Vitronectin deficiency attenuates hepatic fibrosis in a non-alcoholic steatohepatitis-induced mouse model.

Vitronectin (VN), an extracellular matrix protein, is a promising immune biomarker of non-alcoholic steatohepatitis (NASH); however, its precise function remains unclear. This study investigated how VN deficiency contributes to the development of NASH. Towards this aim, wild-type (WT) and VN-/- mice were fed with a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 6 and 10 weeks to induce NASH, and the livers were isolated. In WT mice fed with CDAHFD for 6 and 10 weeks, the expression of Vn mRNA and protein was up-regulated compared with that in mice fed with the MF control diet, indicating that VN is regulated in NASH condition. VN-/- mice showed decreased picrosirius red staining in the liver area and Col1a2 mRNA expression levels, compared with WT mice, indicating that the severity of hepatic fibrosis is attenuated in the CDAHFD-fed VN-/- mice. In addition, VN deficiency did not affect the area of lipid droplets in haematoxylin-eosin staining and the mRNA expression levels of fatty acid synthases, Srebp, Acc and Fas in the CDAHFD-fed mice. Moreover, VN deficiency decreased the inflammation score and the mRNA expression levels of Cd11b and F4/80, macrophage markers, as well as Tnf-α and Il-1ß, inflammatory cytokines in the CDAHFD-fed mice. Furthermore, VN deficiency decreased the protein and mRNA expression levels of α-smooth muscle actin in the CDAHFD-fed mice, suggesting that VN deficiency inhibits the activation of hepatic stellate cells (HSCs). Our findings indicate that VN contributes to the development of fibrosis in the NASH model mice via modulation of the inflammatory reaction and activation of HSCs.

Haemophilus influenzae P4 Interacts With Extracellular Matrix Proteins Promoting Adhesion and Serum Resistance.

Interaction with the extracellular matrix (ECM) is one of the successful colonization strategies employed by nontypeable Haemophilus influenzae (NTHi). Here we identified Haemophilus lipoprotein e (P4) as a receptor for ECM proteins. Purified recombinant P4 displayed a high binding affinity for laminin (Kd = 9.26 nM) and fibronectin (Kd = 10.19 nM), but slightly less to vitronectin (Kd = 16.51 nM). A P4-deficient NTHi mutant showed a significantly decreased binding to these ECM components. Vitronectin acquisition conferred serum resistance to both P4-expressing NTHi and Escherichia coli transformants. P4-mediated bacterial adherence to pharynx, type II alveolar, and bronchial epithelial cells was mainly attributed to fibronectin. Importantly, a significantly reduced bacterial infection was observed in the middle ear of the Junbo mouse model when NTHi was devoid of P4. In conclusion, our data provide new insight into the role of P4 as an important factor for Haemophilus colonization and subsequent respiratory tract infection.

The vitronectin RGD motif regulates TGF-ß-induced alveolar epithelial cell apoptosis.

Transforming growth factor-ß (TGF-ß) is a critical driver of acute lung injury and fibrosis. Injury leads to activation of TGF-ß, which regulates changes in the cellular and matrix makeup of the lung during the repair and fibrosis phase. TGF-ß can also initiate alveolar epithelial cell (AEC) apoptosis. Injury leads to destruction of the laminin-rich basement membrane, which is replaced by a provisional matrix composed of arginine-glycine-aspartate (RGD) motif-containing plasma matrix proteins, including vitronectin and fibronectin. To determine the role of specific matrix proteins on TGF-ß-induced apoptosis, we studied primary AECs cultured on different matrix conditions and utilized mice with deletion of vitronectin (Vtn(-/-)) or mice in which the vitronectin RGD motif is mutated to nonintegrin-binding arginine-glycine-glutamate (RGE) (Vtn(RGE/RGE)). We found that AECs cultured on fibronectin and vitronectin or in wild-type mouse serum are resistant to TGF-ß-induced apoptosis. In contrast, AECs cultured on laminin or in serum from Vtn(-/-) or Vtn(RGE/RGE) mice undergo robust TGF-ß-induced apoptosis. Plasminogen activator inhibitor-1 (PAI-1) sensitizes AECs to greater apoptosis by disrupting AEC engagement to vitronectin. Inhibition of integrin-associated signaling proteins augments AEC apoptosis. Mice with transgenic deletion of PAI-1 have less apoptosis after bleomycin, but deletion of vitronectin or disruption of the vitronectin RGD motif reverses this protection, suggesting that the proapoptotic function of PAI-1 is mediated through vitronectin inhibition. Collectively, these data suggest that integrin-matrix signaling is an important regulator of TGF-ß-mediated AEC apoptosis and that PAI-1 functions as a natural regulator of this interaction.

Notch increased vitronection adhesion protects myeloma cells from drug induced apoptosis.

Notch signaling activation was found in many human cancers including multiple myeloma. It was previously reported that notch contributes to drug resistant of myeloma cells upon chemotherapy treatment, inhibition of notch by inhibitors helped to overcome drug resistance. However, the mechanism of notch developed drug resistance is remained to be fully illustrated. In the current study, we reported that Notch signaling activation up-regulated expression of integrin αvß5 in myeloma cells companied with enhanced cells adhesion on vitronectin. Silencing Notch-1 receptor with siRNA or blocking cells with integrin αvß5 antibody reduced myeloma cells adhesion on vitronectin, importantly, vitronectin mediated adhesion confers protection of myeloma cells from drug induced apoptosis. Thus, we revealed a novel mechanism of myeloma cells resistance to drug induced apoptosis. This study first connected Notch signaling, VTN adhesion and drug resistance together. Therefore, blocking αvß5 receptor with antibody or knock down approach would be a novel promising strategy to treat MM.

Direct evidence of the importance of vitronectin and its interaction with the urokinase receptor in tumor growth.

Extensive evidence implicates the urokinase plasminogen activator receptor (uPAR) in tumor growth, invasion, and metastasis. Recent studies have substantiated the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN) for the signaling activity of the receptor in vitro, however, the possible relevance of this interaction for the activity of uPAR in tumor growth and metastasis has not been assessed. We generated a panel of HEK293 cell lines expressing mouse uPAR (muPAR(WT)), an uPAR mutant specifically deficient in VN binding (muPAR(W32A)), and a truncation variant (muPAR(ΔD1)) deficient in both VN and uPA binding. In vitro cells expressing muPAR(WT) display increased cell adhesion, spreading, migration, and proliferation associated with increased p130Cas and MAPK signaling. Disruption of VN binding or ablation of both VN and uPA binding specifically abrogates these activities of uPAR. When xenografted into SCID (severe combined immunodeficiency) mice, the expression of muPAR(WT), but not muPAR(W32A) or muPAR(ΔD1), accelerates tumor development, demonstrating that VN binding is responsible for the tumor-promoting activity of uPAR in vivo. In an orthotopic xenograft model using MDA-MB-231 cells in RAG1(-/-)/VN(-/-) mice, we document that host deficiency in VN strongly impairs tumor formation. These 2 lines of in vivo experimentation independently demonstrate an important role for VN in tumor growth even if the uPAR dependence of the effect in the MDA-MB-231 model remains to be ascertained.

Studies on primary uveal and cutaneous melanoma cell interaction with vitronectin.

We have examined the diversity between primary uveal (92-1 and Mel202) and cutaneous (FM55P and IGR-39) melanoma cells in their interaction with vitronectin, and established the effect of integrins and ß1,6-branched N-oligosaccharides on this process. The adhesion level of uveal melanoma cells to vitronectin was at least twice lower than that of cutaneous ones, but all cells tested repaired scratch wounds on vitronectin-coated surfaces with similar speed. Swainsonine treatment, by reducing the amount of ß1,6-branches, significantly decreased cell attachment in all cases, but reduction of wound healing efficiency was compromised only in cutaneous melanoma cell. Functional blocking antibodies used in adhesion and migration assays revealed that integrin αvß3 was strongly involved in adhesion and migration only in cutaneous melanoma cells, but its role here was less pronounced than that of integrin αvß5. However, in uveal melanoma the specific anti-αvß5 integrin antibody had no impact on migration speed. Therefore, the anti-α3ß1 integrin antibody was used in order to explain the nature of uveal melanoma interaction with vitronectin, which caused a mild decrease in adhesion efficiency and reduced their motility. Expression of αvß5 integrin differed between the cell lines, but there was no distinct pattern to distinguish uveal melanoma from cutaneous melanoma. In conclusion, αvß5, but not αvß3 integrin is heavily involved in uveal melanoma cell interaction with vitronectin. The role of ß1,6-branched N-glycans in the adhesion, but not during migration, of all cells to vitronectin has been confirmed.

Vitronectin induces phosphorylation of ezrin/radixin/moesin actin-binding proteins through binding to its novel neuronal receptor telencephalin.

Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

Vitronectin inhibits neutrophil apoptosis through activation of integrin-associated signaling pathways.

Vitronectin is present in large concentrations in serum and the extracellular matrix. Although vitronectin is known to modulate neutrophil adhesion and chemotaxis, and to contribute to neutrophil-associated proinflammatory processes, a role in apoptosis has not been demonstrated. In the present studies, we found that neutrophils demonstrated more rapid progression to spontaneous or TNF-related apoptosis-inducing ligand-induced apoptosis when incubated under vitronectin-free conditions than when vitronectin was present. The ability of native vitronectin to delay neutrophil apoptosis was not recapitulated by the vitronectin somatomedin B domain. In contrast, inclusion of the cyclo[Arg-Gly-Asp-D-Phe-Val] peptide in cultures containing vitronectin resulted in enhanced neutrophil apoptosis, showing that the vitronectin RGD motif (Arg-Gly-Asp motif) was responsible for the antiapoptotic effects of vitronectin. Addition of antibodies to ß(1), ß(3), or ß(5), but not to ß(2) or ß(4) integrins, reversed the ability of vitronectin to diminish neutrophil apoptosis. The ability of vitronectin to enhance neutrophil viability was dependent on activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 kinases, but not on the p38 kinase. Increased numbers of apoptotic neutrophils were present in the lungs of LPS-treated transgenic vitronectin-deficient mice, as compared with control mice. These results demonstrate a novel antiapoptotic function for vitronectin.

uPAR-induced cell adhesion and migration: vitronectin provides the key.

Expression of the membrane receptor uPAR induces profound changes in cell morphology and migration, and its expression correlates with the malignant phenotype of cancers. To identify the molecular interactions essential for uPAR function in these processes, we carried out a complete functional alanine scan of uPAR in HEK293 cells. Of the 255 mutant receptors characterized, 34 failed to induce changes in cell morphology. Remarkably, the molecular defect of all of these mutants was a specific reduction in integrin-independent cell binding to vitronectin. A membrane-tethered plasminogen activator inhibitor-1, which has the same binding site in vitronectin as uPAR, replicated uPAR-induced changes. A direct uPAR-vitronectin interaction is thus both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. Collectively these data demonstrate a novel mechanism by which a cell adhesion molecule lacking inherent signaling capability evokes complex cellular responses by modulating the contact between the cell and the matrix without the requirement for direct lateral protein-protein interactions.

Thrombin stimulates integrin ß1-dependent adhesion of human pancreatic cancer cells to vitronectin through protease-activated receptor (PAR)-1.

BACKGROUND/AIMS: The aim of this study was to investigate the effect of thrombin and the thrombin receptor protease-activated receptor (PAR)-1 on adhesion of human pancreatic cancer cell lines to extracellular matrices (ECMs) and to identify related integrins with these effects. METHODOLOGY: Human pancreatic cancer cell lines SUIT-2 and its four sublines, and Panc- 1, AsPC-1 and MiaPaCa-2 were treated with thrombin, PAR-1 agonist TRAP-6, PAR-1 antagonist SCH79797, or anti-integrin ±vß3, ±vß5 and ß1 monoclonal antibodies. Cells were incubated for 45 minutes on micro titer plates that were pre-coated with ECMs (fibronectin, laminin, vitronectin, type IV collagen). The number of adherent cells was measured by the MTT method. RESULTS: Eight human pancreatic cancer cell lines expressed PAR-1. Thrombin significantly enhanced adhesion of SUIT-2 and its sublines and MiaPaCa-2 to vitronectin, especially in the SUIT-2 subline S2-007. We obtained similar results on S2-007 cells through treatment with TRAP-6. However, SCH79797 inhibited the effect of thrombin. Furthermore, anti-integrin ß1 antibody conspicuously inhibited 1U/mL thrombin-induced enhancement of adhesion to vitronectin. CONCLUSIONS: Thrombin significantly enhanced adhesion of pancreatic cancer cells to vitronectin through PAR- 1 depending on the presence of integrin ß1. Suppression of thrombin action by anti-integrin ß1 antibody will become a useful therapy against pancreatic cancer.

Vitronectin in vascular context: facets of a multitalented matricellular protein.

Vitronectin is an abundant adhesive glycoprotein in blood plasma and is found associated with different extracellular matrix sites, the vessel wall, and tumor cells, particularly upon tissue remodeling, injury/repair, or under disease conditions. Plasma vitronectin is a structurally labile molecule that may be converted into a multimeric/multivalent form by interaction with various (hemostatic) factors or through surface binding. Several distinct binding domains along the vitronectin sequence for integrin-type cell adhesion receptors, for urokinase receptor or proteoglycans as well as for growth factors, endow vascular matrix- or fibrin-associated vitronectin with differentiated cell attachment and aggregatory properties. These were found to be relevant for modulation of the cell-matrix interface in angiogenesis, hemostasis and thrombus formation, or wound repair, respectively. Other vitronectin ligands include plasminogen activator inhibitor (PAI)-1 or high molecular weight kininogen that confer strong antiadhesive functions upon integrin- or urokinase receptor-mediated cell interactions with vitronectin. Together, vitronectin acts as a potent matricellular factor, coordinating cell migration with pericellular proteolysis and growth factor signaling at sites of tissue remodeling or in tumors. Structure-function studies of such vitronectin-related ligands and receptors lead to the characterization of their mode of action, also stimulating the search for new antagonists in tumor angiogenesis, platelet aggregation, or atherosclerosis. This review focuses on new developments in vitronectin biology, with particular emphasis on regulatory mechanisms of the protein in the context of cell adhesion/migration/proliferation and cell-dependent proteolysis, relevant for our understanding of hemostasis, thrombosis, tissue repair, and vascular diseases.

Nontypeable Haemophilus influenzae protein E binds vitronectin and is important for serum resistance.

Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a K(D) of 0.4 microM for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84-108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin.

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/alphav integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and alphav integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and alphav integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-alphav integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/alphav integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.

Recombinant plasminogen activator inhibitor-1 inhibits intimal hyperplasia.

OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1) overexpression is implicated in vascular disease. However, the effects of a primary increase in PAI-1 expression on arterial remodeling are poorly defined. We tested the hypothesis that recombinant PAI-1 inhibits intimal hyperplasia after vascular injury. METHODS AND RESULTS: Rats underwent carotid artery injury and received intraperitoneal injections of saline or mutant forms of PAI-1 for 14 days, including an active stable mutant (PAI-1-14-1b), a mutant lacking anti-PA activity (PAI-1-R), or a mutant defective in vitronectin (VN) binding (PAI-1-K). All forms of PAI-1 significantly inhibited neointima formation, whereas elastase-cleaved PAI-1, which lacks both anti-PA and VN-binding functions, did not. Similar effects were observed in a murine model. However, the antiproliferative effect of PAI-1-R was lost in Vn(-/-) mice, suggesting that PAI-1 can inhibit intimal hyperplasia in vivo by a VN-dependent pathway not involving direct inhibition of proteases. In vitro, recombinant PAI-1 inhibited wild-type vascular smooth muscle cell (VSMC) proliferation, promoted apoptosis, and inhibited migration. These effects were lost in VN-deficient VSMCs. CONCLUSIONS: Recombinant PAI-1 inhibits intimal hyperplasia by inhibiting proteases and binding VN. VN is a key determinant of the antiproliferative effect of PAI-1 overexpression. PAI-1-R has therapeutic potential to inhibit vascular restenosis without promoting thrombosis.

Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line.

BACKGROUND: Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible. METHODS: We applied high density SNPa and NGS techniques to in vivo and in vitro models (orthotropic xenograft vitronectin knock-out mice and 3D bioprinted hydrogels with different stiffness) using two representative neuroblastoma cell lines (the MYCN-amplified SK-N-BE(2) and the ALK-mutated SH-SY5Y), to discern how tumor genomics patterns and clonal heterogeneity of the two cell lines are affected. RESULTS: We describe a remarkable subclonal selection of genomic aberrations in SK-N-BE(2) cells grown in knock-out vitronectin xenograft mice that also emerged when cultured for long times in stiff hydrogels. In particular, we detected an enlarged subclonal cell population with chromosome 9 aberrations in both models. Similar abnormalities were found in human high-risk neuroblastoma with MYCN amplification. The genomics of the SH-SY5Y cell line remained stable when cultured in both models. CONCLUSIONS: Focus on heterogeneous intratumor segmental chromosome aberrations and mutations, as a mirror image of tumor microenvironment, is a vital area of future research.

Functional role of vitronectin in breast cancer.

Breast Cancer is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Identification of new biomarkers could be key for early diagnosis and detection. Vitronectin, a glycoprotein that is abundantly found in serum, extracellular matrix, and bone, binds to integrin αvß3, and promotes cell adhesion and migration. Current studies indicate that patients with amplified vitronectin levels have lower survival rates than patients without amplified vitronectin levels. In this study, we focused on the role of vitronectin in breast cancer survival and its functional role as a non-invasive biomarker for early stage and stage specific breast cancer detection. To confirm that the expression of vitronectin is amplified in breast cancer, a total of 240 serum samples (n = 240), 200 from breast cancer patients and 40 controls were analyzed using the Reverse Phase Protein Array (RPPA) technique. Of the 240 samples, 120 samples were of African American (AA) descent, while the other 120 were of White American (WA) descent. Data indicated that there were some possible racial disparities in vitronectin levels and, differences also seen in the recurrent patient samples. Next, we tried to uncover the underlying mechanism which plays a critical role in vitronectin expression. The cellular data from four different breast cancer cell lines- MCF7, MDA-MB-231, MDA-MB-468, and HCC1599 indicated that the PI3K/AKT axis is modulating the expression of vitronectin. We believe that vitronectin concentration levels are involved and connected to the metastasis of breast cancer in certain patients, specifically based on recurrence or ethnicity, which is detrimental for poor prognosis. Therefore, in this current study we showed that the serum vitronectin levels could be an early marker for the breast cancer survival and we also determine the cellular signaling factors which modulate the expression and concentration of vitronectin.

Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression.

Adherent cells assemble fibronectin into a fibrillar matrix on their apical surface. The fibril formation is initiated by fibronectin binding to the integrins alpha5 beta1 and alphav beta3, and is completed by a process that includes fibronectin self-assembly. We found that a 76- amino acid fragment of fibronectin (III1-C) that forms one of the self-assembly sites caused disassembly of preformed fibronectin matrix without affecting cell adhesion. Treating attached fibroblasts or endothelial cells with III1-C inhibited cell migration and proliferation. Rho-dependent stress fiber formation and Rho-dependent focal contact protein phosphorylation were also inhibited, whereas Cdc42 was activated, leading to actin polymerization into filopodia. ACK (activated Cdc42-binding kinase) and p38 MAPK (mitogen-activated protein kinase), two downstream effectors of Cdc42, were activated, whereas PAK (p21-activated kinase) and JNK/SAPK (c-Jun NH2-terminal kinase/ stress-activated protein kinase) were inhibited. III1-C treatment also modulated activation of JNK and ERK (extracellular signal-regulated kinases) in response to growth factors, and reduced the activity of the cyclin E-cdk2 complex. These results indicate that the absence of fibronectin matrix causes activation of Cdc42, and that fibronectin matrix is required for Rho activation and cell cycle progression.

Geometric control of cell life and death.

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

Substrate-bound insulin-like growth factor (IGF)-I-IGF binding protein-vitronectin-stimulated breast cell migration is enhanced by coactivation of the phosphatidylinositide 3-Kinase/AKT pathway by alphav-integrins and the IGF-I receptor.

IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.

Role of alphav integrin in osteoprotegerin-induced endothelial cell migration and proliferation.

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor kappaB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin alpha(v)beta(3) or integrin alpha(v)beta(5) blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-alpha(v)beta(3) antibody or anti-alpha(v)beta(5) antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin alpha(v) activity. Taken together, these results suggest that integrins alpha(v)beta(3) and/or alpha(v)beta(5) contribute to endothelial cell proliferation and migration induced by OPG.