Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules

Influenza A virus pneumonia is characterized by severe lung injury and high mortality. Early infection elicits a strong recruitment of monocytes from the peripheral blood across the endo-/epithelial barrier into the alveolar air space. However, it is currently unclear which of the infected resident lung cell populations, alveolar epithelial cells or alveolar macrophages, elicit monocyte recruitment during influenza A virus infection. In the current study, we investigated whether influenza A virus infection of primary alveolar epithelial cells and resident alveolar macrophages would elicit a basal-to-apical monocyte transepithelial migration in vitro. We found that infection of alveolar epithelial cells with the mouse-adapted influenza A virus strain PR/8 strongly induced the release of monocyte chemoattractants CCL2 and CCL5 followed by a strong monocyte transepithelial migration, and this monocytic response was strictly dependent on monocyte CCR2 but not CCR5 chemokine receptor expression. Analysis of the adhesion molecule pathways demonstrated a role of ICAM-1, VCAM-1, integrin-associated protein (CD47), and junctional adhesion molecule-c on the epithelial cell surface interacting with monocyte beta(1) and beta(2) integrins and integrin-associated protein in the monocyte transmigration process. Importantly, addition of influenza A virus-infected alveolar macrophages further enhanced monocyte transmigration across virus-infected epithelium in a TNF-alpha-dependent manner. Collectively, the data show an active role for virus-infected alveolar epithelium in the regulation of CCL2/CCR2-dependent monocyte transepithelial migration during influenza infection that is essentially dependent on both classical beta(1) and beta(2) integrins but also junctional adhesion molecule pathways.     

Mechanical stretch decreases migration of alveolar epithelial cells through mechanisms involving Rac1 and Tiam1

Mechanical ventilation can overdistend the lungs or generate shear forces in them during repetitive opening/closing, contributing to lung injury and inflammation in patients with acute respiratory distress syndrome (ARDS). Repair of the injured lung epithelium is important for restoring normal barrier and lung function. In the current study, we investigated the effects of cyclic mechanical strain (CS), constant distention strain (CD), and simulated positive end-expiratory pressure (PEEP) on activation of Rac1 and wound closure of rat primary alveolar type 2 (AT2) cells. Cyclic stretch inhibited the migration of wounded AT2 cells in a dose-dependent manner with no inhibition occurring with 5% CS, but significant inhibition with 10% and 15% CS. PEEP conditions were investigated by stretching AT2 cells to 15% maximum strain (at a frequency of 10 cycles/min) with relaxation to 10% strain. AT2 cells were also exposed to 20% CD. All three types of mechanical strain inhibited wound closure of AT2 cells compared with static controls. Since lamellipodial extensions in migrating cells at the wound edge were significantly smaller in stretched cells, we measured Rac1 activity and found it to be decreased in stretched cells. We also demonstrate that Tiam1, a Rac1-specific guanine nucleotide exchange factor, was expressed mainly in the cytosol of AT2 cells exposed to mechanical strain compared with membrane localization in static cells. Downregulation of Tiam1 with 100 microM NSC-23766 inhibited activation of Rac1 and migration of AT2 cells, suggesting its involvement in repair mechanisms of AT2 cells subjected to mechanical strain.     

The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.     

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments.     

CRTAM: A molecule involved in epithelial cell adhesion

Class I‐restricted T cell associated molecule (CRTAM) is a member of the immunoglobulin superfamily that complies with the structural characteristics of the JAM family of proteins and is phylogenetically more closely related to nectin‐like proteins. Here we demonstrate for the first time, that CRTAM is expressed in epithelial cells along the lateral membrane and is important for early cell–cell contacts and cell–substrate interactions. CRTAM is sensitive to intermediate filament disruption and treatment of monolayers with soluble CRTAM enhances cell–cell dissociation and lowers transepithelial electrical resistance. Incubation of newly plated cells with anti‐CRTAM antibody decreases the formation of cell aggregates and promotes cell detachment. Co‐cultures of epithelial cells and fibroblasts that lack CRTAM expression and in vitro binding assays, demonstrate the participation of CRTAM in homotypic and heterotypic trans‐interactions. Hence we conclude that CRTAM is a molecule involved in epithelial cell adhesion. J. Cell. Biochem. 111: 111–122, 2010. © 2010 Wiley‐Liss, Inc.     

Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors

Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.     

Cdk5: A regulator of epithelial cell adhesion and migration

Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.Key words: Cdk5, Src, Rho, stress fibers, epithelial cells, cell adhesion, cell migrationAnchoring of epithelial cells to their basement membrane is essential to maintain their morphology, normal physiological function and survival. Cells attach to extracellular matrix components by means of membrane-spanning integrins, which cluster and link to the actin cytoskeleton via components of focal adhesions. At focal adhesions, actin is bundled into stress fibers, multi-protein cellular contractile machines that strengthen attachment and provide traction during migration.¹ Stress fiber contraction is generated by myosin II, a hexamer containing one pair of each non-muscle heavy chains (NMHCs), essential light chains, and myosin regulatory light chains (MRLC). Myosin motor activity is regulated by phosphorylation of MRLC at Thr18/Ser19 and is required to generate tension on actin filaments and to maintain stress fibers.¹ Although a number of kinases have been identified which phosphorylate MRLC at Thr18/Ser19, the principal kinases in most cells are myosin light chain kinase (MLCK)² and Rho-kinase (ROCK),³ a downstream effector of the small GTPas, RhoA.Rho family small GTPases play a central role in regulating many aspects of cytoskeletal organization and contraction.⁴ These GTPases are subject to both positive regulation by guanine nucleotide exchange factors (GEFs), such as GEF-H1,^5,6 and negative regulation by GTPase-activating proteins (GAPs), such as p190RhoGAP.⁷ As cells spread, the Rho-family GTPase, Cdc42, is activated at the cell periphery, leading to the formation of numerous filapodia. Focal adhesion formation is first seen at the tips of these filapodia as focal adhesion proteins such as talin and focal adhesion kinase (FAK) bind to the intracellular domains of localized integrins.⁸ Src is recruited to activated FAK at the nascent focal adhesion and generates binding sites for additional focal adhesion proteins by phosphorylating FAK and paxillin.⁹ Src activity is essential for the further maturation of the focal adhesion and for activating the Rho-family GTPase, Rac, leading to Arp2/3-dependent actin polymerization, formation of a lamellipodium and extension of the cell boundary. Simultaneously, Src inhibits RhoA by phosphorylating and activating its upstream inhibitor, p190RhoGAP. As the focal adhesion matures, Src is deactivated, allowing the Rho activation necessary for mDia-dependent actin polymerization,¹⁰ myosin-dependent cytoskeletal contraction⁵ and tight adhesion to the extracellular matrix. Since new focal adhesions continually form at the distal boundary of the spreading cell, the most mature and highly contracted stress fibers are localized at the center of the cell.Cell adhesion is an essential component of cell migration: if adhesion is too weak, cells can not generate the traction necessary for migration; if it is too strong, they are unable to overcome the forces that anchor them in place. Thus, the relationship between adhesion force and migration rate is a bell-shaped curve.¹¹ Migration rate increases as adhesive strength increases until an optimum value is reached. Thereafter, increases in adhesive strength decrease migration rate. Since the strength of adhesion depends on extracellular matrix composition as well as the types of integrin expressed in the cell, a decrease in adhesive strength may result in either faster or slower cell migration.Several lines of evidence indicate that the proline-directed serine/threonine kinase cyclin dependent kinase 5 (Cdk5) plays an integral role in regulating cell adhesion and/or migration in epithelial cells.^12–17 Cdk5 is an atypical member of cyclin dependent kinase family, which is activated by the non-cyclin proteins, p35 or p39.¹⁸ Cdk5 is most abundant in neuronal cells where it also regulates migration and cytoskeletal dynamics.¹⁹ In neurons, Cdk5 exerts its effects on migration at least in part by phosphorylating FAK,¹⁹ and the LIS1 associated protein, NDEL1.²⁰ In contrast, recent findings have revealed two novel pathways involved in Cdk5-dependent regulation of migration in epithelial cells.^16,17One of these newly discovered mechanisms links Cdk5 activation to control of stress fiber contraction.¹⁶ We have found that Cdk5 and its activator, p35, co-localize with phosphorylated myosin regulatory light chain (MRLC) on centrally located stress fibers in spreading cells.¹⁶ Moreover, Cdk5 is strongly activated in spreading cells as central stress fiber contraction becomes pronounced.²¹ Since contraction of these central stress fibers is primarily responsible for tight attachment between the cell and the extracellular matrix,⁵ the above findings suggested that Cdk5 might regulate cell adhesion by regulating MRLC phosphorylation. To test this possibility we inhibited Cdk5 activity by several independent means and found that MRLC phosphorylation was likewise inhibited. In addition, we found that inhibiting Cdk5 either prevented the formation of central stress fibers or led to their dissolution. The concave cell boundaries characteristic of contracting cells were also lost.¹⁶ Since MRLC lacks a favorable site for phosphorylation by Cdk5, we asked whether Cdk5 might affect the upstream signaling pathways that regulate MRLC phosphorylation. Experiments with specific pathway inhibitors indicated that the MRLC phosphorylation involved in stress fiber contraction in lens epithelial cells was regulated largely by Rho-kinase (ROCK). Inhibiting Cdk5 activity not only significantly reduced ROCK activity, but also blocked activation of its upstream regulator, Rho. To explore the mechanism behind the Cdk5-dependent regulation of Rho, we turned our attention to p190RhoGAP, which appears to play a major role in regulating Rho-dependent stress fiber contraction.⁷ This RhoGAP must be phosphorylated by Src to be active; as a result, Rho activity is low in the early stages of cell spreading, when Src activity is high. At later times, Src activity falls, p190RhoGAP activity is lost, and Rho-GTP is formed, enabling Rho-dependent myosin phosphorylation and stress fiber contraction.^9,10 We have found that inhibiting Cdk5 activity during this later stage of cell spreading increases Src activity and Src-dependent phosphorylation of its substrate, p190RhoGAP. This in turn leads to decreased Rho activity accompanied by loss of Rho-dependent myosin phosphorylation, dissolution of central stress fibers, and loss of cell contraction (Fig. 1). Moreover, inhibiting Src protects cells from the loss of Rho activation and dissolution of central stress fibers produced by inhibiting Cdk5.¹⁶ Since the effects of Cdk5 on Rho-dependent cytoskeletal contraction appear to be mediated almost entirely through Cdk5-dependent regulation of Src, it will be particularly important to determine how Cdk5 limits Src activity.Open in a separate windowFigure 1Cdk5 inhibition reduces contraction of preformed stress fibers. (A) Cells were spread on fibronectin for 60 min to adhere, allowing them to form focal adhesion and stress fibers (pre-incubation) and then further incubated for 2 h in absence (control) or presence of Cdk5 inhibitor (olomoucine) and stained with phalloidin. The cells without olomoucine (control) had concave boundaries and well-formed stress fibers. Olomoucine treated cells showed loss of central stress fibers and failure to contract. Scale bar = 20 µ. (B) experimental conditions were same as shown in (A). Cdk5 inhibitor, olomoucine, was added after 1 h of spreading (indicated as t = 0) and cells were incubated for an additional 2h in absence or presence of olomoucine. Cell lysates were immunoblotted with antibodies for pMRLC (upper) and MRLC (middle). Tubulin was used as a loading control (lower). Lane 1: untreated (at 0 h); Lane 2: untreated (at 2 h); Lane 3: Cdk5 inhibitor (olomoucine) treated. (C) results of three independent experiments of the type shown in (B) were quantified by densitometry and normalized to determine the relative levels of pMRLC at each time. Statistical analysis demonstrated a significant (p < 0.05) decrease in pMRLC level in olomoucine treated cells compared to untreated cells.The central stress fibers regulated by Cdk5 play a central role in anchoring cells to the substratum, and their loss when Cdk5 is inhibited will reduce adhesion. As discussed above, reduction in cell adhesion may either increase or decrease the rate of cell migration, depending on the cell type and extracellular matrix composition. In lens and corneal epithelial cells, the reduction in adhesion produced by Cdk5 inhibition promotes cell migration.^13,15,16,22 Moreover, regulation of Rho/Rho-kinase signaling by Cdk5 seems to be a major factor in determining the migration rate, since inhibitors of Cdk5 and Rho-kinase increased lens epithelial cell migration rate equivalently and inhibiting both produced no additional effect.¹⁶Interestingly, an independent line of investigation has shown that this is not the only mechanism underlying Cdk5-dependent regulation of cell adhesion and migration. Cdk5 also localizes at focal contacts at the cell periphery and phosphorylates the focal adhesion protein talin.¹⁷ The talin phosphorylation site has been identified as S425, near the FERM domain in the talin head region. Upon focal adhesion disassembly, this region is separated from the talin rod domain by calpain-dependent cleavage.²³ Phosphorylation at S425 by Cdk5 blocks ubiquitylation and degradation of the talin head by inhibiting interaction with the E3 ligase, Smurf1. This leads, ultimately, to greater stability of lamellipodia and newly formed focal adhesions, thus strengthening adhesion to the substrate.¹⁷ Although the exact molecular events involved in this stabilization are not yet clear, it has been suggested that the talin head may “prime” integrins to bind full length talin.²⁴ One possible scenario describing how this might occur is shown in Figure 2. By permitting the isolated head region to escape degradation following calpain cleavage, Cdk5-dependent phosphorylation may stablize a pool of talin head domains to bind focal contacts within the lamellipodium. It is known that the isolated talin head region can bind and activate integrins during cell protrusion.²⁵ The resulting integrin activation would be expected to stabilize the lamellipodium by strengthening integrin-dependent adhesion. Since the head domain lacks sites for actin binding, which are located in the talin rod domain,²⁶ the bound head domain would have to be replaced by full length talin to enable focal adhesion attachment to the cytoskeleton.²⁵ The head domain might promote this replacement by recruiting the PIP-kinase needed to generate PI(4,5) P₂,²³ which facilitates binding of full length talin to integrin by exposing the auto-inhibited integrin binding sites.²⁷ The binding of full length talin and the resulting link between the integrins and the actin cytoskeleton would then further strengthen adhesion.²⁵ This model predicts that full length talin would bind poorly in the absence of Cdk5 activity, due to degradation of the talin head and the resulting limited availability of PI(4,5)P₂, and thus provides a possible explanation for the observed rapid turnover of peripheral focal adhesions.¹⁷ Clearly, other models may be proposed to explain the increase in adhesion produced by talin head phosphorylation, and deciding among them will be an active area for future investigation. Nonetheless, it is now certain that talin is a key substrate for Cdk5 at focal adhesions.Open in a separate windowFigure 2Mechanism of Cdk5-dependent regulation of cell adhesion and migration. Binding of p35 to Cdk5 forms the active Cdk5/p35 kinase, which regulates cell adhesion and migration in two distinct ways. Cdk5-dependent phosphorylation of the talin head domain at Ser425 prevents its ubiquitylation and degradation, allowing it to persist following calpain cleavage. The phosphorylated talin head may then bind to integrin at peripheral sites and recruit PIP-K, which converts PI(4)P to PI(4,5)P₂. PI(4,5)P₂ may promote replacement of the talin head by full length talin. Full length talin recruits other focal adhesion proteins to form the mature focal adhesion. The talin tail provides the site for the actin binding and polymerization. Polymerized actin is subsequently bundled into stress fibers. Cdk5/p35 also regulates the Rho-dependent myosin phosphorylation necessary for stress fiber stability and cytoskeletal contraction by limiting Src activity. This in turn decreases Src-dependent phosphorylation of p190RhoGAP, favoring Rho-GTP formation, Rho-dependent stress fiber polymerization, stabilization and contraction. Both pathways modulate cell migration by increasing adhesive strength.In summary, the presently available data indicate that Cdk5 has at least two distinct functions in cell adhesion (Fig. 2). On the one hand, it stabilizes peripheral focal adhesions and promotes their attachment to the cytoskeleton by phosphorylating the talin head. On the other hand, once the actin cytoskeleton has been organized into stress fibers, Cdk5 enhances the Rho activation essential for stability and contraction of central stress fibers by limiting Src activity. The discovery that Cdk5 is involved in two separate events required for efficient migration, suggests that it may coordinate multiple signaling pathways. The known involvement of Cdk5 and its activator, p35, in regulating microtubule stability suggests yet another mechanism by which Cdk5 activity may regulate cytoskeletal function. Microtubules are closely associated with stress fibers²⁸ and their depolymerization has been shown to release the Rho activating protein, GEF-H1, leading to Rho activation and Rho-dependent myosin contraction.⁶ Since cell adhesion and migration play an important role in the progression of many pathological conditions, Cdk5, its substrates and its downstream effectors involved in cell adhesion may provide novel targets for therapeutic intervention.^15,29     

Entry of viruses through the epithelial barrier: pathogenic trickery

Mucosal surfaces--such as the lining of the gut or the reproductive tract--are the main point of entry for viruses into the body. As such, almost all viruses interact with epithelial cells, and make use of the normal epithelial signalling and trafficking pathways of the host cell. In addition to protein receptors, carbohydrate chains of proteoglycans and epithelial-membrane glycosphingolipids have emerged as a new class of receptors for viral attachment to the host cell.     

Monocyte attachment and migration through collagen IV in diabetes mellitus

The interactions between monocytes and extracellular matrix proteins have been implicated in atherosclerosis pathophysiology. In the present study we evaluated monocyte attachment and migration through oxidized and non-oxidized collagen IV. Monocyte attachment was tested on microwells coated with either native or oxidized collagen IV. Monocyte migration through collagen IV was examined on transwells. Monocytes derived from patients with diabetes mellitus showed an increased ability to attach and migrate through collagen IV as compared to those derived from healthy volunteers. Moreover, control monocytes attached to oxidized collagen at a higher degree, while they migrated through oxidized collagen at a lower degree, as compared to the native protein. Our results also showed the involvement of the alpha2 integrin subunit in the above phenomena suggesting a modified interaction between monocytes and collagen IV in diabetes mellitus.     

Integrin activation by chemokines: relevance to inflammatory adhesion cascade during T cell migration

The adhesive function of integrins is regulated through cytoplasmic signaling induced by several stimuli, whose process is designated "inside-out signaling". A large number of leukocytes are rapidly recruited to the sites of inflammation where they form an essential component of the response to infection, injury, autoimmune disorders, allergy, tumor invasion, atherosclerosis and so on. The recruitment of leukocytes into tissue is regulated by a sequence of interactions between the circulating leukocytes and the endothelial cells. Leukocyte integrins play a pivotal role in leukocyte adhesion to endothelial cells. During the process, the activation of integrins by various chemoattractants, especially chemokines, is essential for integrin-mediated adhesion in which a signal transduced to the leukocyte converts the functionally inactive integrin to an active adhesive configuration. We have proposed that H-Ras-sensitive activation of phosphoinositide 3 (PI 3)-kinase and subsequent profilin-mediated actin polymerization, can be involved in chemokine-induced integrin-dependent adhesion of T cells. The present review documents the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemoattractants including chemokines during inflammatory processes. In contrast, various adhesion molecules are known to transduce extracellular information into cytoplasm, which leads to T cell activation and cytokine production from the cells, designated "outside-in signaling". Such a bi-directional "cross-talking" among adhesion molecules and cytokines is most relevant to inflammatory processes by augmenting immune cell migration from circulation into inflamed tissue such as rheumatoid arthritis, tumor invasion, Beh?et's disease and atherosclerosis.     

CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.     

Ep-CAM: a human epithelial antigen is a homophilic cell-cell adhesion molecule

The epithelial glycoprotein 40 (EGP40, also known as GA733-2, ESA, KSA, and the 17-1A antigen), encoded by the GA-733-2 gene, is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates, in a Ca(2+)- independent manner, a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations, in particular, causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs), although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate.     

Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms

Background

Mesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. Furthermore, MSC can ameliorate pulmonary fibrosis in animal models although mechanisms of action remain unclear. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration.

Methods

To investigate the paracrine role of human MSC (hMSC) on pulmonary epithelial repair, hMSC-conditioned media (CM) and a selected cohort of hMSC-secretory proteins (identified by LC-MS/MS mass spectrometry) were tested on human type II alveolar epithelial cell line A549 cells (AEC) and primary human small airway epithelial cells (SAEC) using an in vitro scratch wound repair model. A 3D direct-contact wound repair model was further developed to assess the migratory properties of hMSC.

Results

We demonstrate that MSC-CM facilitates AEC and SAEC wound repair in serum-dependent and –independent manners respectively via stimulation of cell migration. We also show that the hMSC secretome contains an array of proteins including Fibronectin, Lumican, Periostin, and IGFBP-7; each capable of influencing AEC and SAEC migration and wound repair stimulation. In addition, hMSC also show a strong migratory response to AEC injury as, supported by the observation of rapid and effective AEC wound gap closure by hMSC in the 3D model.

Conclusion

These findings support the notion for clinical application of hMSCs and/or their secretory factors as a pharmacoregenerative modality for the treatment of idiopathic pulmonary fibrosis (IPF) and other fibrotic lung disorders.     

Polysialylation of the neural cell adhesion molecule: interfering with polysialylation and migration in neuroblastoma cells

Polysialic acid represents a unique posttranslational modification of the neural cell adhesion molecule (NCAM). It is built as a homopolymer of up to 150 molecules of alpha 2-8-linked sialic acids on N-glycans of the fifth immunoglobulin-like domain of NCAM. Besides its role in cell migration and axonal growth during development, polysialic acids are closely related to tumor malignancy as they are linked to the malignant potential of several tumors, such as undifferentiated neuroblastoma. Polysialic acid expression is significantly more frequent in high-grade tumors than in low-grade tumors. It is synthesized in the Golgi apparatus by the activity of two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV. Interestingly, polysialylation of tumors is not equally synthesized by both polysialyltransferases. It has been shown that especially the ST8SiaII gene is not expressed in some normal tissue, but is strongly expressed in tumor tissue. Here we summarize some knowledge on the role of polysialic acid in cell migration and tumor progression and present novel evidence that interfering with polysialylation using unnatural sialic acid precursors decreases the migration of neuroblastoma cells.     

Adducins inhibit lung cancer cell migration through mechanisms involving regulation of cell-matrix adhesion and cadherin-11 expression

Cell migration is a critical mechanism controlling tissue morphogenesis, epithelial wound healing and tumor metastasis. Migrating cells depend on orchestrated remodeling of the plasma membrane and the underlying actin cytoskeleton, which is regulated by the spectrin-adducin-based membrane skeleton. Expression of adducins is altered during tumorigenesis, however, their involvement in metastatic dissemination of tumor cells remains poorly characterized. This study investigated the roles of α-adducin (ADD1) and γ-adducin (ADD3) in regulating migration and invasion of non-small cell lung cancer (NSCLC) cells. ADD1 was mislocalized, whereas ADD3 was markedly downregulated in NSCLC cells with the invasive mesenchymal phenotype. CRISPR/Cas9-mediated knockout of ADD1 and ADD3 in epithelial-type NSCLC and normal bronchial epithelial cells promoted their Boyden chamber migration and Matrigel invasion. Furthermore, overexpression of ADD1, but not ADD3, in mesenchymal-type NSCLC cells decreased cell migration and invasion. ADD1-overexpressing NSCLC cells demonstrated increased adhesion to the extracellular matrix (ECM), accompanied by enhanced assembly of focal adhesions and hyperphosphorylation of Src and paxillin. The increased adhesiveness and decreased motility of ADD1-overexpressing cells were reversed by siRNA-mediated knockdown of Src. By contrast, the accelerated migration of ADD1 and ADD3-depleted NSCLC cells was ECM adhesion-independent and was driven by the upregulated expression of pro-motile cadherin-11. Overall, our findings reveal a novel function of adducins as negative regulators of NSCLC cell migration and invasion, which could be essential for limiting lung cancer progression and metastasis.     

Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes.

Monocytes play a critical role in defending the host against foreign organisms and in regulating the behavior of other cells. Monocytes circulate as nonadherent cells in the blood and migrate as adherent cells through tissues. Adhesion molecules mediate not only cell adhesion, but also migration, phagocytosis, and many other adhesion-dependent functions. Monocyte chemoattractant protein-1 (MCP-1) is thought to be responsible for monocyte recruitment in acute inflammatory conditions and may be an important mediator in chronic inflammation. In this study, immunofluorescence flow cytometry was used to determine whether MCP-1 can regulate the cell surface expression of adhesion molecules, particularly beta-2 and alpha-4 integrins and the leukocyte adhesion molecule-1. We found that MCP-1 induced expression of CD11c (p150,95 alpha-subunit) and CD11b (Mac-1 alpha-subunit), and caused little or no change of CD11a (lymphocyte function-associated Ag-1 alpha-subunit), very late activation Ag-4, or leukocyte adhesion molecule-1. We demonstrated that antibodies to beta-2 and alpha-4 integrins inhibited MCP-1-induced monocyte chemotaxis. We also showed that MCP-1 is capable of inducing IL-1 and IL-6, but not TNF production of monocytes. These results indicate that MCP-1 is not only a chemoattractant but also a novel cytokine with the capacity to regulate several parameters of monocyte function.     

Molecular mechanisms underlying adhesion and migration of hematopoietic stem cells

Hematopoietic stem cell transplantation is the most powerful treatment modality for a large number of hematopoietic malignancies, including leukemia. Successful hematopoietic recovery after transplantation depends on homing of hematopoietic stem cells to the bone marrow and subsequent lodging of those cells in specific niches in the bone marrow. Migration of hematopoietic stem cells to the bone marrow is a highly regulated process that requires correct regulation of the expression and activity of various molecules including chemoattractants, selectins and integrins. This review will discuss recent studies that have extended our understanding of the molecular mechanisms underlying adhesion, migration and bone marrow homing of hematopoietic stem cells.     

Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity

Epithelial tight junctions form a selectively permeable barrier to ions and small molecules. Junctional adhesion molecule 1 (JAM1/JAM-A/F11R) is a tight junction-associated transmembrane protein that has been shown to participate in the regulation of epithelial barrier function. In a recent study, we presented evidence suggesting that JAM1 homodimer formation is critical for epithelial barrier function (Mandell, K. J., McCall, I. C., and Parkos, C. A. (2004) J. Biol. Chem. 279, 16254-16262). Here we have used small interfering RNA to investigate the effect of the loss of JAM1 expression on epithelial cell function. Consistent with our previous study, knockdown of JAM1 was observed to increase paracellular permeability in epithelial monolayers. Interestingly, knockdown of JAM1 also produced dramatic changes in cell morphology, and a similar effect was observed with expression of a JAM1 mutant lacking the putative homodimer interface. Further studies revealed that JAM1 knockdown decreased cell-matrix adhesion and spreading on matrix proteins that are ligands of beta1 integrins. These changes were characterized by a decrease in beta1 integrin protein levels and loss of beta1 integrin staining at the cell surface. Immunolabeling of cells for the small GTPase Rap1, a known activator of beta1 integrins, revealed colocalization of Rap1 with JAM1 at intercellular junctions, and knockdown of JAM1 resulted in decreased Rap1 activity. Lastly, knockdown of Rap1b resulted in diminished beta1 integrin expression and altered cell morphology analogous to that observed with knockdown of JAM1. Together, these results suggest that JAM1 regulates epithelial cell morphology and beta1 integrin expression by modulating activity of the small GTPase Rap1.