RGD motif of lipoprotein T, involved in adhesion of Mycoplasma conjunctivae to lamb synovial tissue cells

Lipoprotein T (LppT), a membrane-located 105-kDa lipoprotein of Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC) of domestic sheep and wild Caprinae, was characterized. LppT was shown to promote cell attachment to LSM 192 primary lamb joint synovial cells. Adhesion of M. conjunctivae to LSM 192 cells is inhibited by antibodies directed against LppT. The RGD (Arg-Gly-Asp) motif of LppT was found to be a specific site for binding of M. conjunctivae to these eukaryotic host cells. Recombinant LppT fixed to polymethylmethacrylate slides binds LSM 192 cells, whereas LppT lacking the RGD site is deprived of binding capacity to LSM 192, and LppT containing RGE rather than RGD shows reduced binding. Synthetic nonapeptides derived from LppT containing RGD competitively inhibit binding of LSM 192 cells to LppT-coated slides, whereas nonapeptides containing RAD rather than RGD do not inhibit. RGD-containing, LppT-derived nonapeptides are able to directly inhibit binding of M. conjunctivae to LSM 192 cells by competitive inhibition, whereas the analogous nonapeptide containing RAD rather than RGD or the fibronectin-derived RGD hexapeptide has no inhibitory effect. These results reveal LppT as the first candidate of a RGD lectin in Mycoplasma species that is assumed to bind to beta integrins.

Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM)

Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion.

eIF4E is an important determinant of adhesion and pseudohyphal growth of the yeast S. cerevisiae

eIF4E, the cytoplasmatic cap-binding protein, is required for efficient cap-dependent translation. We have studied the influence of mutations that alter the activity and/or expression level of eIF4E on haploid and diploid cells in the yeast S. cerevisiae. Temperature-sensitive eIF4E mutants with reduced levels of expression and reduced cap-binding affinity clearly show a loss in haploid adhesion and diploid pseudohyphenation upon starvation for nitrogen. Some of these mutations affect the interaction of the cap-structure of mRNAs with the cap-binding groove of eIF4E. The observed reduction in adhesive and pseudohyphenating properties is less evident for an eIF4E mutant that shows reduced interaction with p20 (an eIF4E-binding protein) or for a p20-knockout mutant. Loss of adhesive and pseudohyphenating properties was not only observed for eIF4E mutants but also for knockout mutants of components of eIF4F such as eIF4B and eIF4G1. We conclude from these experiments that mutations that affect components of the eIF4F-complex loose properties such as adhesion and pseudohyphal differentiation, most likely due to less effective translation of required mRNAs for such processes.

Junctional adhesion molecule (JAM)-C deficient C57BL/6 mice develop a severe hydrocephalus

The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C(-/-) mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C(-/-) mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C(-/-) C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C(-/-) mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3(rd) ventricle in JAM-C(-/-) C57BL/6 mice. Taken together, our study suggests that JAM-C(-/-) C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.

Connexin37 protects against atherosclerosis by regulating monocyte adhesion

A genetic polymorphism in the human gene encoding connexin37 (CX37, encoded by GJA4, also known as CX37) has been reported as a potential prognostic marker for atherosclerosis. The expression of this gap-junction protein is altered in mouse and human atherosclerotic lesions: it disappears from the endothelium of advanced plaques but is detected in macrophages recruited to the lesions. The role of CX37 in atherogenesis, however, remains unknown. Here we have investigated the effect of deleting the mouse connexin37 (Cx37) gene (Gja4, also known as Cx37) on atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice, an animal model of this disease. We find that Gja4(-/-)Apoe(-/-) mice develop more aortic lesions than Gja4(+/+)Apoe(-/-) mice that express Cx37. Using in vivo adoptive transfer, we show that monocyte and macrophage recruitment is enhanced by eliminating expression of Cx37 in these leukocytes but not by eliminating its expression in the endothelium. We further show that Cx37 hemichannel activity in primary monocytes, macrophages and a macrophage cell line (H36.12j) inhibits leukocyte adhesion. This antiadhesive effect is mediated by release of ATP into the extracellular space. Thus, Cx37 hemichannels may control initiation of the development of atherosclerotic plaques by regulating monocyte adhesion. H36.12j macrophages expressing either of the two CX37 proteins encoded by a polymorphism in the human GJA4 gene show differential ATP-dependent adhesion. These results provide a potential mechanism by which a polymorphism in CX37 protects against atherosclerosis.

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.

Opioids trigger alpha 5 beta 1 integrin-mediated monocyte adhesion

Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.

Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells

We present evidence for differential roles of Rho-kinase and myosin light chain kinase (MLCK) in regulating shape, adhesion, migration, and chemotaxis of human fibrosarcoma HT1080 cells on laminin-coated surfaces. Pharmacological inhibition of Rho-kinase by Y-27632 or inhibition of MLCK by W-7 or ML-7 resulted in significant attenuation of constitutive myosin light chain phosphorylation. Rho-kinase inhibition resulted in sickle-shaped cells featuring long, thin F-actin-rich protrusions. These cells adhered more strongly to laminin and migrated faster. Inhibition of MLCK in contrast resulted in spherical cells and marked impairment of adhesion and migration. Inhibition of myosin II activation with blebbistatin resulted in a morphology similar to that induced by Y-27632 and enhanced migration and adhesion. Cells treated first with blebbistatin and then with ML-7 also rounded up, suggesting that effects of MLCK inhibition on HT1080 cell shape and motility are independent of inhibition of myosin activity.

Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

The effect of gamma-tocopherol on proliferation, integrin expression, adhesion, and migration of human glioma cells

The effect of vitamin E on proliferation, integrin expression, adhesion, and migration in human glioma cells has been studied. gamma-tocopherol at 50 microM concentration exerted more inhibitory effect than alpha-tocopherol at the same concentration on glioma cell proliferation. Integrin alpha5 and beta1 protein levels were increased upon both alpha- and gamma-tocopherol treatments. In parallel, an increase in the alpha5beta1 heterodimer cell surface expression was observed. The tocopherols inhibited glioma cell-binding to fibronectin where gamma-tocopherol treatment induced glioma cell migration. Taken together, the data reported here are consistent with the notion that the inhibition of glioma cell proliferation induced by tocopherols may be mediated, at least in part, by an increase in integrin alpha5 and beta1 expression. Cell adhesion is also negatively affected by tocopherols, despite a small increase in the surface appearance of the alpha5beta1 heterodimer. Cell migration is stimulated by gamma-tocopherol. It is concluded that alpha5 and beta1 integrin expression and surface appearance are not sufficient to explain all the observations and that other integrins or in general other factors may be associated with these events.

Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to promote thrombus formation in high shear environments. We found that thrombospondin-1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin-1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s(-1). Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin-1. Platelet adhesion to thrombospondin-1 is not mediated via beta3-integrins or GPIa. CD36 partially mediates the adhesion of pre-activated platelets. We identified GPIb as high shear adhesion-receptor for thrombospondin-1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin-1-GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.

Platelet receptors for adhesion and activation. Variability as a factor in susceptibility to cardiovascular diseases

Polymorphisms in coagulation factors leading to altered susceptibility to cardiovascular diseases have been known for some time and some are now well-established risk factors. More recently, an increasing number of polymorphisms have been identified in platelet receptors and a series of studies indicate that these too may play a role as individual risk factors for stroke and myocardial infarction. The effect of these platelet polymorphisms appears less clear-cut than some of the coagulation factor effects and other, associated, risk factors may be important in defining their role. In this review platelet receptor polymorphisms and their role as risk factors are surveyed and their possible relevance discussed.

Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system

Site-specific delivery of anticancer agents to tumors represents a promising therapeutic strategy because it increases efficacy and reduces toxicity to normal tissues compared with untargeted drugs. Sterically stabilized immunoliposomes (SIL), guided by antibodies that specifically bind to well internalizing antigens on the tumor cell surface, are effective nanoscale delivery systems capable of accumulating large quantities of anticancer agents at the tumor site. The epithelial cell adhesion molecule (EpCAM) holds major promise as a target for antibody-based cancer therapy due to its abundant expression in many solid tumors and its limited distribution in normal tissues. We generated EpCAM-directed immunoliposomes by covalently coupling the humanized single-chain Fv antibody fragment 4D5MOCB to the surface of sterically stabilized liposomes loaded with the anticancer agent doxorubicin. In vitro, the doxorubicin-loaded immunoliposomes (SIL-Dox) showed efficient cell binding and internalization and were significantly more cytotoxic against EpCAM-positive tumor cells than nontargeted liposomes (SL-Dox). In athymic mice bearing established human tumor xenografts, pharmacokinetic and biodistribution analysis of SIL-Dox revealed long circulation times in the blood with a half-life of 11 h and effective time-dependent tumor localization, resulting in up to 15% injected dose per gram tissue. These favorable pharmacokinetic properties translated into potent antitumor activity, which resulted in significant growth inhibition (compared with control mice), and was more pronounced than that of doxorubicin alone and nontargeted SL-Dox at low, nontoxic doses. Our data show the promise of EpCAM-directed nanovesicular drug delivery for targeted therapy of solid tumors.

Mechanotransduction in striated muscle via focal adhesion kinase

Contractile tissues demonstrate a pronounced capacity to remodel their composition in response to mechanical challenges. Descriptive evidence suggests the upstream involvement of the phosphotransfer enzyme FAK (focal adhesion kinase) in the molecular control of load-dependent muscle plasticity. Thereby FAK evolves as a myocellular transducer of mechanical signals towards downstream transcript expression in myofibres. Recent advances in somatic gene therapy now allow the exploration of the functional involvement of this enzyme in mechanotransduction in intact muscle.

Brain endothelial PPARgamma controls inflammation-induced CD4+ T cell adhesion and transmigration in vitro

An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells (EC) which strongly relies on interaction with EC-expressed adhesion molecules. We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of brain EC inflammation. The PPARgamma agonist pioglitazone reduces transendothelial migration of encephalitogenic T cells across TNFalpha-stimulated brain EC. This effect is clearly PPARgamma mediated, as lentiviral PPARgamma overexpression in brain EC results in selective abrogation of inflammation-induced ICAM-1 and VCAM-1 upregulation and subsequent adhesion and transmigration of T cells. We therefore propose that PPARgamma in brain EC may be exploited to target detrimental EC-T cell interactions under inflammatory conditions.