prominin-1 (CD133), yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). We thus propose a new CSC signalling pathway downstream of mtp53 in which Akt regulates WIP and controls YAP/TAZ stability. WIP drives a mechanism that stimulates growth signals, promoting YAP/TAZ and β-catenin stability in a Hippo-independent fashion, which allows cells to coordinate processes such as proliferation, stemness and invasiveness, which are key factors in cancer progression. Based on this multistep tumourigenic model, it is tantalizing to propose that WIP inhibitors may be applied as an effective anti-cancer therapy.

Lacking of palladin leads to multiple cellular events changes which contribute to NTD

BackgroundThe actin cytoskeleton-associated protein palladin plays an important role in cell motility, morphogenesis and adhesion. In mice, Palladin deficient embryos are lethal before embryonic day (E) 15.5, and exhibit severe cranial neural tube and body wall closure defects. However, the mechanism how palladin regulates the process of cranial neural tube closure (NTC) remains unknown.MethodsIn this paper, we use gene knockout mouse to elucidate the function of palladin in the regulation of NTC process.ResultsWe initially focuse on the expression pattern of palladin and found that in embryonic brain, palladin is predominantly expressed in the neural folds at E9.5. We further check the major cellular events in the neural epithelium that may contribute to NTC during the early embryogenesis. Palladin deficiency leads to a disturbance of cytoskeleton in the neural tube and the cultured neural progenitors. Furthermore, increased cell proliferation, decreased cell differentiation and diminished apical cell apoptosis of neural epithelium are found in palladin deficient embryos. Cell cycle of neural progenitors in Palladin-/- embryos is much shorter than that in wt ones. Cell adhesion shows a reduction in Palladin-/- neural tubes.ConclusionsPalladin is expressed with proper spatio-temporal pattern in the neural folds. It plays a crucial role in regulating mouse cranial NTC by modulating cytoskeleton, proliferation, differentiation, apoptosis, and adhesion of neural epithelium. Our findings facilitate further study of the function of palladin and the underlying molecular mechanism involved in NTC.

PDLIM5 links kidney anion exchanger 1 (kAE1) to ILK and is required for membrane targeting of kAE1

Anion exchanger 1 (AE1) mediates Cl−/HCO3− exchange in erythrocytes and kidney intercalated cells where it functions to maintain normal bodily acid-base homeostasis. AE1’s C-terminal tail (AE1C) contains multiple potential membrane targeting/retention determinants, including a predicted PDZ binding motif, which are critical for its normal membrane residency. Here we identify PDLIM5 as a direct binding partner for AE1 in human kidney, via PDLIM5’s PDZ domain and the PDZ binding motif in AE1C. Kidney AE1 (kAE1), PDLIM5 and integrin-linked kinase (ILK) form a multiprotein complex in which PDLIM5 provides a bridge between ILK and AE1C. Depletion of PDLIM5 resulted in significant reduction in kAE1 at the cell membrane, whereas over-expression of kAE1 was accompanied by increased PDLIM5 levels, underscoring the functional importance of PDLIM5 for proper kAE1 membrane residency, as a crucial linker between kAE1 and actin cytoskeleton-associated proteins in polarized cells.

WIP Drives Tumor Progression through YAP/TAZ-Dependent Autonomous Cell Growth

In cancer, the deregulation of growth signaling pathways drives changes in the cell's architecture and its environment that allow autonomous growth of tumors. These cells then acquire a tumor-initiating "stemness" phenotype responsible for disease advancement to more aggressive stages. Here, we show that high levels of the actin cytoskeleton-associated protein WIP (WASP-interacting protein) correlates with tumor growth, both of which are linked to the tumor-initiating cell phenotype. We find that WIP controls tumor growth by boosting signals that stabilize the YAP/TAZ complex via a mechanism mediated by the endocytic/endosomal system. When WIP levels are high, the β-catenin Adenomatous polyposis coli (APC)-axin-GSK3 destruction complex is sequestered to the multi-vesicular body compartment, where its capacity to degrade YAP/TAZ is inhibited. YAP/TAZ stability is dependent on Rac, p21-activated kinase (PAK) and mammalian diaphanous-related formin (mDia), and is Hippo independent. This close biochemical relationship indicates an oncogenic role for WIP in the physiology of cancer pathology by increasing YAP/TAZ stability.

Deletion of calponin 2 in macrophages attenuates the severity of inflammatory arthritis in mice.

Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of calponin are present in vertebrates, among which calponin 2 encoded by the Cnn2 gene is expressed in multiple types of cells, including blood cells from the myeloid lineage. Our previous studies demonstrated that macrophages from Cnn2 knockout (KO) mice exhibit increased migration and phagocytosis. Intrigued by an observation that monocytes and macrophages from patients with rheumatoid arthritis had increased calponin 2, we investigated anti-glucose-6-phosphate isomerase serum-induced arthritis in Cnn2-KO mice for the effect of calponin 2 deletion on the pathogenesis and pathology of inflammatory arthritis. The results showed that the development of arthritis was attenuated in systemic Cnn2-KO mice with significantly reduced inflammation and bone erosion than that in age- and stain background-matched C57BL/6 wild-type mice. In vitro differentiation of calponin 2-null mouse bone marrow cells produced fewer osteoclasts with decreased bone resorption. The attenuation of inflammatory arthritis was confirmed in conditional myeloid cell-specific Cnn2-KO mice. The increased phagocytotic activity of calponin 2-null macrophages may facilitate the clearance of autoimmune complexes and the resolution of inflammation, whereas the decreased substrate adhesion may reduce osteoclastogenesis and bone resorption. The data suggest that calponin 2 regulation of cytoskeleton function plays a novel role in the pathogenesis of inflammatory arthritis, implicating a potentially therapeutic target.

Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) induces constitutive signaling in EBV-infected cells to ensure the survival of the latently infected cells. LMP1 is localized to lipid raft domains to induce signaling. In the present study, a genome-wide screen based on bimolecular fluorescence complementation (BiFC) was performed to identify LMP1-binding proteins. Several actin cytoskeleton-associated proteins were identified in the screen. Overexpression of these proteins affected LMP1-induced signaling. BiFC between the identified proteins and LMP1 was localized to lipid raft domains and was dependent on LMP1-induced signaling. Proximity biotinylation assays with LMP1 induced biotinylation of the actin-associated proteins, which were shifted in molecular mass. Together, the findings of this study suggest that the association of LMP1 with lipid rafts is mediated at least in part through interactions with the actin cytoskeleton. LMP1 signaling requires oligomerization, lipid raft partitioning, and binding to cellular adaptors. The current study utilized a genome-wide screen to identify several actin-associated proteins as candidate LMP1-binding proteins. The interaction between LMP1 and these proteins was localized to lipid rafts and dependent on LMP1 signaling. This suggests that the association of LMP1 with lipid rafts is mediated through interactions with actin-associated proteins.

Abstract LB-70: BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression

Abstract Functional loss of BRCA1 has been implicated in genomic instability and cancer progression. A recent study demonstrated that BRCA1 plays a role in cancer cell migration. Using a random cell motility assay, we showed that BRCA1 promotes ovarian cancer cell migration. A proteomics study of BRCA1 +/- cell lines and tissues identified Profilin1 (Pfn1) as one of the differentially expressed proteins. This was further confirmed by Western blot and IHC analyses with tissue samples and cell lines. Pfn1 is an actin cytoskeleton-associated protein which promotes breast cancer cell motility/invasion and dissemination when downregulated. To further study whether BRCA1 impacts cell migration through Pfn1, Pfn1 was silenced which revealed enhanced motility in multiple BRCA1-WT ovarian cancer cell lines. Furthermore, overexpression of Pfn1 in BRCA1 cell lines reduced random motility which further implicates the importance of Pfn1 in ovarian cancer motility. This is the first evaluation of both motility and the functional role of Pfn1 in ovarian cancer cells. While BRCA1 has been extensively studied in the context of DNA repair, there is emerging evidence that BRCA1 proteins may play a role in cell migration. The increased motility seen in wild-type cells compared to BRCA1-mutated cells may portend increased metastatic potential in wild-type cancers. Our findings demonstrate that 1) BRCA1 positively influences ovarian cancer cell motility, and 2) BRCA1 status determines Pfn1's role in ovarian cancer cell motility suggesting a possible novel mechanism association between Pfn1 and BRCA1 and ovarian cancer cell motility. Future studies should explore whether Pfn1 downregulation enhances the metastatic potential of ovarian cancer cells with intact BRCA1 function and whether Pfn1 may serve as a possible therapeutic target. Citation Format: David M. Gau, Jamie Lesnock, Thomas Krivak, Robert Edwards, Partha Roy. BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2014-LB-70

Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells

The actin cytoskeleton is critically involved in the regulation of neurite outgrowth. The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner. Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth. Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury. The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.

LMX1B is Essential for the Maintenance of Differentiated Podocytes in Adult Kidneys

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

GRAIL (Gene Related to Anergy in Lymphocytes) Regulates Cytoskeletal Reorganization through Ubiquitination and Degradation of Arp2/3 Subunit 5 and Coronin 1A

Anergy is an important mechanism for the maintenance of peripheral tolerance and avoidance of autoimmunity. The up-regulation of E3 ubiqitin ligases, including GRAIL (gene related to anergy in lymphocytes), is a key event in the induction and preservation of anergy in T cells. However, the mechanisms of GRAIL-mediated anergy induction are still not completely understood. We examined which proteins serve as substrates for GRAIL in anergic T cells. Arp2/3-5 (actin-related protein 2/3 subunit 5) and coronin 1A were polyubiquitinated by GRAIL via Lys-48 and Lys-63 linkages. In anergic T cells and GRAIL-overexpressed T cells, the expression of Arp2/3-5 and coronin 1A was reduced. Furthermore, we demonstrated that GRAIL impaired lamellipodium formation and reduced the accumulation of F-actin at the immunological synapse. GRAIL functions via the ubiquitination and degradation of actin cytoskeleton-associated proteins, in particular Arp2/3-5 and coronin 1A. These data reveal that GRAIL regulates proteins involved in the actin cytoskeletal organization, thereby maintaining the unresponsive state of anergic T cells.

Interleukin-6 (IL-6) Regulates Claudin-2 Expression and Tight Junction Permeability in Intestinal Epithelium

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.

Targeted Proteomics of Isolated Glomeruli from the Kidneys of Diabetic Rats: Sorbin and SH3 Domain Containing 2 Is a Novel Protein Associated with Diabetic Nephropathy

To evaluate proteins associated with the development of diabetic nephropathy, a major cause of the end-stage renal disease, we analyzed protein expression in isolated glomeruli from spontaneous type 2 diabetic (OLETF) rats and their age-matched control littermates (LETO) in the early and proteinuric stages of diabetic nephropathy using QSTAR Elite LC-MS/MS. Among the 191 and 218 proteins that were altered significantly in the OLETF rats, twenty-four were actin cytoskeleton-associated proteins implicated in the formation of stress fibers, and the impairment of actin polymerization, intermediate filaments and microtubules. Importantly, sorbin and SH3 domain containing 2 (SORBS2), which is involved in the formation of stress fibers, was significantly upregulated in both stages of diabetic nephropathy (1.49- and 1.97-fold, resp.). Immunohistochemical and quantitative-PCR analyses revealed upregulation of SORBS2 in podocytes of glomeruli of OLETF rats. Our findings suggested that SORBS2 may be associated with the development of diabetic nephropathy possibility by reorganization of actin filaments.