Marching at the front and dragging behind: differential αVβ3-integrin turnover regulates focal adhesion behavior

Integrins are cell–substrate adhesion molecules that provide the essential link between the actin cytoskeleton and the extracellular matrix during cell migration. We have analyzed αVβ3-integrin dynamics in migrating cells using a green fluorescent protein–tagged β3-integrin chain. At the cell front, adhesion sites containing αVβ3-integrin remain stationary, whereas at the rear of the cell they slide inward. The integrin fluorescence intensity within these different focal adhesions, and hence the relative integrin density, is directly related to their mobility. Integrin density is as much as threefold higher in sliding compared with stationary focal adhesions. High intracellular tension under the control of RhoA induced the formation of high-density contacts. Low-density adhesion sites were induced by Rac1 and low intracellular tension. Photobleaching experiments demonstrated a slow turnover of β3-integrins in low-density contacts, which may account for their stationary nature. In contrast, the fast β3-integrin turnover observed in high-density contacts suggests that their apparent sliding may be caused by a polarized renewal of focal contacts. Therefore, differential acto-myosin–dependent integrin turnover and focal adhesion densities may explain the mechanical and behavioral differences between cell adhesion sites formed at the front, and those that move in the retracting rear of migrating cells

LPS differentially regulates adhesion and transendothelial migration of human monocytes under static and flow conditions

One of the key components of the innate immune response is the recognition of microbial products such as LPS by Toll-like receptors on monocytes and neutrophils. We show here that short-term stimulation of primary human monocytes with LPS led to an increase in adhesion of monocytes to endothelial cells and a dramatic decrease in transendothelial migration under static conditions. In contrast, under normal physiological flow, monocyte adhesion and migration across a human umbilical vein endothelial cell monolayer appeared to be unaffected by LPS treatment. LPS stimulation of monocytes activated β1 and β2 integrins, but did not increase their surface expression levels. During septic shock, reduction in blood flow as a result of vasodilation and vascular permeability leads to adhesion and accumulation of LPS-stimulated circulating monocytes onto the blood vessel walls. The different findings of monocyte migration under static and flow conditions in our study may offer one explanation for this phenomenon. The rapid engagement of LPS-activated monocytes preventing transendothelial migration could represent a novel mechanism of bacterial exclusion from the vasculature. This occurs during the early stages of sepsis, and in turn may modulate the severity of the pathophysiolog

Glycoprotein IIb-IIIa Is Expressed on Avian Multilineage Hematopoietic Progenitor Cells

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The mechanisms and dynamics of αvβ3 integrin clustering in living cells

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility

The spatiotemporal localization of JAM-C following sciatic nerve crush in adult rats

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The pyrazolyl-urea GeGe3 inhibits tumor angiogenesis and reveals dystrophia myotonica protein Kinase (DMPK)1 as a novel angiogenesis target

The limitation of targeting VEGF/VEGFR2 signalling to stop angiogenesis in cancer therapy has been blamed on re-activation of alternative receptor tyrosine kinases by compensatory angiogenic factors. Targeting MAPK and PI3K signaling pathways in endothelial cells may be an alternative or complementary approach. Herein we aimed to evaluate the antitumor and antiangiogenic potential of a novel pyrazolyl-urea kinase inhibitor, GeGe3, and to identify its kinase targets. We found GeGe3 to inhibit the proliferation of HUVEC and endothelial tube formation. GeGe3 impaired inter-segmental angiogenesis during development of zebrafish embryos. In mice, GeGe3 blocked angiogenesis and tumor growth in transplanted subcutaneous Lewis Lung Carcinomas. Screening for GeGe3-targeted kinases revealed Aurora B, Aurora C, NEK10, polo-like kinase (PLK)2, PLK3, DMPK1 and CAMK1 as candidate targets. Biochemical analysis of these kinases showed DMPK1 regulation upon VEGF challenge. Investigation of the role of DMPK1 in endothelial cells revealed DMPK1 as a novel mediator of angiogenesis that controls the activation of MAPK signaling, proliferation and migration. GeGe3 alters angiogenesis by targeting DMPK in tumor endothelial cells and pericytes. The pyrazolyl-urea GeGe3, a novel blocker of MAPK and PI3K pathways, strongly inhibits physiological and tumor angiogenesis. We also report GeGe3-targeted kinase DMPK as a novel mediator of angiogenesis

Blockade but not overexpression of the junctional adhesion molecule C influences virus-induced type 1 diabetes in mice

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans

Urokinase Receptor (CD87) Regulates Leukocyte Recruitment via β2 Integrins In Vivo

The urokinase receptor (CD87; uPAR) is found in close association with β2 integrins on leukocytes. We studied the functional consequence of this association for leukocyte adhesion and migration. In vivo, the β2 integrin–dependent recruitment of leukocytes to the inflamed peritoneum of uPAR-deficient mice was significantly reduced as compared with wild-type animals. In vitro, β2 integrin–mediated adhesion of leukocytes to endothelium was lost upon removal of uPAR from the leukocyte surface by phosphatidyl-inositol–specific phospholipase C. Leukocyte adhesion was reconstituted when soluble intact uPAR, but not a truncated form lacking the uPA-binding domain, was allowed to reassociate with the cell surface. uPAR ligation with a monoclonal antibody induced adhesion of monocytic cells and neutrophils to vascular endothelium by six- to eightfold, whereas ligation with inactivated uPA significantly reduced cell-to-cell adhesion irrespective of the β2 integrin–stimulating pathway. These data indicate that β2 integrin–mediated leukocyte–endothelial cell interactions and recruitment to inflamed areas require the presence of uPAR and define a new phenotype for uPAR-deficient mice. Moreover, uPAR ligation differentially modulates leukocyte adhesion to endothelium and provides novel targets for therapeutic strategies in inflammation-related vascular pathologies

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 3rd 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