Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single-cell tracking and traction force microscopy

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but instead a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion, and crossover during cell–cell and cell–matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo receptor complex and that their migration is blocked by myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over myelin. Our data relate the decrease of traction force of OEC with lower migratory capacity over myelin, which correlates with changes in the F-actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo receptor inhibitor NEP1-40.     

The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.     

The pleiotropic roles of ADAM9 in the biology of solid tumors

A disintegrin and a metalloprotease (ADAM) 9 is a metzincin cell-surface protease involved in several biological processes such as myogenesis, fertilization, cell migration, inflammatory response, proliferation, and cell–cell interactions. ADAM9 has been found over-expressed in several solid tumors entities such as glioma, melanoma, prostate cancer, pancreatic ductal adenocarcinoma, gastric, breast, lung, and liver cancers. Immunohistochemical analyses highlight ADAM9 expression by actual cancer cells and associate its abundant presence with clinicopathological features such as shortened overall survival, poor tumor grade, de-differentiation, therapy resistance, and metastasis formation. In each of these tumors, ADAM9 may contribute to tumor biology via proteolytic or non-proteolytic mechanisms. For example, in liver cancer, ADAM9 has been found to shed MHC class I polypeptide-related sequence A, contributing towards the evasion of tumor immunity. ADAM9 may also contribute to tumor biology in non-proteolytic ways probably through interaction with different integrins. For example, in melanoma, the interaction between ADAM9 and β1 integrins facilitates tumor stroma cross talks, which then promotes invasion and metastasis via the activation of MMP1 and MMP2. In breast cancer, the interaction between β1 integrins on endothelial cells and ADAM9 on tumor cells facilitate tumor cell extravasation and invasion to distant sites. This review summarizes the present knowledge on ADAM9 in solid cancers, and the different mechanisms which it employ to drive tumor progression.     

Cellular communications in bone homeostasis and repair

Cellular communication between the bone component cells osteoblasts, osteocytes and (pre-)osteoclasts is essential for bone remodeling which maintains bone integrity. As in the remodeling of other organs, cell death is a trigger for remodeling of bone. During the systematic process of bone remodeling, direct or indirect cell–cell communication is indispensable. Thus, osteoblasts induce migration and differentiation of preosteoclasts, which is followed by bone resorption (by mature multinuclear osteoclasts). After completion of bone resorption, apoptosis of mature osteoclasts and differentiation of osteoblasts are initiated. At this time, the osteoblasts do not support osteoclast differentiation but do support bone formation. Finally, osteoblasts differentiate to osteocytes in bone or to bone lining cells on bone surfaces. In this way, old bone areas are regenerated as new bone. In this review the role of cell–cell communication in bone remodeling is discussed.     

Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner

During mitosis, cells detach, and the cell–matrix interactions become restricted. At the completion of cytokinesis, the two daughter cells are still connected transiently by an intercellular bridge (ICB), which is subjected to abscission, as the terminal step of cytokinesis. Cell adhesion to the matrix is mediated by syndecan-4 (SDC4) transmembrane heparan sulfate proteoglycan. Our present work demonstrated that SDC4 promotes cytokinesis in a phosphorylation-dependent manner in MCF-7 breast adenocarcinoma cells. The serine179-phosphorylation and the ectodomain shedding of SDC4 changed periodically in a cell cycle-dependent way reaching the maximum at G2/M phases. On the contrary, the phospho-resistant Ser179Ala mutant abrogated the shedding. The phosphorylated full-length and shed remnants enriched along the mitotic spindles, and subsequently in the ICBs, however, proper membrane insertion was necessary for midbody localization. Expression of phosphomimicking Ser179Glu SDC4 resulted in incomplete abscission, whereas expression of the phospho-resistant SDC4 led to giant, multinucleated cells.     

Intra-brain microinjection of human mesenchymal stem cells decreases allodynia in neuropathic mice

Neuropathic pain is a very complex disease, involving several molecular pathways. Current available drugs are usually not acting on the several mechanisms underlying the generation and propagation of pain. We used spared nerve injury model of neuropathic pain to assess the possible use of human mesenchymal stem cells (hMSCs) as anti-neuropathic tool. Human MSCs were transplanted in the mouse lateral cerebral ventricle. Stem cells injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once transplanted in cerebral ventricle. Anti-nociceptive effect was detectable from day 10 after surgery (6 days post cell injection). Human MSCs reduced the mRNA levels of the pro-inflammatory interleukin IL-1β mouse gene, as well as the neural β-galactosidase over-activation in prefrontal cortex of SNI mice. Transplanted hMSCs were able to reduce astrocytic and microglial cell activation.     

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-β exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-β^? MSCs. Notably, PDGFR-β⁺ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 10⁶ GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-β⁺ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 10⁵ remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-β^? MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 10⁵) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-β⁺ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-β⁺ MSCs migrated eightfold more than PDGFR-β^? MSCs toward PDGF-BB. Intriguingly, PDGFR-β⁺ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-β^? MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-β identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.     

Megasatellites: a new class of large tandem repeats discovered in the pathogenic yeast Candida glabrata

Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell–cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.     

Functions of skin-resident γδ T cells

The murine epidermis contains resident T cells that express a canonical γδ TCR and arise from fetal thymic precursors. These cells are termed dendritic epidermal T cells (DETC) and use a TCR that is restricted to the skin in adult animals. DETC produce low levels of cytokines and growth factors that contribute to epidermal homeostasis. Upon activation, DETC can secrete large amounts of inflammatory molecules which participate in the communication between DETC, neighboring keratinocytes and langerhans cells. Chemokines produced by DETC may recruit inflammatory cells to the epidermis. In addition, cell–cell mediated immune responses also appear important for epidermal–T cell communication. Information is provided which supports a crucial role for DETC in inflammation, wound healing, and tumor surveillance.     

Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.     

Genes involved in breast cancer metastasis to bone

Metastasis to bone occurs frequently in advanced breast cancer and is accompanied by debilitating skeletal complications. Current treatments are palliative and new therapies that specifically prevent the spread of breast cancer to bone are urgently required. While our understanding of interactions between breast cancer cells and bone cells has greatly improved, we still know little about the molecular determinants that regulate specific homing of breast cancer cells to the bone. In this review, we focus on genes that have been implicated in migration and adhesion of breast cancer cells to bone, as well as genes that promote tumor cell proliferation in the bone microenvironment. In addition, the review discusses new technologies, including better animal models, that will further assist with the identification of the molecular determinants of bone metastasis and will guide the development of new therapies. Received 25 January 2002; received after revision 27 March 2002; accepted 5 April 2002 RID="*" ID="*"Corresponding author.     

GSK-3β is essential for physiological electric field-directed Golgi polarization and optimal electrotaxis

Endogenous electrical fields (EFs) at corneal and skin wounds send a powerful signal that directs cell migration during wound healing. This signal therefore may serve as a fundamental regulator directing cell polarization and migration. Very little is known of the intracellular and molecular mechanisms that mediate EF-induced cell polarization and migration. Here, we report that Chinese hamster ovary (CHO) cells show robust directional polarization and migration in a physiological EF (0.3–1 V/cm) in both dissociated cell culture and monolayer culture. An EF of 0.6 V/cm completely abolished cell migration into wounds in monolayer culture. An EF of higher strength (≥1 V/cm) is an overriding guidance cue for cell migration. Application of EF induced quick phosphorylation of glycogen synthase kinase 3β (GSK-3β) which reached a peak as early as 3 min in an EF. Inhibition of protein kinase C (PKC) significantly reduced EF-induced directedness of cell migration initially (in 1–2 h). Inhibition of GSK-3β completely abolished EF-induced GA polarization and significantly inhibited the directional cell migration, but at a later time (2–3 h in an EF). Those results suggest that GSK-3β is essential for physiological EF-induced Golgi apparatus (GA) polarization and optimal electrotactic cell migration.     

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM–ALCAM) or heterophilic (ALCAM–CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.     

E-cadherin plays an essential role in collective directional migration of large epithelial sheets

In wound healing and development, large epithelial sheets migrate collectively, in defined directions, and maintain tight cell-cell adhesion. This type of movement ensures an essential function of epithelia, a barrier, which is lost when cells lose connection and move in isolation. Unless wounded, epithelial sheets in cultures normally do not have overall directional migration. Cell migration is mostly studied when cells are in isolation and in the absence of mature cell-cell adhesion; the mechanisms of the migration of epithelial sheets are less well understood. We used small electric fields (EFs) as a directional cue to instigate and guide migration of epithelial sheets. Significantly, cells in monolayer migrated far more efficiently and directionally than cells in isolation or smaller cell clusters. We demonstrated for the first time the group size-dependent directional migratory response in several types of epithelial cells. Gap junctions made a minimal contribution to the directional collective migration. Breaking down calcium-dependent cell-cell adhesion significantly reduced directional sheet migration. Furthermore, E-cadherin blocking antibodies abolished migration of cell sheets. Traction force analysis revealed an important role of forces that cells in the leading rows exert on the substratum. With EF, the traction forces of the leading edge cells coordinated in directional re-orientation. Our study thus identifies a novel mechanism--E-cadherin dependence and coordinated traction forces of leading cells in collective directional migration of large epithelial sheets.     

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study

In the past, clinical trials transplanting bone marrow–derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit⁺ CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit⁺ CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.     

The multifaceted role of periostin in tumorigenesis

Periostin, also called osteoblast-specific factor 2 (OSF-2), is a member of the fasciclin family and a disulfide-linked cell adhesion protein that has been shown to be expressed preferentially in the periosteum and periodontal ligaments, where it acts as a critical regulator of bone and tooth formation and maintenance. Furthermore, periostin plays an important role in cardiac development. Recent clinical evidence has also revealed that periostin is involved in the development of various tumors, such as breast, lung, colon, pancreatic, and ovarian cancers. Periostin interacts with multiple cell-surface receptors, most notably integrins, and signals mainly via the PI3-K/Akt and other pathways to promote cancer cell survival, epithelial–mesenchymal transition (EMT), invasion, and metastasis. In this review, aspects related to the function of periostin in tumorigenesis are summarized.     

Marrow mesenchymal stem cells transduced with TPO/FL genes as support for ex vivo expansion of hematopoietic stem/progenitor cells

A new marrow-derived mesenchymal stem cell (hMSC) line that could support expansion of hematopoietic stem/progenitor cells (HSPCs) was developed. Primary hMSCs were infected with retrovirus containing Flt-3 ligand and thrombopoietin genes. CD34+ cells from cord blood were expanded with primary hMSCs or transduced hMSCs. The expansion of total nucleated cells, CD34+ cells and mixed colonies containing erythroid and myeloid cells and megakaryocytes for 2 weeks coculture with transduced hMSCs was remarkably increased. The outputs of long-term culture-initiating cells for 2 and 4 weeks coculture with transduced hMSCs were also largely increased. The expansion rates of HSPCs with transduced hMSCs were unchanged for 6 weeks. In contrast, the expansion rates of HSPCs with primary hMSCs declined drastically through 6 weeks. SCID-repopulating cell expansion with transduced hMSCs for 4 weeks was significantly higher than that of uncultured CD34+ cells and HSPCs expanded with primary hMSCs. Received 21 June 2005; received after revision 30 July 2005; accepted 24 August 2005     

TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates,Notch activation and ADAM10 membrane compartmentalization

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aβ, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.