The coxsackie adenovirus receptor inhibits cancer cell migration

The coxsackie and adenovirus receptor (CAR) is a key factor in adenoviral cancer gene therapy. Reduced expression of CAR during progression of prostate and bladder cancer has been reported. In embryonic development and tissue differentiation, CAR is also differentially expressed. This study suggests a role of CAR expression in cell adhesion and cell motility of human cancer cells. Stable CAR-expressing clones from E-cadherin-deficient A2780 ovarian and CaSki cervical cancer cells with originally low and high CAR expression levels, respectively, were established. CAR reexpression in otherwise singularly growing A2780 parental cells resulted in formation of cell-cell contacts and aggregation in cell clusters. CAR overexpression in cell adhesion-forming CaSki cells did not result in morphological changes. Migration of the A2780 CAR clones was strongly reduced as characterized by using spread-off assays. Using migration chambers, formation of satellite colonies was reduced by 97% in CAR-expressing A2780 cell clones and by 23% in CAR-expressing CaSki cell clones. Parental A2780 and CaSki cells selected for high migratory ability by using migration chambers expressed endogenous CAR on lower levels associated with lower adenoviral transduction efficiency. Our data suggest CAR as a new inhibitory factor for cancer cell migration.     

The tumor suppressor CYLD regulates microtubule dynamics and plays a role in cell migration

The familial cylindromatosis tumor suppressor CYLD is known to contain three cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, which exist in a number of microtubule-binding proteins and are responsible for their association with microtubules. However, it remains elusive whether CYLD interacts with microtubules and, if so, whether the interaction is mediated by the CAP-Gly domains. In this study, our data demonstrate that CYLD associates with microtubules both in cells and in vitro, and the first CAP-Gly domain of CYLD is mainly responsible for the interaction. Knockdown of cellular CYLD expression dramatically delays microtubule regrowth after nocodazole washout, indicating an activity for CYLD in promoting microtubule assembly. Our data further demonstrate that CYLD enhances tubulin polymerization into microtubules by lowering the critical concentration for microtubule assembly. In addition, we have identified by wound healing assay a critical role for CYLD in mediating cell migration and found that its first CAP-Gly domain is required for this activity. Thus CYLD joins a growing list of CAP-Gly domain-containing proteins that regulate microtubule dynamics and function.     

Aquaporin-1 plays a key role in erythropoietin-induced endothelial cell migration

Water influx through aquaporin-1 (AQP-1) has been linked to the ability of different cell types to migrate, and therefore plays an important part in processes like metastasis and angiogenesis. Since the erythroid growth factor erythropoietin (Epo) is now recognized as an angiogenesis promoter, we investigated the participation of AQP-1 as a downstream effector of this cytokine in the migration of endothelial cells. Inhibition of AQP-1 with either mercury ions (Hg²⁺) or a specific siRNA led to an impaired migration of EA.hy926 endothelial cells exposed to Epo (wound-healing assays). Epo also induced the expression of AQP-1 at mRNA and protein levels, an effect which was dependent on the influx of extracellular calcium through L-type calcium channels as well as TRPC3 channels.The relationship between Epo and AQP-1 was further confirmed at shorter exposure times, as the cytokine was unable to trigger calcium influxes in cells where AQP-1 had previously been knocked down. Moreover, Epo promoted changes in the subcellular localization of AQP-1 as well as rearrangements in the actin cytoskeleton, which are consistent with a migratory phenotype. Worthy of note, carbamylated erythropoietin (cEpo), the non-erythropoietic and non-promigratory derivative of Epo, was incapable of AQP-1 modulation.The therapeutical implications of aquaporin targeting in angiogenesis-related diseases highlight the importance of the present results in the context of the relationship between AQP-1 and Epo.     

Angiopoietin 2 signaling plays a critical role in neural crest cell migration

Background

Collective neural crest cell migration is critical to the form and function of the vertebrate face and neck, distributing bone, cartilage, and nerve cells into peripheral targets that are intimately linked with head vasculature. The vasculature and neural crest structures are ultimately linked, but when and how these patterns develop in the early embryo are not well understood.

Results

Using in vivo imaging and sophisticated cell behavior analyses, we show that quail cranial neural crest and endothelial cells share common migratory paths, sort out in a dynamic multistep process, and display multiple types of motion. To better understand the underlying molecular signals, we examined the role of angiopoietin 2 (Ang2), which we found expressed in migrating cranial neural crest cells. Overexpression of Ang2 causes neural crest cells to be more exploratory as displayed by invasion of off-target locations, the widening of migratory streams into prohibitive zones, and differences in cell motility type. The enhanced exploratory phenotype correlates with increased phosphorylated focal adhesion kinase activity in migrating neural crest cells. In contrast, loss of Ang2 function reduces neural crest cell exploration. In both gain and loss of function of Ang2, we found disruptions to the timing and interplay between cranial neural crest and endothelial cells.

Conclusions

Together, these data demonstrate a role for Ang2 in maintaining collective cranial neural crest cell migration and suggest interdependence with endothelial cell migration during vertebrate head patterning.

     

The wnt receptor ryk plays a role in Mammalian planar cell polarity signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.     

Regulation of microtubules in cell migration

Directional cell migration is a fundamental process in all organisms that is stringently regulated during tissue development, chemotaxis and wound healing. Migrating cells have a polarized morphology with an asymmetrical distribution of signaling molecules and the cytoskeleton. Microtubules are indispensable for the directional migration of certain cells. Recent studies have shown that Rho family GTPases, which are key regulators of cell migration, affect microtubules, in addition to the actin cytoskeleton and adhesion. Rho family GTPases capture and stabilize microtubules through their effectors at the cell cortex, leading to a polarized microtubule array; in turn, microtubules modulate the activities of Rho family GTPases. In this article, we discuss how a polarized microtubule array is established and how microtubules facilitate cell migration.     

The PP2A-associated protein alpha4 plays a critical role in the regulation of cell spreading and migration

Compared with kinases, the role of protein phosphatases in regulating biological functions is less well understood. Here we show that alpha4, a non-catalytic subunit of the protein phosphatase 2A, plays a major role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblasts lacking alpha4 were impaired in their ability to spread and migrate compared with wild-type cells, whereas enforced expression of alpha4 promoted cell spreading and migration. These effects were not restricted to fibroblasts. Using a T cell-specific alpha4 transgenic mouse model, increased alpha4 expression was found to increase lymphocyte motility and chemotaxis. Elevated alpha4 expression results in an increase in the GTP-bound state of Rac1, and GTP-bound Rac1 was dramatically reduced in alpha4-deficient cells. A constitutively active mutant of Rac1 rescued the defects of cell spreading and migration caused by alpha4 deletion, while inhibition of Rac1 blocked the ability of alpha4 to promote cell migration. Together, these data define a novel role for the protein phosphatase 2A regulatory subunit alpha4 in the regulation of cell spreading and migration.     

The role of receptor/channel activity in neuronal cell migration

Confocal laser microscopy, in conjunction with carbocyanine dyes and calcium-sensitive fluorescent indicators, was used in slices and explant cultures of developing cerebellum to study cellular mechanisms underlying a motility of neuronal cell migration. The results indicate that a combination of voltage- and ligand-activated ion channels cooperatively regulates Ca²⁺ influx into the migrating cells. We suggest that molecules, present in the local cellular milieu, affect cell motility by activating specific ion channels and second messengers that influence polymerization of stiff and contractile cytoskeletal proteins. This early interaction between postmitotic neurons and surrounding cells controls the rate of their movements, sculpts their shapes, establishes their positions, and, therefore, indirectly determines their identities to prior formation of synaptic connections. © 1995 John Wiley Sons, Inc.     

The role of microtubules in guard cell function

Guard cells are able to sense a multitude of environmental signals and appropriately adjust the stomatal pore to regulate gas exchange in and out of the leaf. The role of the microtubule cytoskeleton during these stomatal movements has been debated. To help resolve this debate, in vivo stomatal aperture assays with different microtubule inhibitors were performed. We observed that guard cells expressing the microtubule-binding green fluorescent fusion protein (green fluorescent protein::microtubule binding domain) fail to open for all major environmental triggers of stomatal opening. Furthermore, guard cells treated with the anti-microtubule drugs, propyzamide, oryzalin, and trifluralin also failed to open under the same environmental conditions. The inhibitory conditions caused by green fluorescent protein::microtubule binding domain and these anti-microtubule drugs could be reversed using the proton pump activator, fusicoccin. Therefore, we conclude that microtubules are involved in an upstream event prior to the ionic fluxes leading to stomatal opening. In a mechanistic manner, evidence is presented to implicate a microtubule-associated protein in this putative microtubule-based signal transduction event.     

Hepatocyte growth factor plays a dual role in tendon-derived stem cell proliferation,migration, and differentiation

Heterotopic ossification is common in tendon healing after trauma, but the detailed mechanisms remain unknown. Tendon-derived stem cells (TDSCs) are a type of progenitor cell found in the tendon niche, and their incorrect differentiation after trauma may lead to tendon calcification. The expression of hepatocyte growth factor (HGF) presents drastic fluctuations in serum/tissue after trauma and was found to activate quiescent stellate cells and contribute to wound healing; however, its potential role in TDSCs remains elusive. In this study, TDSCs isolated from rats were cultured in media containing HGF with or without a signaling inhibitor, and the proliferation, migration, and differentiation ability of TDSCs were measured to determine the role and mechanism of HGF in TDSCs. We showed that HGF promotes TDSC proliferation and migration but inhibits TDSC osteogenic differentiation ability. HGF activated-HGF/c-Met, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling, which was positively correlated with TDSCs proliferation and migration but negatively related to TDSC osteogenic differentiation ability. The phosphorylation of Smad1/5/8 was also negatively related to HGF/c-Met, MAPK/ERK1/2, and PI3K/AKT signaling, which demonstrated that the inhibition of osteogenic differentiation was dependent on BMP/Smad1/5/8 signaling. Overall, we showed that HGF could promote TDSCs proliferation and migration and inhibit osteogenic differentiation in vitro, suggesting a potential role for HGF as a cytokine treatment of tendon trauma.     

Coxsackievirus and adenovirus receptor (CAR) binds immunoglobulins.

The coxsackievirus and adenovirus receptor protein (CAR) serves as the cell surface receptor for group B coxsackieviruses and most adenoviruses, but the physiological function and ligand for this protein remain to be described. An affinity column was constructed with the recombinant extracellular domain of the CAR (rECAR) to isolate potential ligands by affinity chromatography. Immunoglobulins G and M were consistently isolated from human sera passed through the column, suggesting that the CAR may be an immunoglobulin-binding protein. Further investigation revealed that the affinity-purified immunoglobulins bound to rECAR-coated immunoassay plates, and the peroxidase-labeled rECAR bound the immunoglobulins on ligand-overlay blots. The peroxidase-labeled rECAR was incorporated into immunoprecipitates formed between the affinity-purified immunoglobulins and rabbit antibodies against human immunoglobulins, but not into immunoprecipitates formed between mouse IgG and rabbit antibodies against mouse IgG. The CAR present in HeLa cell lysates also bound to the affinity-purified immunoglobulins on Immobilon membranes, showing that the association is not limited to the recombinant protein. These results demonstrate that the CAR binds IgG and IgM present in serum, and reveal a direct interaction between the coxsackievirus and adenovirus receptor and the immune system.     

Regulation of cell migration by dynamic microtubules

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.     

Dok-R plays a pivotal role in angiopoietin-1-dependent cell migration through recruitment and activation of Pak.

Tek/Tie-2 is an endothelial cell (EC)-specific receptor tyrosine kinase that plays a critical role in angiogenesis via its regulation by the angiopoietin family of growth factor ligands. Angiopoietin-1 (Ang1) can promote EC migration; however, the signaling mechanisms underlying this process remain elusive. Here we demonstrate that Dok-R/Dok-2 can associate with Tek in ECs following Ang1 stimulation, resulting in tyrosine phosphorylation of Dok-R and the subsequent recruitment of Nck and the p21-activating kinase (Pak/Pak1) to the activated receptor. Ang1-mediated migration is increased upon Dok-R overexpression and this requires a functional Nck binding site on Dok-R. Localization of this Dok-R-Nck-Pak complex to the activated Tek receptor at the cellular membrane is coincident with activation of Pak kinase. The ability of Dok-R to bind Nck is required for maximal activation of Pak and overexpression of Pak results in increased Ang1-mediated cell motility. Our study outlines a novel signaling pathway underlying Ang1-driven cell migration that involves Dok-R and its recruitment of Nck and the subsequent activation of Pak.     

Scavenger receptor BI plays a role in facilitating chylomicron metabolism

The function of scavenger receptor class B type I (SR-BI) in mediating the selective uptake of high density lipoprotein (HDL) cholesterol esters is well established. However, the potential role of SR-BI in chylomicron and chylomicron remnant metabolism is largely unknown. In the present investigation, we report that the cell association of 160 nm-sized triglyceride-rich chylomicron-like emulsion particles to freshly isolated hepatocytes from SR-BI-deficient mice is greatly reduced (>70%), as compared with wild-type littermate mice. Competition experiments show that the association of emulsion particles with isolated hepatocytes is efficiently competed for (>70%) by the well established SR-BI ligands, HDL and oxidized low density lipoprotein (LDL), whereas LDL is ineffective. Upon injection into SR-BI-deficient mice the hepatic association of emulsion particles is markedly decreased ( approximately 80%) as compared with wild-type mice. The relevance of these findings for in vivo chylomicron (remnant) metabolism was further evaluated by studying the effect of SR-BI deficiency on the intragastric fat load-induced postprandial triglyceride response. The postprandial triglyceride response is 2-fold higher in SR-BI-deficient mice as compared with wild-type littermates (area-under-the-curve 39.6 +/- 1.2 versus 21.1 +/- 3.6; p < 0.005), with a 4-fold increased accumulation of chylomicron (remnant)-associated triglycerides in plasma at 6 h after intragastric fat load. We conclude that SR-BI is important in facilitating chylomicron (remnant) metabolism and might function as an initial recognition site for chylomicron remnants whereby the subsequent internalization can be exerted by additional receptor systems like the LDL receptor and LDL receptor-related protein.     

The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM’s role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM–microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.     

Borrelia burgdorferi ftsZ plays a role in cell division

ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZ(Bbu)). Its gene product (FtsZ(Bbu)) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZ(Bbu) might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZ(Bbu) antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZ(Bbu) could interfere with the function of E. coli FtsZ, ftsZ(Bbu) was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZ(Bbu) generated a filamentous phenotype. This suggested interference of ftsZ(Bbu) with E. coli FtsZ function and confirmed the role of ftsZ(Bbu) in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.