Melanoma cell extravasation under flow conditions is modulated by leukocytes and endogenously produced interleukin 8

Attachment of tumor cells to the endothelium (EC) under flow conditions is critical for the migration of tumor cells out of the vascular system to establish metastases. Innate immune system processes can potentially promote tumor progression through inflammation dependant mechanisms. White blood cells, neutrophils (PMN) in particular, are being studied to better understand how the host immune system affects cancer cell adhesion and subsequent migration and metastasis. Melanoma cell interaction with the EC is distinct from PMN-EC adhesion in the circulation. We found PMN increased melanoma cell extravasation, which involved initial PMN tethering on the EC, subsequent PMN capture of melanoma cells and maintaining close proximity to the EC. LFA-1 (CD11a/CD18 integrin) influenced the capture phase of PMN binding to both melanoma cells and the endothelium, while Mac-1 (CD11b/CD18 integrin) affected prolonged PMN-melanoma aggregation. Blocking E-selectin or ICAM-1 (intercellular adhesion molecule) on the endothelium or ICAM-1 on the melanoma surface reduced PMN-facilitated melanoma extravasation. Results indicated a novel finding that PMN-facilitated melanoma cell arrest on the EC could be modulated by endogenously produced interleukin-8 (IL-8). Functional blocking of the IL-8 receptors (CXCR1 and CXCR2) on PMN, or neutralizing soluble IL-8 in cell suspensions, significantly decreased the level of Mac-1 up-regulation on PMN while communicating with melanoma cells and reduced melanoma extravasation. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines, and PMN-melanoma adhesion in the recruitment of metastatic cancer cells to the endothelium in the microcirculation, which are significant in fostering new approaches to cancer treatment through anti-inflammatory therapeutics.     

Shear stress and shear rate differentially affect the multi-step process of leukocyte-facilitated melanoma adhesion

Previous studies have shown that neutrophils (PMNs) facilitate melanoma cell extravasation [M.J. Slattery, C. Dong, Neutrophils influence melanoma adhesion and migration under flow conditions, Intl. J. Cancer 106 (2003) 713–722] Little is known, however, about the specific interactions between PMNs, melanoma and the endothelium (EC) or the molecular mechanism involved under flow conditions. The aim of this study is to investigate a “two-step adhesion” hypothesis that involves initial PMN tethering on the EC and subsequent melanoma cells being captured by tethered PMNs. Different effects of hydrodynamic shear stress and shear rate were analyzed using a parallel-plate flow chamber. Results indicate a novel finding that PMN-facilitated melanoma cell arrest on the EC is modulated by shear rate, which is inversely-proportional to cell–cell contact time, rather than by the shear stress, which is proportional to the force exerted on formed bonds. β₂ integrins/ICAM-1 adhesion mechanisms were examined and the results indicate LFA-1 and Mac-1 cooperate to mediate the PMN–EC–melanoma interactions under shear conditions. In addition, endogenously produced IL-8 contributes to PMN-facilitated melanoma arrest on the EC through the CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) on PMN. These results provide new evidence for the complex role of hemodynamic forces, secreted chemokines and PMN–melanoma adhesion in the recruitment of metastatic cancer cells to the EC.     

Kinetics analysis of binding between melanoma cells and neutrophils

It has been determined previously that polymorphonuclear leukocytes, or PMNs, can facilitate melanoma cell extravasation through the endothelium under shear conditions. The interactions between melanoma cells and PMNs are mediated by the beta2-integrins expressed by PMNs and intercellular adhesion molecules (ICAM-1) expressed on melanoma cells. In this study, the kinetics of these interactions was studied using a parallel plate flow chamber. The dissociation rates were calculated under low force conditions for ICAM-1 interactions with both beta2-integrins, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), together and separately by using functional blocking antibodies on PMNs. The kinetics of PMNs stimulated with IL-8 was also determined. It was concluded that the small number of constitutively expressed active beta2-integrins on PMNs are sufficient to bind to ICAM-1 expressed on melanoma cells and that the intrinsic dissociation rate for these adhesion molecules appear to be more dependent on what method is used to determine them than on what cells express them.     

ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release

Interaction ofthe ₂-integrin complex on thepolymorphonuclear neutrophil (PMN) with intercellular adhesionmolecule-1 (ICAM-1) has been implicated in PMN-mediated cytotoxicity.This study examined interaction of the CD11a, CD11b, and CD18 subunitsof the ₂-integrin with ICAM-1,transfected into Chinese hamster ovarian (CHO) cells to avoid effectsof other adhesion molecules. Incubation of quiescent PMNs withwild-type and ICAM-1-transfected CHO cells produced nominal cell lysis.Similarly, when phorbol myristate acetate (PMA)-activated PMNs wereincubated with wild-type CHO cells, minimal cytotoxicity was produced.However, when ICAM-1-transfected CHO cells were incubated withPMA-activated PMNs, 40% cell lysis occurred. Blockade with amonoclonal antibody (MAb) to ICAM-1 or MAbs to CD11a, CD11b, or CD18reduced PMN-mediated cytotoxicity to baseline. To examine the role ofadhesion in cytotoxicity, we studied₂-integrin-mediated PMNadhesion to ICAM-1-transfected CHO cells and found that MAbs for CD11a,CD11b, and CD18 all abrogated PMN cytotoxicity despite disparateeffects on adhesion. To assess the role of CD18,₂-integrin subunits werecross-linked, and CD18 alone mediated protease release. Moreover,ICAM-1 was immunoprecipitated from transfected CHO cells and incubatedwith PMNs. This soluble ICAM-1 provoked elastase release, similar toPMA, which could be inhibited by MAbs to CD18 but not MAbs to other₂-integrin subunits. Inaddition, coincubation with protease inhibitors eglin C and AAPVCKreduced PMN-mediated cytotoxicity to control levels. Finally,ICAM-1-transfected CHO cells were exposed to activated PMNs from apatient with chronic granulomatous disease that caused significant celllysis, equivalent to that of PMNs from normal donors. Collectively,these data suggest that ICAM-1 provokes PMN-mediated cytotoxicity viaCD18-mediated protease release.

     

Two-dimensional kinetics of beta 2-integrin and ICAM-1 bindings between neutrophils and melanoma cells in a shear flow

Cell adhesion, mediated by specific receptor-ligand interactions, plays an important role in biological processes such as tumor metastasis and inflammatory cascade. For example, interactions between beta 2-integrin (lymphocyte function-associated antigen-1 and/or Mac-1) on polymorphonuclear neutrophils (PMNs) and ICAM-1 on melanoma cells initiate the bindings of melanoma cells to PMNs within the tumor microenvironment in blood flow, which in turn activate PMN-melanoma cell aggregation in a near-wall region of the vascular endothelium, therefore enhancing subsequent extravasation of melanoma cells in the microcirculations. Kinetics of integrin-ligand bindings in a shear flow is the determinant of such a process, which has not been well understood. In the present study, interactions of PMNs with WM9 melanoma cells were investigated to quantify the kinetics of beta 2-integrin and ICAM-1 bindings using a cone-plate viscometer that generates a linear shear flow combined with a two-color flow cytometry technique. Aggregation fractions exhibited a transition phase where it first increased before 60 s and then decreased with shear durations. Melanoma-PMN aggregation was also found to be inversely correlated with the shear rate. A previously developed probabilistic model was modified to predict the time dependence of aggregation fractions at different shear rates and medium viscosities. Kinetic parameters of beta 2-integrin and ICAM-1 bindings were obtained by individual or global fittings, which were comparable to respectively published values. These findings provide new quantitative understanding of the biophysical basis of leukocyte-tumor cell interactions mediated by specific receptor-ligand interactions under shear flow conditions.     

VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration

Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the ₁- and ₂-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation. human umbilical vein endothelial cells; vascular-endothelial cadherin; F-actin; reactive oxygen species; p38 mitogen-activated protein kinase; vascular cell adhesion molecule     

Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin alphavbeta3

Interaction of tumor cells with the vascular wall is required for metastasis from the bloodstream. The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPS-treated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s^–1. At a venous shear rate of 400 s^–1, 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor "activation" occurred at a significantly lower shear rate of 50 s^–1. This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s^–1. The integrin _v3 has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of _v3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s^–1. Collectively, our data suggest that _v3 promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions. platelet releasate; vitronectin; fibronectin     

Fibrin serves as a divalent ligand that regulates neutrophil-mediated melanoma cells adhesion to endothelium under shear conditions

Elevated soluble fibrin (sFn) levels are characteristic of melanoma hematogeneous dissemination, where tumor cells interact intimately with host cells. Melanoma adhesion to the blood vessel wall is promoted by immune cell arrests and tumor-derived thrombin, a serine protease that converts soluble fibrinogen (sFg) into sFn. However, the molecular requirement for sFn-mediated melanoma-polymorphonuclear neutrophils (PMNs) and melanoma-endothelial interactions under physiological flow conditions remain elusive. To understand this process, we studied the relative binding capacities of sFg and sFn receptors e.g., α(v)β(3) integrin and intercellular adhesion molecule-1 (ICAM-1) expressed on melanoma cells, ICAM-1 on endothelial cells (EC), and CD11b/CD18 (Mac-1) on PMNs. Using a parallel-plate flow chamber, highly metastatic melanoma cells (1205Lu and A375M) and human PMNs were perfused over an EC monolayer expressing ICAM-1 in the presence of sFg or sFn. It was found that both the frequency and lifetime of direct melanoma adhesion or PMN-facilitated melanoma adhesion to the EC in a shear flow were increased by the presence of sFn in a concentration-dependent manner. In addition, sFn fragment D and plasmin-treated sFn failed to increase melanoma adhesion, implying that sFn-bridged cell adhesion requires dimer-mediated receptor-receptor cross-linking. Finally, analysis of the respective kinetics of sFn binding to Mac-1, ICAM-1, and α(v)β(3) by single bond cell tethering assays suggested that ICAM-1 and α(v)β(3) are responsible for initial capture and firm adhesion of melanoma cells. These results provide evidence that sFn enhances melanoma adhesion directly to ICAM-1 on the EC, while prolonged shear-resistant melanoma adhesion requires interactions with PMNs.     

Peptides based on alphaV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation

Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds V-integrins, including V3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on V-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and V-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to V3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via V3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential. sickle cell disease; intercellular adhesion molecule-4; V3-integrin; peripheral resistance unit; endothelium; erythrocytes     

Effects of IL-8, Gro-alpha, and LTB(4) on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils

Firm adhesion ofrolling neutrophils on inflamed endothelium is dependent on₂ (CD18)-integrins and activating stimuli. LFA-1 (CD11a/CD18) appears to be more important than Mac-1 (CD11b/CD18) inneutrophil emigration at inflammatory sites, but little is known of therelative binding characteristics of these two integrins underconditions thought to regulate firm adhesion. The present studyexamined the effect of chemoattractants on the kinetics of LFA-1 andMac-1 adhesion in human neutrophils. We found that subnanomolarconcentrations of interleukin-8, Gro-, and leukotriene B₄ (LTB₄) induced rapid and optimal rates ofLFA-1-dependent adhesion of neutrophils to intercellular adhesionmolecule (ICAM)-1-coated beads. These optimal rates of LFA-1 adhesionwere transient and decayed within 1 min after chemoattractantstimulation. Mac-1 adhesion was equally rapid initially but continuedto rise for 6 min after stimulation. A fourfold higher density ofICAM-1 on beads markedly increased the rate of binding to LFA-1 but did not change the early and narrow time window for the optimal rate ofadhesion. Using well-characterized monoclonal antibodies, we showedthat activation of LFA-1 and Mac-1 by Gro- was completely blocked byanti-CXC chemokine receptor R2, but activation of these integrins byinterleukin-8 was most effectively blocked by anti-CXC chemokinereceptor R1. The topographical distribution of beads also reflectedsignificant differences between LFA-1 and Mac-1. Beads bound to Mac-1translocated to the cell uropod within 4 min, but beads bound to LFA-1remained bound to the lamellipodial regions at the same time. Thesekinetic and topographical differences may indicate distinct functionalcontributions of LFA-1 and Mac-1 on neutrophils.

     

Platelet-induced enhancement of LS174T colon carcinoma and THP-1 monocytoid cell adhesion to vascular endothelium under flow

This study was undertaken to characterize the adhesion of LS174T colon adenocarcinoma cells to 4-h TNF--stimulated human umbilical vein endothelial cells (HUVECs) under flow in the presence and absence of platelets and erythrocytes. Cell binding to HUVECs was significantly enhanced by simultaneous perfusion of thrombin-activated, but not resting, platelets. This increase was achieved via a platelet bridging mechanism whereby a previously tethered LS174T cell (primary tether) captures a free-flowing cell (secondary tether) that subsequently attaches to the endothelium downstream of the already adherent cell. The total number of tumor cells tethering to HUVECs and the percentage of secondary tethers relative to the total amount of cell tethering depended on platelet concentration and wall shear stress. At 0.8 dyn/cm² and a platelet-to-LS174T cell ratio of 25:1, the total amount of cell tethering nearly doubled as a result of platelet-induced enhancement compared with the amount without platelet perfusion. Moreover, the percentage of secondary tethers increased from background levels (<5%) in the absence of platelets to 60% at a platelet-to-LS174T cell ratio of 25:1. Platelet-mediated secondary tethering is not limited to LS174T colon carcinoma cells, as THP-1 monocytoid cells also displayed this pattern of interaction. Secondary tethering was dependent on both platelet P-selectin and _IIb3-integrin for LS174T cells and P-selectin alone for THP-1 cells. Furthermore, platelet-mediated secondary tethering of both cell types occurred in the presence of red blood cells. Altogether, these results reveal a novel role for platelets in promoting cell binding to endothelium through a secondary tethering mechanism. P-selectin; _IIb3-integrins; shear stress     

CYP450 dietary inhibitors attenuate TNF-alpha-stimulated endothelial molecule expression and leukocyte adhesion

Enhanced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and other endothelial cell adhesion molecules (ECAMs) are associated with the onset and progression of inflammatory bowel disease (IBD). We show in this study that two cytochrome P-450 (CYP450) inhibitors from Citrus paradis (grapefruit), bergamottin, and 6',7'-dihydroxybergamottin (DHB) block tumor necrosis factor (TNF)--stimulated expression of MAdCAM-1 in cultured endothelial cells and also reduce ₄₇-dependent lymphocyte adhesion. Bergamottin (20–50 µM) or DHB (10–30 µM) pretreatment dose-dependently reduced TNF--mediated expression of MAdCAM-1 and lymphocyte adhesion. Bergamottin and DHB also prevented expression of two other ECAMs, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (but not E-selectin). SKF-525a, a specific CYP450 inhibitor, also blocked the expression of MAdCAM-1 mediated by TNF-. Similar to SKF-525a (20 µM), bergamottin (20 µM) and DHB (20 µM) directly inhibited the activity of CYP450 3A4. These results suggest that natural CYP450 inhibitors may be effective in reducing ECAM expression and leukocyte adhesion and therefore be useful in the clinical treatment of inflammatory states like IBD. cytochrome P-450; inflammatory bowel disease; lymphocytes; mucosal adhesion cell adhesion molecule-1     

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its -subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin _IIb3 is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on _IIb3, we examined _IIb3/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that -actinin, myosin heavy chain, and Syk coimmunoprecipitate with _IIb3 in resting platelets and that 120 dyn/cm² shear stress leads to their disassociation from _IIb3. Shear-induced disassociation of -actinin and myosin heavy chain from the ₃ tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to _IIb3. Syk's disassociation from ₃ is inhibited when VWF binding to either GpIb-IX-V or _IIb3 is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to _IIb3 but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing _IIb3 with ₃ truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates _IIb3 function and suggest that shear-induced _IIb3-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the ₃-tail. platelets; mechanoreceptor; integrin; shear stress; signal transduction     

High-temporal-resolution analysis demonstrates that ICAM-1 stabilizes WEHI 274.1 monocytic cell rolling on endothelium

Leukocyte rolling, adhesion, and migration on vascular endothelium involve several sets of adhesion molecules that interact simultaneously. Each of these receptor-ligand pairs may play multiple roles. We examined the role of ICAM-1 in adhesive interactions with mouse aortic endothelial cells (MAECs) in an in vitro flow system. Average rolling velocity of the monocytic cell line WEHI 274.1 was increased on ICAM-1-deficient MAECs compared with wild-type MAECs, both with and without TNF- stimulation. High-temporal-resolution analysis provided insights into the underlying basis for these differences. Without TNF- stimulation, average rolling velocity was slower on wild-type than on ICAM-1-deficient endothelium because of brief (<1 s) pauses. On TNF--stimulated ICAM-1-deficient endothelium, cells rolled faster because of transient accelerations, producing "jerky" rolling. Firm adhesion to ICAM-1-deficient MAECs was significantly reduced compared with wild-type MAECs, although the number of rolling cells was similar. These results demonstrate directly that ICAM-1 affects rolling velocity by stabilizing leukocyte rolling. intercellular adhesion molecule-1; cell adhesion; leukocytes; vascular endothelium; videomicroscopy     

Regulatory effects of estrogen on acute lung inflammation in mice

The role of estrogen in the regulation of the inflammatory response is not well defined. In this study, we investigated the effects of ovarian hormones on the acute inflammatory response in mouse lungs. Acute lung injury was induced by intratracheal instillation of bacterial lipopolysaccharide (LPS) in male, female, and ovariectomized (OVX) mice. End points of injury were polymorphonuclear neutrophil (PMN) content in bronchoalveolar lavage (BAL) fluids, myeloperoxidase activity in whole lung, and leak of albumin into the lung. After intratracheal instillation of LPS, all end points of injury were substantially increased in male and OVX mice compared with the female mice with intact ovaries. BAL fluids of all mice showed similar levels of chemokines (macrophage inflammatory protein MIP-2, KC, and monocyte chemoattractant proteins MCP-1 and MCP-3) and TNF-, but enhanced levels of IL-1 were found in OVX and male mice. Serum levels of IL-6 and ICAM-1 levels in lung homogenates from OVX and male mice, compared with those in female mice with intact ovaries, were also enhanced after instillation of LPS. Albumin and PMN content in LPS-injured lungs were reduced to levels found in female mice after administration of estradiol in OVX mice and corresponded to reduced IL-1, IL-6, and ICAM-1 levels. These data suggest that estrogen suppresses lung inflammatory responses in mice through an effect on vascular cell adhesion molecules and proinflammatory mediators. lipopolysaccharide; vascular cell adhesion molecule-1; interleukin-1; interleukin-6     

Laminin-10 and Lutheran blood group glycoproteins in adhesion of human endothelial cells

Laminin 5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of 5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin 5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed M_r 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins ₁ and _v3, whereas in the adhesion to laminin-10/11, Lu and integrin ₁ were involved. In the cells adhering to the 5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that 5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to 5 laminins in collaboration with integrins ₁ and _v3. integrin; cycloheximide     

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and 86Rb+ uptake

The carboxy terminus (CT) of the colonic H⁺-K⁺-ATPase is required for stable assembly with the -subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HK₂, we selected 84 amino acids in the CT of the -subunit of mouse colonic H⁺-K⁺-ATPase (CT-HK₂) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HK₂ with CD63, recombinant CT-HK₂ and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HK₂ protein (but not CT-HK₁) coprecipitated with CD63, confirming stable assembly of HK₂ with CD63. In HEK-293 transfected with HK₂ plus ₁-Na⁺-K⁺-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HK₂/NK₁ and ⁸⁶Rb⁺ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HK₂-NK₁ complex in the cell membrane. protein assembly; cell surface localization     

Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.     

Tumor Cell Extravasation Mediated by Leukocyte Adhesion is Shear Rate Dependent on IL-8 Signaling^*

To complete the metastatic journey, cancer cells have to disseminate through the circulation and extravasate to distal organs. However, the extravasation process, by which tumor cells leave a blood vessel and invade the surrounding tissue from the microcirculation, remains poorly understood at the molecular level. In this study, tumor cell adhesion to the endothelium (EC) and subsequent extravasation were investigated under various flow conditions. Results have shown polymorphonuclear neutrophils (PMNs) facilitate melanoma cell adhesion to the EC and subsequent extravasation by a shear-rate dependent mechanism. Melanoma cell-PMN interactions are mediated by the binding between intercellular adhesion molecule-1 (ICAM-1) on melanoma cells andb2integrins on PMNs. In addition, the fluid convection affects the extent of activation ofb2integrins on PMNs by endogenously secreted interleukin 8 (IL-8) within the tumor microenvironment. Results also indicate that shear rate affects the binding kinetics between PMNs and melanoma cells, which may contribute to the shear-rate dependence of melanoma extravasation in a shear flow when mediated by PMNs.     

beta-Adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2-mediated disruption of beta1 integrin signaling and mitochondrial pathway

Stimulation of -adrenergic receptors (-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of matrix metalloproteinase-2 (MMP-2) inhibits -AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with ₁ integrins. Herein we tested the hypothesis that MMP-2 impairs ₁ integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, whereas treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited -AR-stimulated activation of JNKs and levels of cytosolic cytochrome c. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated -AR-stimulated decreases in mitochondrial membrane potential. Overexpression of ₁ integrins using adenoviruses expressing the human _1A-integrin decreased -AR-stimulated cytochrome c release and apoptosis. Overexpression of ₁ integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the ₁ integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis. matrix metalloproteinases; focal adhesion kinase; c-Jun NH₂-terminal kinase; cytochrome c