Cell migration to the chemokine CXCL8: Paxillin is activated and regulates adhesion and cell motility

The chemokine CXCL8 is a powerful inducer of directional cell motility, primarily during inflammation. In this study, we found that CXCL8 stimulation led to paxillin phosphorylation in normal neutrophils, and that both CXCL8 receptors (CXCR1 and CXCR2) mediated CXCL8-induced paxillin phosphorylation. In CXCR2-transfected cells, the process depended on G_αi and G_αs coupling to CXCR2. Dominant negative (DN) paxillin increased CXCL8-induced adhesion and migration, indicating that endogenous paxillin keeps migration at submaximal levels. Furthermore, using activating antibodies to β1 integrins, analyses with focal adhesion kinase (FAK) DN variant (FRNK) and co-immunoprecipitations of FAK and paxillin, we found that β1 integrin ligation cooperates with CXCL8-induced stimulation, leading to FAK activation and thereafter to FAK-mediated paxillin phosphorylation. Our findings indicate that paxillin keeps directional motility at a restrained magnitude, and suggest that perturbations in its activation may lead to chemotactic imbalance and to pathological conditions associated with excessive or reduced leukocyte migration. R. Mintz, T. Meshel: These authors contributed equally to this work. Received 31 July 2008; received after revision 14 December 2008; accepted 16 December 2008     

The plasminogen activation system in tumor growth, invasion, and metastasis

Generation of the serine proteinase plasmin from the extracellular zymogen plasminogen can be catalyzed by either of two other serine proteinases, the urokinase- and tissue-type plasminogen activators (uPA and tPA). The plasminogen activation system also includes the serpins PAI-1 and PAI-2, and the uPA receptor (uPAR). Many findings, gathered over several decades, strongly suggest an important and causal role for uPA-catalyzed plasmin generation in cancer cell invasion through the extracellular matrix. Recent evidence suggests that the uPA system is also involved in cancer cell-directed tissue remodeling. Moreover, the system also supports cell migration and invasion by plasmin-independent mechanisms, including multiple interactions between uPA, uPAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors, and growth factors. These interactions seem to allow temporal and spatial reorganizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. The increased knowledge about the plasminogen activation system may allow utilization of its components as targets for anti-invasive therapy.     

The urokinase receptor and integrins in cancer progression

Enhanced levels of expression of urokinase receptor (uPAR) and certain integrins have been linked to cancer cell progression. This has classically been attributed to matrix degradation via the activation of the urokinase (uPA)/plasmin system and modulation of cell motility and survival through integrin engagement. More recently, uPAR has been shown to play multiple roles independent of protease activity. Specifically, uPAR has been shown to be intimately involved in the regulation of cell adhesion, migration and proliferation in part through interactions with other membrane partners, including integrins. The goal of this review is to summarize recent insights in the function of uPAR/integrin interactions, to provide a framework for understanding the importance of these interactions in the context of cancer, and to highlight its potential as a target for therapeutic intervention.     

The urokinase receptor: a ligand or a receptor? Story of a sociable molecule

In this last decade, the structure and functions of the receptor for the urokinase-type plasminogen activator have been extensively studied and characterized. This interesting receptor plays a key role in cell adhesion, migration and proliferation. It was identified 20 years ago as the specific cell-surface molecule that could bind and concentrate urokinase on the cell membrane, thus initiating the proteolytic cascade promoted by the activation of plasminogen. The identification of new extracellular ligands, such as vitronectin, and of cell-surface interactors, such as integrins and fMet-Leu-Phe receptors, shed new light on its possible roles, totally independent of the enzymatic properties of its ligand. uPAR ligands and interactors and the functional consequences of the multiple binding capability of this intriguing receptor are reviewed here. Received 19 September 2005; received after revision 4 December 2005; accepted 6 December 2005     

The role of VEGF receptors in angiogenesis; complex partnerships

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis but also have profound effects on neural cells. VEGFs are predominantly produced by endothelial, hematopoietic and stromal cells in response to hypoxia and upon stimulation with growth factors such as transforming growth factors, interleukins or platelet-derived growth factor. VEGFs bind to three variants of type III receptor tyrosine kinases, VEGF receptor 1, 2 and 3. Each VEGF isoform binds to a particular subset of these receptors giving rise to the formation of receptor homo- and heterodimers that activate discrete signaling pathways. Signal specificity of VEGF receptors is further modulated upon recruitment of coreceptors, such as neuropilins, heparan sulfate, integrins or cadherins. Here we summarize the knowledge accumulated since the discovery of these proteins more than 20 years ago with the emphasis on the signaling pathways activated by VEGF receptors in endothelial cells during cell migration, growth and differentiation. Received 15 September 2005; received after revision 11 November; accepted 24 November 2005     

Activation of protease-activated receptor (PAR) 1 by frog trefoil factor (TFF) 2 and PAR4 by human TFF2

Trefoil factors (TFFs) promote epithelial cell migration to reseal superficial wounds after mucosal injury, but their receptors and the molecular mechanisms underlying this process are poorly understood. In this study, we showed that frog TFF2 activates protease-activated receptor (PAR) 1 to induce human platelet aggregation. Based on this result, we further tested the involvement of PARs in human TFF2 (hTFF2)-promoted mucosal healing. hTFF2-stimulated migration of epithelial HT-29 cells was largely inhibited by PAR4 depletion with small interfering RNAs but not by PAR1 or PAR2 depletion. The PAR4-negative epithelial cell lines AGS and LoVo were highly responsive to hTFF2 as assessed by phosphorylation of ERK1/2 and cell migration upon PAR4 expression. Our findings suggest that hTFF2 promotes cell migration via PAR4. These findings will be helpful in further investigations into the functions and molecular mechanisms of TFFs and PARs in physiology and disease.     

Receptor communication within the lymphocyte plasma membrane: a role for the thrombospondin family of matricellular proteins

Lymphocytes, the principal cells of the immune system, carry out immune surveillance throughout the body by their unique capacity to constantly reposition themselves between a free-floating vascular state and a tissue state characterized by migration and frequent adhesive interactions with endothelial cells and components of the extracellular matrix. Therefore, mechanisms co-ordinating adhesion and migration with signals delivered through antigen recognition probably play a pivotal role for the regulation of lymphocyte behaviour and function. Endogenous thrombospondin-1 (TSP-1) seems to be the hub in such a mechanism for autocrine regulation of T cell adhesion and migration. TSP-1 functions as a mediator of cis interaction of vital receptors within the T lymphocyte plasma membrane, including integrins, low density lipoprotein receptor-related protein, calreticulin and integrin-associated protein. Received 1 June 2006; received after revision 28 June 2006; accepted 11 October 2006     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.     

Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported, in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was dependent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization. Moreover, we observed that blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.     

Molecular mechanisms of lipoprotein receptor signalling

The low-density lipoprotein (LDL) receptor is the prototype of a classical endocytosis receptor that mediates the uptake of extracellular ligands. Other members of the LDL receptor gene family, on the other hand, have been shown to regulate intracellular signalling cascades. Among these are the LDL receptor-related protein 1, LRP1, a promiscuous and ubiquitously expressed receptor which is critically involved in a multitude of diverse physiological processes; the Reelin receptors ApoER2 and VLDL receptor, which participate in neuronal development; and megalin, a multifunctional receptor expressed in various epithelia. In this review, we focus on recent developments that highlight similarities and differences between these related receptors and their biological function, and discuss open questions as to the underlying molecular mechanisms.     

P2Y<Subscript>2</Subscript> receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y₂ receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y₂ receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y₂ Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y₂ Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y₂ receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y₂ RGE mutant receptors. Consistent with the role of P2Y₂ receptors in vasodilation, receptor knockdown and overexpression of P2Y₂ RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y₂ receptor in shear stress-induced cytoskeletal alterations in HUVECs.     

The biology of cell locomotion within three-dimensional extracellular matrix

Cell migration in three-dimensional (3-D) extracellular matrix (ECM) is not a uniform event but rather comprises a modular spectrum of interdependent biophysical and biochemical cell functions. Haptokinetic cell migration across two-dimensional (2-D) surfaces consists of at least three processes: (i) the protrusion of the leading edge for adhesive cell-substratum interactions is followed by (ii) contraction of the cell body and (iii) detachment of the trailing edge. In cells of flattened morphology migrating slowly across 2-D substrate, contact-dependent clustering of adhesion receptors including integrins results in focal contact and stress fiber formation. While haptokinetic migration is predominantly a function of adhesion and deadhesion events lacking spatial barriers towards the advancing cell body, the biophysics of the tissues require a set of cellular strategies to overcome matrix resistance. Matrix barriers force the cells to adapt their morphology and change shape and/or enzymatically degrade ECM components, either by contact-dependent proteolysis or by protease secretion. In 3-D ECM, in contrast to 2-D substrate, the cell shape is mostly bipolar and the cytoskeletal organization is less stringent, frequently lacking discrete focal contacts and stress fibers. Morphologically large spindle-shaped cells (i.e., fibroblasts, endothelial cells, and many tumor cells) of high integrin expression and strong cytoskeletal contractility utilize integrin-dependent migration strategies that are coupled to the capacity to reorganize ECM. In contrast, a more dynamic ameboid migration type employed by smaller cells expressing low levels of integrins (i.e., T lymphocytes, dendritic cells, some tumor cells) is characterized by largely integrin-independent interaction strategies and flexible morphological adaptation to preformed fiber strands, without structurally changing matrix architecture. In tumor invasion and angiogenesis, migration mechanisms further comprise the migration of entire cell clusters or strands maintaining stringent cell-cell adhesion and communication while migrating. Lastly, cellular interactions, enzyme and cytokine secretion, and tissue remodeling provided by reactive stroma cells (i.e. fibroblasts and macrophages) contribute to cell migration. In conclusion, depending on the cellular composition and tissue context of migration, diverse cellular and molecular migration strategies can be developed by different cell types.     

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.     

Agonist-induced trafficking of the low-affinity formyl peptide receptor FPRL1

The formyl peptide-like receptor FPRL1 is a member of the chemoattractant subfamily of G protein- coupled receptors involved in regulating leukocyte migration in inflammation. To elucidate mechanisms underlying the internalization of ligand-bound FPRL1 and possible receptor recycling, we characterized the endocytic itinerary of FPRL1. We show that agonist-triggered internalization from the plasma membrane into intracellular compartments is prevented by perturbation of clathrin-mediated endocytosis, such as expression of the dominant-negative clathrin Hub mutant, siRNA-mediated depletion of cellular clathrin and expression of a dominant-negative mutant of the large GTPase dynamin. Internalized FPRL1 co-localized with endocytosed transferrin and the small GTPases Rab4 and Rab11 in vesicular structures most resembling recycling endosomes. Recycling of FPRL1 was significantly reduced by pretreatment with PI3-kinase inhibitors. Thus, ligand-bound FPRL1 undergoes primarily clathrin-mediated and dynamin-dependent endocytosis and the receptor recycles via a rapid PI3-kinase-sensitive route as well as pathways involving perinuclear recycling endosomes.Received 19 March 2004; received after revision 26 April 2004; accepted 12 May 2004     

EP4 receptor stimulation down-regulates human eosinophil function

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE₂ receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca²⁺ mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE₂ and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.     

CCN1: a novel inflammation-regulated biphasic immune cell migration modulator

In this study, we performed a comprehensive analysis of the effect of CCN1 on the migration of human immune cells. The molecule CCN1, produced by fibroblasts and endothelial cells, is considered as an important matrix protein promoting tissue repair and immune cell adhesion by binding various integrins. We recently reported that CCN1 therapy is able to suppress acute inflammation in vivo. Here, we show that CCN1 binds to various immune cells including T cells, B cells, NK cells, and monocytes. The addition of CCN1 in vitro enhances both actin polymerization and transwell migration. Prolonged incubation with CCN1, however, results in the inhibition of migration of immune cells by a mechanism that involves downregulation of PI3Kγ, p38, and Akt activation. Furthermore, we observed that immune cells themselves produce constitutively CCN1 and secretion is induced by pro-inflammatory stimuli. In line with this finding, patients suffering from acute inflammation had enhanced serum levels of CCN1. These findings extend the classical concept of CCN1 as a locally produced cell matrix adhesion molecule and suggest that CCN1 plays an important role in regulating immune cell trafficking by attracting and locally immobilizing immune cells.     

Ethanol and opioid receptor signalling

Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate mu-opioid receptor binding by increasing receptor Bmax, and, in some cases, chronic ethanol exposure decreases the density or affinity of the mu-opioid receptors. By contrast, high concentrations of ethanol acutely decrease delta-opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the delta-opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the delta-opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the delta-opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of G alpha s and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the delta-opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in G alpha s or G alpha i. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the delta-opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in G alpha s and increases in G alpha i. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.     

Distribution of the 1,25 dihydroxy-vitamin D3 receptor in the bursa of Fabricius of chicken.

The vitamin D3 metabolite 1,25(OH)2D3 is probably involved in B lymphocyte ontogeny. We therefore determined the distribution of the 1,25(OH)2D3 receptor in the bursa of Fabricius and spleen cells of 7-day-old chicks, by immunohistochemistry using a monoclonal antibody against the chick intestinal cell 1,25(OH)2D3 receptor. The bursa cells of young (7-day-old) chicks contained large amounts of receptor while the spleen cells did not. The bursa cells of older (35-day-old) chicks contained fewer receptors, but the number of receptors in the spleen increased.     

Regulation and interactions in the activation of cell-associated plasminogen

The main components in plasminogen activation include plasminogen, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), and plasminogen activator inhibitors-1 and –2 (PAI-1, PAI-2). These components are subject to extensive regulation and interactions with for example, pericellular adhesion molecules. Although uPA and tPA are quite similar in structure and have common inhibitors and physiological substrates, their physiological roles are distinct. Traditionally, the role of tPA has been in fibrinolysis and that of uPA in cell migration, especially in cancer cells. Recently several targets for tPA/plasmin have been found in neuronal tissues. The functional role of the PAIs is no longer simply to inhibit overexpressed plasminogen activators, and PAI-2 has an unidentified role in the regulation of cell death.Received 2 June 2004; received after revision 30 June 2004; accepted 20 July 2004     

Chromogranin A binds to αvβ6-integrin and promotes wound healing in mice

Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, neurons, cardiomyocytes, and keratinocytes, is the precursor of various peptides that regulate the carbohydrate/lipid metabolism and the cardiovascular system. We have found that CgA, locally administered to injured mice, can accelerate keratinocyte proliferation and wound healing. This biological activity was abolished by the Asp(45)Glu mutation. CgA and its N-terminal fragments, but not the corresponding Asp(45)Glu mutants, could selectively recognize the αvβ6-integrin on keratinocytes (a cell-adhesion receptor that is up-regulated during wound healing) and regulate keratinocyte adhesion, proliferation, and migration. No binding was observed to other integrins such as αvβ3, αvβ5, αvβ8, α5β1, α1β1, α3β1, α6β4, α6β7 and α9β1. Structure-activity studies showed that the entire CgA(39-63) region is crucial for αvβ6 recognition (K(i) = 7 nM). This region contains an RGD site (residues CgA(43-45)) followed by an amphipathic α-helix (residues CgA(47-63)), both crucial for binding affinity and selectivity. These results suggest that the interaction of the RGD/α-helix motif of CgA with αvβ6 regulates keratinocyte physiology in wound healing.