Actin Cytoskeletal Rearrangement Research Articles

Localization of the RhoGEF Effector Activating Surface of Gα 13 to its Carboxy Terminus

The monomeric GTPase Rho regulates a variety of cellular processes, such as actin cytoskeletal rearrangement and gene expression. Two members of the heterotrimeric GTP-binding protein (G-protein) family, Gα12 and Gα13, have been shown to mediate signals from G-protein coupled receptors leading to Rho activation. In this novel signaling pathway, one of the guanine nucleotide exchange factors (GEFs) for Rho, p115RhoGEF, serves as a direct link between Gα13 and Rho. The goal of the present study is to isolate the region of Gα13 responsible for stimulating p115’s RhoGEF activity. To do this, three sets of Gα12/13 and Gα13/12 chimeras were constructed, in which the fusion point between Gα12 and Gα13 (or Gα13 and Gα12) was moved progressively towards the C-terminus of the chimera. The resulting constructs represent chimeras with fusion points before and after the switch regions of Gα12 and Gα13, as well as in the α4 helix. The results of in vitro experiments suggest that the region of Gα13 responsible for stimulation of RhoGEF activity is localized to the C-terminus of the switch regions. A precise delineation of the signaling pathway involving Gα12 and Gα13, p115, and Rho will allow for a more complete understanding of the biochemical mechanism and physiological significance of Rho activation through G-proteins. sources of support: National Institutes of Health (grant Gm 61454) and National Institutes of Health Pharmacological Sciences Training Program (grant Gm 070388)

Intersectin Regulates Endothelial Cell Junction Integrity

Our recent work indicated that the intersectin-2L (ITSN-2L), via its DH-PH domain with Cdc42-specific guanine nucleotide exchange factor activity, promotes actin polymerization and decrease in caveolae internalization. Expression of the DH-PH domain of ITSN-2L in cultured endothelial cells revealed a selective activation of Cdc42. Biochemical analyses of endothelial cell lysates and double fluorescent labeling of cultured endothelial cells indicate that ITSN interacts and co-localizes with members of the Cdc42-N-WASp-Arp2/3 actin polymerization pathway. Moreover, phalloidin staining of ECs expressing the DH-PH domain of ITSN-2L showed actin cytoskeletal changes such as increases in filopodia and cortical actin, while a biochemical assay for actin polymerization indicated a shift from G- to F-actin. We now show that these actin cytoskeletal rearrangements caused by ITSN-2L also have an effect on the integrity of interendothelial junctions. Morphological analysis of ECs transfected with the DH-PH domain of ITSN-2L demonstrated the appearance of gaps between cells. Moreover, functional studies in mouse lungs, upon activation of Cdc42, indicated increased vascular permeability via the interendothelial junctions. When coupled to our previous data indicating a decrease in caveolae internalization with ITSN overexpression, these current data show the involvement of ITSN in the regulation of both transcellular and paracellular transport pathways in the endothelium. In this sense, ITSN may serve to regulate endothelial permeability by controlling both pathways. Thus, the regulation of Cdc42 activity and actin polymerization by ITSN may be crucial for the loss of endothelial junctional integrity. This research is supported in part by NIH T32 HL07289.

Engagement of P‐selectin Glycoprotein Ligand‐1 (PSGL‐1) induces interactions with the actin cytoskeleton

P-selectin glycoprotein ligand-1 (PSGL-1) is a well-studied P-selectin counter receptor. Its role as a rolling receptor has been well established, and interactions between PSGL-1 and P-selectin are known to be critical for a strong inflammatory response. Involvement of PSGL-1 in signal transduction after P-selectin engagement is not well known. The purpose of this study was to investigate potential downstream events following PSGL-1 ligation by either a function blocking monoclonal antibody (KPL-1) or P-selectin. Crosslinking PSGL-1 on HL60 cells with KPL-1, which binds to the P-selectin binding site on PSGL-1, induces PSGL-1 association with the actin cytoskeleton. Conversely, crosslinking of PSGL-1 with KPL-2, an antibody that recognizes a sequence on PSGL-1 near the trans-membrane domain, did not. We have shown that interactions between PSGL-1 and the actin cytoskeleton are required for rolling on P-selectin. To determine the potential pathway that induced associations with the actin cytoskeleton, cells were pre-incubated with various pharmacological inhibitors prior to crosslinking. Reagents tested included inhibitors of tyrosine-specific protein kinases (Genistein), p38 kinase (SB 203580), MAP kinase kinase (PD 98059), protein kinase C (Calphostin C), and Syk (Piceatannol). The results from this investigation will help us define the intracellular signaling pathway required for actin cytoskeletal rearrangement following PSGL-1 ligation. [This work was supported by NIH R01 GM60563 and T32HL007829].

Functional Role of Chemokine Receptor 10 in Melanoma Metastasis

Chemokines and their receptors are best known for their ability to initiate cellular signaling cascades that mediate leukocyte migration. Chemokine receptors have also been shown to play a role in cancer metastasis, potentially through similar mechanisms. Chemokine Receptor 10 (CCR10) is expressed at significantly higher levels in human metastatic melanomas as compared with normal melanocytes, suggesting a role in melanoma metastasis. To address the functional significance of CCR10 activation in melanoma metastasis, we have engineered the mouse melanoma cell line B16.F1 to express high levels of CCR10 to generate a model system to elucidate the cellular signaling pathways induced by CCR10 activation. Elevated expression of CCR10 in transgenic melanoma cells was confirmed both at the mRNA and protein level. Transgenic melanoma cells exhibit a greater proliferation rate in response to CCR10 ligands (CTACK and MEC) as compared with control melanoma cells. Treatment of transgenic melanoma cells with CTACK induces actin cytoskeletal rearrangements as assayed by fluorescently labeled phalloidin binding. Transgenic melanoma cells demonstrate an increased rate of migration on a fibronectin matrix after treatment with CTACK in an in vitro migration assay. Together these data suggest that CCR10 activation induces signaling pathways, which lead to cellular proliferation and migration in melanoma cells.

Role of ERM (ezrin-radixin-moesin) proteins in T lymphocyte polarization, immune synapse formation and in T cell receptor-mediated signaling

Following antigen recognition, T lymphocytes undergo strong actin cytoskeletal rearrangements. These play a crucial role in the molecular reorganization at the contact site between the T lymphocyte and the antigen presenting cell, termed the immunological synapse. Moreover, they are necessary for T cell activation that leads to cytokine secretion, T cell proliferation and effector function. Little is known on how membrane and signaling molecules interact with the actin cytoskeleton during these processes. Here we review the function of the ERM family of membrane-microfilament linkers, making emphasis on the role of these proteins in T lymphocyte physiology. We discuss how ERM proteins are involved in membrane reorganization during T lymphocyte polarization and immune synapse formation, and how these proteins may contribute to T cell receptor-mediated intracellular signaling that leads to T cell activation.

Galectin-8 binds specific β1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells

Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a β-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound α1β1, α3β1 and α5β1 but not α2β1 and α4β1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that α5β1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to α1 and α5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific β1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.

Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.

Tumor Suppressor in Lung Cancer (TSLC)1 Suppresses Epithelial Cell Scattering and Tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.

Arfs, phosphoinositides and membrane traffic

Arf (ADP-ribosylation factor) GTP-binding proteins function in cells to regulate membrane traffic and structure. Arfs accomplish this task through modification of membrane lipids and the recruitment of proteins, including coat proteins and actin, to membrane surfaces. Arf1 and Arf6 are the most divergent and most studied human Arf proteins that localize predominantly to the Golgi complex and plasma membrane respectively. We have been studying the targeting of Arf1 and Arf6 to these specific compartments and the common and divergent activities that they exert on these membranes. We have found that Arf6 acts through activation of type I phosphatidylinositol 4-phosphate 5-kinases to generate phosphatidylinositol 4,5-bisphosphate and that this activity is instrumental in facilitating the actin cytoskeletal rearrangements and alterations in endosomal membrane trafficking observed with increased Arf6 activation. Arf1 can also stimulate the activity of phosphatidylinositol kinases and recruit coat proteins and actin cytoskeletal elements to the Golgi complex.

Arfs, phosphoinositides and membrane traffic.

Arf (ADP-ribosylation factor) GTP-binding proteins function in cells to regulate membrane traffic and structure. Arfs accomplish this task through modification of membrane lipids and the recruitment of proteins, including coat proteins and actin, to membrane surfaces. Arf1 and Arf6 are the most divergent and most studied human Arf proteins that localize predominantly to the Golgi complex and plasma membrane respectively. We have been studying the targeting of Arf1 and Arf6 to these specific compartments and the common and divergent activities that they exert on these membranes. We have found that Arf6 acts through activation of type I phosphatidylinositol 4-phosphate 5-kinases to generate phosphatidylinositol 4,5-bisphosphate and that this activity is instrumental in facilitating the actin cytoskeletal rearrangements and alterations in endosomal membrane trafficking observed with increased Arf6 activation. Arf1 can also stimulate the activity of phosphatidylinositol kinases and recruit coat proteins and actin cytoskeletal elements to the Golgi complex.

Rho family GTPases are activated during HGF-stimulated prostate cancer-cell scattering

An important process in embryogenesis and cancer-cell metastasis is the conversion of epithelial cells to a migratory phenotype, a phenomenon known as epithelial-mesenchymal transition (E-MT). To achieve E-MT, cells dissociate from neighbouring cells and adopt a migratory morphology. This transition requires remodelling of their cell shape and substratum adhesions; activities that require extensive reorganisation of the actin cytoskeleton. Hepatocyte growth factor (HGF)-induced scattering of Madin Darby canine kidney (MDCK) cells is a routinely used model of E-MT, in which actin cytoskeletal rearrangement is known to be dependent on Rho family GTPases. We have developed a novel model of HGF-induced E-MT using the human prostate cancer cell line, DU145. This model overcomes the limitation of using a canine cell line and facilitates the study of E-MT in human cancer. We demonstrate for the first time the scattering response of individual DU145 cells to HGF in real time and have characterised changes in actin cytoskeletal organisation and cell adhesions as these cells respond to HGF. HGF-induced scattering of DU145 cells is dependent on the activity of Rho family GTPases, and using this model, we are able to demonstrate for the first time that endogenous Cdc42 is activated downstream of HGF. Furthermore we have also shown that the response of DU145 cells to HGF is dependent on a phosphatidylinositide 3-kinase pathway.

Mechanism of Redox-mediated Guanine Nucleotide Exchange on Redox-active Rho GTPases

Rho GTPases regulate multiple cellular processes including actin cytoskeletal rearrangements, transcriptional regulation, and oxidant production. The studies described herein demonstrate that small molecule redox agents, in addition to protein regulatory factors, can regulate the activity of redox-active Rho GTPases. A novel (GXXXXGK(S/T)C) motif, conserved in a number of Rho GTPases, appears critical for redox-mediated guanine nucleotide dissociation in vitro. A detailed molecular mechanism for redox regulation of GXXXXGK(S/T)C motif-containing Rho GTPases is proposed.

The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β

IntroductionTransforming growth factor-β (TGF-β) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-β type II receptor antagonist that inhibits TGF-β binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-β, particularly its stimulation of mammary epithelial–mesenchymal transition (EMT), can be prevented by CystC.MethodRetroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Δ14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-β stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-β stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-β.ResultsCystC and Δ14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-β by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-β-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Δ14CystC also inhibited TGF-β signaling in two tumorigenic human breast cancer cell lines.ConclusionOur findings show that TGF-β stimulation of initiating metastatic events, including decreased cell polarization, reduced cell–cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-β.

NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein.

The Helicobacter pylori immunodominant protein, CagA, is associated with severe gastritis and carcinoma. Injection of CagA into gastric epithelial cells by type IV secretion leads to actin-cytoskeletal rearrangements and cell scattering. CagA has been reported to have no role in the induction of transcription factor NF-kappaB and IL-8, which are crucial determinants for chronic inflammation. Here, we provide several lines of evidence showing that CagA is able to induce IL-8 in a time- and strain-dependent manner. We also show that by exchanging specific cagA genes, high IL-8-inducing H. pylori strains could be converted into low inducing strains and vice versa. Our results suggest that IL-8 release induced by CagA occurs via a Ras-->Raf-->Mek-->Erk-->NF-kappaB signaling pathway in a Shp-2- and c-Met-independent manner. Thus, CagA is a multifunctional protein capable of effecting both actin remodeling and potentiation of chemokine release.

341. Lentiviral Vector-Mediated Gene Therapy as Treatment for Wiskott-Aldrich Syndrome (WAS): Pre-Clinical Studies in Human Cell Lines and WASp -/- Mice

WAS is an immunodeficiency characterized by recurrent infection, thrombocytopenia, eczema, and increased susceptibility to autoimmunity and malignancy. WAS results from deficient function of the WAS protein (WASp) which functions in receptor-mediated actin cytoskeletal rearrangement. Gene therapy represents an attractive potential therapeutic approach for WAS patients.

The Wiskott-Aldrich Syndrome Protein Regulates Nuclear Translocation of NFAT2 and NF-κB (RelA) Independently of Its Role in Filamentous Actin Polymerization and Actin Cytoskeletal Rearrangement

The Wiskott-Aldrich Syndrome Protein Regulates Nuclear Translocation of NFAT2 and NF-κB (RelA) Independently of Its Role in Filamentous Actin Polymerization and Actin Cytoskeletal Rearrangement

The Wiskott-Aldrich Syndrome Protein Regulates Nuclear Translocation of NFAT2 and NF-κB (RelA) Independently of Its Role in Filamentous Actin Polymerization and Actin Cytoskeletal Rearrangement

Effector functions mediated by NK cells involve cytotoxicity and transcription-dependent production and release of cytokines and chemokines. Although the JAK/STAT pathway mediates lymphokine-induced transcriptional regulation in NK cells, very little is known about transcriptional regulation induced during cell-cell contact. We demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an important component for integration of signals leading to nuclear translocation of NFAT2 and NF-kappaB (RelA) during cell-cell contact and NKp46-dependent signaling. This WASp function is independent of its known role in F-actin polymerization and cytoskeletal rearrangement. Absence of WASp results in decreased accumulation of calcineurin, WASp-interacting protein, and molecules upstream of calcium mobilization, i.e., activated ZAP70 and phospholipase C-gamma1, in the disorganized NK cell immune synapse. Production of GM-CSF, but not IFN-gamma, is decreased, while natural cytotoxicity of Wiskott-Aldrich syndrome-NK cells is maintained. Our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcription in NK cells.

αPIX Associates with Calpain 4, the Small Subunit of Calpain, and Has a Dual Role in Integrin-mediated Cell Spreading

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

Tetraspanin CD82 Attenuates Cellular Morphogenesis through Down-regulating Integrin α6-Mediated Cell Adhesion

Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.

Stimulation of Vascular Smooth Muscle Cell Proliferation and Migration by Apolipoprotein(a) Is Dependent on Inhibition of Transforming Growth Factor-β Activation and on the Presence of Kringle IV Type 9

Elevated plasma concentrations of lipoprotein(a) are a risk factor for the development of a variety of atherosclerotic disorders. Despite intensive study, the mechanisms by which lipoprotein(a) promotes these disorders remain to be unequivocally defined. It has been demonstrated that lipoprotein(a), through its unique constituent apolipoprotein(a) (apo(a)), stimulates vascular smooth muscle cell (SMC) migration and proliferation. These effects arise from the ability of apo(a) to inhibit the formation of active transforming growth factor beta (TGF-beta) from its latent precursor, which in turn is caused by the ability of apo(a) to decrease the formation of plasmin from its precursor plasminogen. We utilized a battery of recombinant apo(a) variants that represent systematic deletions of the various domains in the molecule to further probe the mechanism underlying the effect of apo(a) on SMC responses. All recombinant apo(a) variants that contained kringle IV type 9 were able to stimulate SMC proliferation and migration and to decrease the formation of active TGF-beta; conversely all recombinant apo(a) variants lacking kringle IV type 9 had no effect on these parameters. The kringle IV type 9-dependent effects of apo(a) on SMC proliferation required the presence of plasminogen, suggesting for the first time that this kringle mediates the ability of apo(a) to inhibit pericellular plasmin formation.