Impaired Cell Adhesion Research Articles

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1−/− and ROCK2−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.

RSK2 Protein Suppresses Integrin Activation and Fibronectin Matrix Assembly and Promotes Cell Migration

Modulation of integrin activation is important in many cellular functions including adhesion, migration, and assembly of the extracellular matrix. RSK2 functions downstream of Ras/Raf and promotes tumor cell motility and metastasis. We therefore investigated whether RSK2 affects integrin function. We report that RSK2 mediates Ras/Raf inactivation of integrins. As a result, we find that RSK2 impairs cell adhesion and integrin-mediated matrix assembly and promotes cell motility. Active RSK2 appears to affect integrins by reducing actin stress fibers and disrupting focal adhesions. Moreover, RSK2 co-localizes with the integrin activator talin and is present at integrin cytoplasmic tails. It is thereby in a position to modulate integrin activation and integrin-mediated migration. Activation of RSK2 promotes filamin phosphorylation and binding to integrins. We also find that RSK2 is activated in response to integrin ligation to fibronectin. Thus, RSK2 could participate in a feedback loop controlling integrin function. These results reveal RSK2 as a key regulator of integrin activity and provide a novel mechanism by which it may promote cell migration and cancer metastasis.

Interferon-γ inhibits integrin-mediated extracellular signal-regulated kinase activation stimulated by fibronectin binding in thyroid cells.

Hashimoto's thyroiditis (HT) is an autoimmune disorder characterized by the presence of specific antibodies and by a lymphocytic infiltration of the thyroid secreting inflammatory cytokines. Macrophages, lymphocytes, and cytokines play a pivotal role in both development and progression of Th1-mediated autoimmune diseases, and a direct role in the destruction of thyroid follicles and follicular cell function in autoimmune thyroiditis. Integrins are integral membrane receptors involved in cell-extra-cellular matrix (ECM) interaction with both structural and signaling functions. The integrin- ECM interaction is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of cytokine stimulation on integrin expression and signaling in the thyroid cell. Primary cultures from normal thyroids were treated with interferon-γ (IFN-γ), INF-α, tumor necrosis factor-α, interleukin 1a or these cytokines all together. Integrin expression, cell adhesion to fibronectin (FN) and FN-stimulated extracellular signal-regulated kinase (ERK) phosphorylation were determined after cytokine treatment. IFN-γ and IFN-α were the most effective, reducing the expression of the integrin αvβ3 and slightly increasing the α3β1. Cell treatment with IFN-γ strongly impaired cell adhesion to FN. At the same time, the treatment with IFN-γ dramatically inhibited the stimulation of ERK phosphorylation induced by cell adhesion to FN. In conclusion, IFN-γ inhibits the expression of the integrin αvβ3, reducing the cell adhesion to FN and the following intracellular signaling in thyroid cells in culture. These results suggest that integrins may be a target of the infiltrating lymphocytes and have a role in the pathogenesis of autoimmune thyroiditis.

The histone demethylase PHF8 is essential for cytoskeleton dynamics

PHF8 is a histone demethylase associated with X-linked mental retardation. It has been described as a transcriptional co-activator involved in cell cycle progression, but its physiological role is still poorly understood. Here we show that PHF8 controls the expression of genes involved in cell adhesion and cytoskeleton organization such as RhoA, Rac1 and GSK3β. A lack of PHF8 not only results in a cell cycle delay but also in a disorganized actin cytoskeleton and impaired cell adhesion. Our data demonstrate that PHF8 directly regulates the expression of these genes by demethylating H4K20me1 at promoters. Moreover, c-Myc transcription factor cooperates with PHF8 to regulate the analysed promoters. Further analysis in neurons shows that depletion of PHF8 results in down-regulation of cytoskeleton genes and leads to a deficient neurite outgrowth. Overall, our results suggest that the mental retardation phenotype associated with loss of function of PHF8 could be due to abnormal neuronal connections as a result of alterations in cytoskeleton function.

Enhanced radiosensitivity of head and neck squamous cell carcinoma cells by β1 integrin inhibition

PurposeIntegrin-mediated adhesion to extracellular matrix (ECM) contributes to the regulation of the cellular radiation response in various tumor entities. To evaluate whether targeting of β1 integrin enhances the radiosensitivity of head and neck SCC cell lines (HNSCC) was assessed using either inhibitory anti-β1 integrin antibodies or specific β1 integrin small interfering RNA (siRNA). Materials and methodsThe HNSCC cell lines FaDu, UTSCC15 and UTSCC14 were used. Upon β1 integrin inhibition, colony formation, proliferation, DNA double strand breaks, adhesion, and migration as well as protein expression and phosphorylation of integrin downstream targets like Focal Adhesion Kinase and AKT were determined. ResultsWe found that siRNA- and antibody-mediated targeting of β1 integrin result in a dose- and cell line-dependent radiosensitization that was accompanied by a decreased cell proliferation and an increased number of radiogenic DNA double strand breaks. Analysis of signal transduction events revealed a dephosphorylation of focal adhesion proteins, prevention of radiation-induced phosphorylation of pro-survival protein kinases and impaired cell adhesion and migration upon blocking of β1 integrins. ConclusionsOur data suggest that β1 integrin critically contributes to the cellular radioresistance of HNSCC. Further studies are warranted to evaluate whether targeting β1 integrin emerges as novel approach to improve radiotherapy patients’ outcome.

An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

IntroductionIn humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and MethodsHere, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. ResultsMice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. ConclusionMaintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declared

An Essential Role for B1 Integrin Mediated Cell-Matrix Interactions in the Development and Maintenance of the Nucleus Pulposus

Introduction In humans, the almost total disappearance of notochordal cells in the nucleus pulposus (NP) correlates with early degenerative changes in the intervertebral disk which include changes in the extracellular matrix (ECM). Recent cell lineage studies in mice suggest cells within the NP are derived from the early embryonic notochord. Since notochordal cells are surrounded by a rich ECM, the functions of the notochord and the NP are likely mediated via cell-matrix interactions. Integrin heterodimers are transmembrane proteins and are the major adhesion receptors that mediate cell-matrix interactions. They connect the ECM to the intracellular cytoskeleton and transduce bidirectional signals between the extracellular matrix and intracellular compartments. At least 24 integrin heterodimers have been identified, and among them receptors containing b 1 integrin comprise the largest integrin-subfamily. Materials and Methods Here, we used Cre recombinase to specifically inactivate b 1 integrin gene in the notochord in mice, to understand the role of b 1 integrin-mediated cell-matrix interactions in the development and maintenance of the notochord and NP. The development and fate of the notochordal cells were analyzed at embryonic stages E8.5-E10.5 using key expression markers such as Shh, Brachyury, and Noto and cell apoptotic assay. NP formation was studied by histological analysis. The function of b 1 integrin in cell-matrix interactions in notochord cells isolated by FACS were analyzed using a functional blocking antibody specific to b 1 integrin. Fibronectin matrix and Cadherin-2 expression pattern were investigated by immunofluorescence and confocal microscopy. Results Mice lacking b 1 integrin subunit in notochordal cells display abnormal vertebral column development with reduced size or total absence of the notochord descendents and NP in the intervertebral disks (IVD). This abnormality is accompanied with disorganized kinked patterning of the tail vertebrae and hemivertebrae. These defects are related to defects in the development and maintenance of the notochord at early embryonic stages from E8.25. Abnormalities in localization of the notochord were found, such as displacement from D-V or L-R midline axes, and failure to separate from the foregut endoderm. Assembly of ECM components was also impaired. Impaired cell matrix assembly and cell-cell contacts found in the mutants may possibly contribute to the notochord mallocalization. Increased cell apoptosis together with impaired cell-cell adhesion may potentially contribute to the defective notochord tissue integrity. b 1 integrin antibody blocking assays on notochordal cells display impaired cell adhesion, delayed maturation of focal adhesion complexes and decreased migration ability on matrix substrates. Conclusion Maintenance of b 1 integrin mediated cell-matrix interactions is essential for the proper development and maintenance of the notochord and NP, and later the proper patterning of the axial skeleton. Impaired cell-matrix interactions which affect cell survival, cell polarity, and migration may contribute to the progressive loss of NP cells during disk degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared

Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin.

Network analysis and proteomic identification of vimentin as a key regulator associated with invasion and metastasis in human hepatocellular carcinoma cells

Poor prognoses have long been associated with the high relapse and metastasis of human hepatocellular carcinoma (HCC). To achieve long-term survival, it is necessary to identify new HCC biomarkers and investigate their roles in cell mobility and invasiveness. Of note, overexpression of vimentin (Vim) was significantly correlated with tumor nuclear grade (p=0.01) and the invasive potential, indicating that Vim may be a promising candidate in regulating HCC metastasis. RNA interference-mediated silencing of Vim (siVim) suppressed the invasive and migratory propensity, and matrix metalloproteinase (MMP)-9 activity, and elicited morphological changes in poorly differentiated SK-Hep-1 cells. Moreover, we performed a comprehensive proteomic analysis to survey global protein changes mediated by siVim in SK-Hep-1 cells. Significant changes in cytoskeleton protein but not messenger RNA levels encoding these targeted proteins were observed. All of the data in the current study and a network analysis implied that abolition of Vim may disturb the expression and stability of various cytoskeletal proteins through promoting the ubiquitin system, resulting in impaired cell adhesion and motility. Collectively, an integrated approach represents a modality to explore novel relationships in a proteome complex and highlights the functional roles of Vim in HCC metastasis. This article is part of a Special Issue entitled: Translational Proteomics.

Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP⁻/⁻ cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP⁻/⁻ Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

TGFβ modulates cell-to-cell communication in early epithelial-to-mesenchymal transition.

A key pathology in diabetic nephropathy is tubulointerstitial fibrosis. The condition is characterised by increased deposition of the extracellular matrix, fibrotic scar formation and declining renal function, with the prosclerotic cytokine TGF-β1 mediating many of these catastrophic changes. Here we investigated whether TGF-β1-induced epithelial-to-mesenchymal transition (EMT) plays a role in alterations in cell adhesion, cell coupling and cell communication in the human renal proximal tubule. Whole-cell and cell compartment abundance of E-cadherin, N-cadherin, snail, vimentin, β-catenin and connexin-43 was determined in human kidney cell line (HK)2 and human proximal tubule cells with or without TGF-β1, using western blotting and immunocytochemistry, followed by quantification by densitometry. The contribution of connexin-43 in proximal tubule cell communication was quantified using small interfering RNA knockdown, while dye-transfer was used to assess gap junctional intercellular communication (GJIC). Functional tethering was assessed by single-cell force spectroscopy with or without TGF-β1, or by immunoneutralisation of cadherin ligation. High glucose (25mmol/l) increased the secretion of TGF-β1 from HK2 cells. Analysis confirmed early TGF-β1-induced morphological and phenotypical changes of EMT, with altered levels of adhesion and adherens junction proteins. These changes correlated with impaired cell adhesion and decreased tethering between coupled cells. Impaired E-cadherin-mediated adhesion reduced connexin-43 production and GJIC, these effects being mimicked by neutralisation of E-cadherin ligation. Upregulation of N-cadherin failed to restore adhesion or connexin-43-mediated GJIC. We provide compelling evidence that TGF-β1-induced EMT instigates a loss of E-cadherin, cell adhesion and ultimately of connexin-mediated cell communication in the proximal tubule under diabetic conditions; these changes occur ahead of overt signs of renal damage.

A role for collagen XXIII in cancer cell adhesion, anchorage-independence and metastasis

Collagen XXIII is a transmembrane collagen previously shown to be upregulated in metastatic prostate cancer that has been used as a tissue and fluid biomarker for non-small cell lung cancer and prostate cancer. To determine whether collagen XXIII facilitates cancer cell metastasis in vivo and to establish a function for collagen XXIII in cancer progression, collagen XXIII knockdown cells were examined for alterations in in vivo metastasis as well as in vitro cell adhesion. In experimental and spontaneous xenograft models of metastasis, H460 cells expressing collagen XXIII shRNA formed fewer lung metastases than control cells. Loss of collagen XXIII in H460 cells also impaired cell adhesion, anchorage-independent growth and cell seeding to the lung, but did not affect cell proliferation. Corroborating a role for collagen XXIII in cell adhesion, overexpression of collagen XXIII in H1299 cells, which do not express endogenous collagen XXIII, enhanced cell adhesion. Consequent reduction in OB-cadherin, alpha-catenin, gamma-catenin, beta-catenin, vimentin and galectin-3 protein expression was also observed in response to loss of collagen XXIII. This study suggests a potential role for collagen XXIII in mediating metastasis by facilitating cell-cell and cell-matrix adhesion as well as anchorage-independent cell growth.

Advanced glycation end products and diabetic retinopathy.

Retinopathy is a serious microvascular complication of diabetes and a major cause of blindness in young adults, worldwide. Early diabetic retinopathy is characterized by a loss of pericytes from retinal capillaries, the appearance of acellular capillaries and microaneurysms, and a breakdown of the blood-retinal barrier. In later stages, this can evolve into the proliferative phase in which there is neovascularization of the retina, which greatly increases the probability of vision loss. Advanced glycation end products (AGEs) which accumulate under hyperglycemic conditions are thought to play an important role in the pathogenesis of diabetic retinopathy. AGEs arise primarily by the modification of amine groups of proteins by reactive dicarbonyls such as methylglyoxal. Intracellular proteins including anti-oxidant enzymes, transcription factors and mitochondrial proteins are targets of dicarbonyl modification and this can modify their functional properties and thus compromise cellular physiology. Likewise, modification of extracellular proteins by dicarbonyls can impair cell adhesion and can generate ligands that can potentially bind to cell surface AGE receptors that activate pro-inflammatory signaling pathways. AGE inhibitors have been shown to provide protection in animal models of diabetic retinopathy and currently are being evaluated in clinical trials.

398 EXPRESSION OF RICTOR IS ASSOCIATED WITH METASTASIS AND POOR PROGNOSIS OF RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Rictor is a key member of mTORC2, which is crucial to various carcinoma. We measured Rictor expression levels in tumor tissues and six RCC cell lines and evaluated its significance and prognostic use in renal cell carcinoma (RCC). Then we evaluated the change of cell migration, invation and adhesion in RCC cell with downregulation of Rictor. METHODS: Expression of Rictor was examined through immunohistochemistry in a tissue microarray constructed from 217 renal cancer patients to assess the correlation between Rictor expression level and RCC progression. We subsequently analyzed Rictor expression levels in several RCC cell lines through western blotting. Rictor expression in ACHN cell line was down-regulated by SiRNA and the change of cell migration, invision and adhesion wan examined. RESULTS: Rictor positive staining was noted in 74% of RCC patients with 86% in CCRCC and 62 % in PRCC respectively, whereas staining of Rictor expression was remarkably weak in most normal tubules.(FigA). Further statistical analysis showed that Rictor expression was detected in 24 out of 31 long metastasis cases (p 0.000) and in 10 out of 14 lymph node metastasis cases (p 0.003) (Table1). Rictor expression is significantly higher in RCC cell lines with high metastatic potential (ACHN°¢SN12C°¢HTB47) than in primary RCC cell lines(786-O and 769-P )(Fig2A). The Rictor expression level was significantly higher in cases with a symptomatic presentation,as well as in higher TNM stage and higher Furhman grade compared with those with lower stage and lower grade. (Table1) Patients with positive Rictor expression showed lower probability of disease-free survival compared with patients with negative Rictor expression. Knockdown of Rictor by small interference RNA inhibits cancer cell migration and invision and impaired EGF-induced cell adhesion. CONCLUSIONS: The Rictor expression may correlate with cancer migration and metastasis, and the detection of expression levels provides useful prognostic information for cancer-specific survival in patients with RCC. Down-regulation of Rictor inhibits cancer cell migration and invision impaired EGF-induced cell adhesion,so Rictor is a potential target for the treatment of RCC metastasis.

Retinal Pigment Epithelium Cell Alignment on Nanostructured Collagen Matrices

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α₂ were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α₂ was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α₂ expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α₂-mediated matrix binding was verified by preincubation with an α₂-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers.

Controlling Fibroblast Adhesion with Ph Modified Polyelectrolyte Multilayers

Tissue cells need to adhere to a biomaterial surface to promote their growth and differentiation and, thus, foster the integration of implants. As a result, surface features and their modification play an important role in biomedical applications. In this study, the layer-by-layer (LbL) technique was used to design self-assembled polyelectrolyte multilayer (PEM) coatings of polyethyleneimine (PEI) and heparin (HEP) on glass, which will control the adhesion of primary human dermal fibroblasts in a model system. The study showed that, among other surface features, the wettability of surfaces can be controlled by changing the conditions during multilayer self-assembly. Here, the pH value of the HEP solution was adjusted to acidic or alkaline values for terminal layers, which also led to a change in multilayer growth. Further, the study revealed that plain terminal layers were rather cytophobic. Upon pre-adsorption of fibronectin (FN), a clear effect on cell adhesion and morphology in dependence on the pH setup was evident. Proliferation studies clearly showed that terminal layers, which impaired cell adhesion, also inhibited growth of human fibroblasts under serum-conditions. On the other hand, on layers with pronounced cell adhesion an elevated cell growth was also observed. As a result, HEP terminated multilayers are interesting for applications requiring cell repellent properties, whereas PEI terminated multilayers could be used to promote cell adhesion and growth on implant surfaces.

α-Actinin-4 Is Essential for Maintaining the Spreading, Motility and Contractility of Fibroblasts

Backgroundα-Actinins cross-link actin filaments, with this cross-linking activity regulating the formation of focal adhesions, intracellular tension, and cell migration. Most non-muscle cells such as fibroblasts express two isoforms, α-actinin-1 (ACTN1) and α-actinin-4 (ACTN4). The high homology between these two isoforms would suggest redundancy of their function, but recent studies have suggested different regulatory roles. Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin.Methodology/Principal FindingsUsing molecular, biochemical and cellular techniques, we probed the cellular functions of ACTN4 in fibroblasts. Knockdown of ACTN4 expression in murine lung fibroblasts significantly impaired cell migration, spreading, adhesion, and proliferation. Surprisingly, knockdown of ACTN4 enhanced cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. These results, except the increased contractility, are consistent with a putative role of ACTN4 in cytokinesis. For the transcellular tension, knockdown of ACTN4 significantly increased the expression of myosin light chain 2, a element of the contractility machinery. Re-expression of wild type human ACTN4 in ACTN4 knockdown murine lung fibroblasts reverted cell spreading, cellular and nuclear cross-sectional area, and contractility back towards baseline, demonstrating that the defect was due to absence of ACTN4.SignificanceThese results suggest that ACTN4 is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion.

Kindlin‐2 is expressed in malignant mesothelioma and is required for tumor cell adhesion and migration

Kindlin-2 is a novel integrin-interacting focal adhesion protein that belongs to the Kindlin family. Focal adhesion proteins control cytoskeleton dynamics and promote cancer cell growth, survival, migration and metastasis. Little is known, however, about expression of Kindlin-2 in association with human cancer. We now reveal high Kindlin-2 expression in malignant mesothelioma (MM) cell lines using an affinity-purified anti-Kindlin-2 antibody. Furthermore, we show by immunohistochemistry that Kindlin-2 is highly expressed in 92 of 102 (90%) MMs with epitheliod; sarcomatoid, biphasic and poorly differentiated morphologies. In addition, Kindlin-2 expression correlates to cell proliferation, suggesting a role for Kindlin-2 in tumor growth. We also detect increased expression of Kindlin-2 at the invasion front of tumors concurrent with increased expression of integrin-linked kinase, a Kindlin-binding protein. Besides the high expression of Kindlin-2 in pleural MMs, pleural metastases of lung adenocarcinoma also express large amounts of Kindlin-2, but not Kindlin-1. Notably, in vitro, when endogenous Kindlin-2 was knocked down with RNAi in MM cells, this impaired cell spreading, adhesion and migration. Overall, our study suggests that heightened expression of Kindlin-2 might contribute to tumor progression in MM.

Angiotensin II Type 1a–Deficient Bone Marrow–Derived Dendritic Cells Produce Higher Levels of Monocyte Chemoattractant Protein 1

To the Editor: We read with interest the article from Crowley et al,1 who studied the role of angiotensin II type 1 (AT1) receptors (AT1aR) on immune cells in the pathogenesis of angiotensin II–induced hypertension by generating bone marrow chimeras with wild-type (WT) donors or donors lacking AT1aR. Interestingly, they found that the group of donors lacking AT1aR had more albuminuria and higher expression of a number of inflammatory mediators, including monocyte chemoattractant protein 1 (MCP-1), with persistent infiltration of macrophages in the kidney, concluding that AT1aR on bone marrow–derived cells had protective actions. The absence of a functional renin-angiotensin system, like in mice genetically lacking renin, angiotensinogen, angiotensin-converting enzyme, or AT1aR, is associated with microvascular disease and tubulointerstitial inflammation. Ouyang et al2 described that AT1a knockout (KO) mice spontaneously develop glomerular and tubulointerstitial diseases. They observed increased …

A Profile of Differentially Abundant Proteins at the Yeast Cell Periphery during Pseudohyphal Growth

Yeast filamentous growth is a stress response to conditions of nitrogen deprivation, wherein yeast colonies form pseudohyphal filaments of elongated and connected cells. As proteins mediating adhesion and transport are required for this growth transition, we expect that the protein complement at the yeast cell periphery plays a critical and tightly regulated role in pseudohyphal filamentation. To identify proteins differentially abundant at the yeast cell periphery during pseudohyphal growth, we generated quantitative proteomic profiles of plasma membrane protein preparations under conditions of vegetative growth and filamentation. By isobaric tags for relative and absolute quantification chemistry and two-dimensional liquid chromatography-tandem mass spectrometry, we profiled 2463 peptides and 356 proteins, identifying 11 differentially abundant proteins that localize to the yeast cell periphery. This protein set includes Ylr414cp, herein renamed Pun1p, a previously uncharacterized protein localized to the plasma membrane compartment of Can1. Pun1p abundance is doubled under conditions of nitrogen stress, and deletion of PUN1 abolishes filamentous growth in haploids and diploids; pun1Delta mutants are noninvasive, lack surface-spread filamentation, grow slowly, and exhibit impaired cell adhesion. Conversely, overexpression of PUN1 results in exaggerated cell elongation under conditions of nitrogen stress. PUN1 contributes to yeast nitrogen signaling, as pun1Delta mutants misregulate amino acid biosynthetic genes during nitrogen stress. By chromatin immunoprecipitation and reverse transcription-PCR, we find that the filamentous growth factor Mss11p directly binds the PUN1 promoter and regulates its transcription. In total, this study provides the first profile of differential protein abundance during pseudohyphal growth, identifying a previously uncharacterized membrane compartment of Can1 protein required for wild-type nitrogen signaling and filamentous growth.