Modulation of Myoepithelial-Associated α6β4 Integrin in a Breast Cancer Cell Line Alters Invasive Potential

In normal breast, cell–stromal contact is mediated by myoepithelial cells which strongly express α2β1, α3β1, and α6β4 integrins, while epithelial cells exhibit α2β1 and α3β1 integrins at cell–cell borders, but do not express α6β4 integrin. Breast carcinomas consistently show down-regulation of all integrins. We have investigated the modulatory effect of stromal proteins, hormones, and transforming growth factor β (TGF-β) on integrin expression in breast cancer cell lines MCF-7, T47-D, and MDA-MB 231 using indirect immunofluorescence and confocal laser scanning microscopy. MCF-7 and T47-D cells displayed low levels of both α2β1 and α3β1 integrins, and no α6β4 integrin, and this profile remained unchanged by modulatory agents. The MDA-MB 231 cells exhibited stronger staining for α2β1 and α3β1 integrins and focal staining for α6β4 integrin under control conditions, but markedly enhanced reactivity for the α6β4 complex in the presence of TGF-β. This was associated with acquisition of a spread cellular morphology and localization of α6β4 at the cell periphery in a discrete punctate distribution. There was associated enhanced expression of epiligrin, the ligand for α6β4, with similar localization to the cell periphery. Cell invasion assays through a Matrigel barrier revealed significantly reduced invasive potential of TGF-β-treated cells, an effect largely reversed following preincubation of the treated cells with anti-β4 integrin antibody. We conclude that α6β4 integrin can be up-regulated by TGF-β and has an anti-invasive effect on MDA-MB 231 cells. In addition to α6β4, MDA-MB 231 cells exhibit other myoepithelial markers including cytokeratin 14, vimentin, and weak expression of CALLA. These findings support the concept of a subgroup of breast carcinomas displaying features of myoepithelial differentiation.     

Tubulointerstitial De Novo Expression of the α8 Integrin Chain in a Rodent Model of Renal Fibrosis – A Potential Target for Anti-Fibrotic Therapy?

In the normal kidney, the α8 integrin chain is expressed only on mesangial cells and vascular smooth muscle cells. α8 integrin ligates several matrix molecules including fibronectin, osteopontin and fibrillin-1. Recently, we detected de novo expression of α8 integrin on epithelial cells in renal cysts. We hypothesized that the α8 integrin chain is induced in tubular epithelia undergoing dedifferentiation and contributes to the fibrotic response in the tubulointerstitium (TI) after unilateral ureteral obstruction (UUO). After induction of UUO in rats by ligation of the right ureter, increased expression of the α8 integrin chain and its ligands was observed. In the TI, α8 integrin was localized to cytokeratin-positive epithelial cells and to interstitial fibroblasts; and colocalized with its ligands. In mice underexpressing α8 integrin UUO led to collagen deposition and fibroblast activation comparable to wild types. Mice lacking α8 integrin showed even more TI damage, fibroblast activation and collagen deposition after UUO compared to wild type mice. We conclude that the expression of the α8 integrin chain and its ligands is strongly induced in the TI after UUO, but underexpression of α8 integrin does not attenuate TI fibrosis. Mice lacking the α8 integrin chain are even more susceptible to TI damage than wild type mice. Thus, interactions of α8 integrin with its ligands do not seem to contribute to the development or progression of TI fibrosis in UUO. Targeting α8 integrin might not be a useful approach for anti-fibrotic therapy.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.     

Dual Control by Divalent Cations and Mitogenic Cytokines of α4β1 and α5β1 Integrin Avidity Expressed by Human Hemopoietic Cells

Beta-1 integrins have essential functions in hemopoietic and immune systems by controlling phenomenons such as cell homing and cell activation. The function α4β1 and α5β1 integrins is regulated by divalent cations and, as demonstrated more recently, by mitogenic cytokines which activate them by “inside-out” mechanisms. Using the adhesive interaction of a cytokine-dependent human hemopoietic cell line to immobilized fibronectin, we have analyzed the requirements in divalent cations Mn²⁺, Mg²⁺ and Ca²⁺ for α4β1 and α5β1 activation by “inside-out” mechanisms triggered by cytokines such as granulocyte-macrophage colony stimulating factor or KIT ligand, or by external conformational constraints with the function-activating anti-β₁ integrin monoclonal antibody 8A2. The intrinsic difference between these two modes of β₁ integrin activation was revealed by their different requirements in divalent cations. We found that in the absence of any divalent cations, α4β1 and α5β1 were non-functional even after further stimulation by cytokines or 8A2. However, whilst either Ca²⁺, Mg²⁺ or Mn²⁺ were able to restore adhesive functions of α4β1 and α5β1 when activated by 8A2, only Mg²⁺ and Mn²⁺ were able to support activation of α5β1 and α5β1 by cytokines. Furthermore, high concentrations of Ca²⁺ exceeding 20 mM dramatically inhibited cell adhesion to fibronectin induced by Mn²⁺ and cytokines but not by 8A2. On the contrary, in the presence of both Ca²⁺ and Mg²⁺, Mn²⁺ had an additive effect on the activation of α5β1 and α5β1 by mitogenic cytokines. The presence of the absence of these divalent cations did not inhibit early tyrosine phosphorylation induced by the binding of KIT ligand to its tyrosine-kinase receptor KIT. Therefore, we propose that in hemopoietic cells, Ca²⁺, Mg²⁺ and Mn²⁺ may modulate in vivo α4β1 and α5β1 regulation by mitogenic cytokines, a phenomenon involved in the regulation of hemopoietic progenitor cell homing within the bone marrow.     

BMP-7 Enhances Cell Migration and αvβ3 Integrin Expression via a c-Src-Dependent Pathway in Human Chondrosarcoma Cells

Bone morphogenic protein (BMP)-7 is a member of the transforming growth factor (TGF)-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.     

Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

The α(6)β(4) integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α(6)β(4) integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α(6)β(4) expression status. Integrin α(6)β(4)-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α(6)β(4)-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α(6)β(4) signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α(6)β(4) cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β(1) integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α(6)β(4) facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.     

RGDechi-hCit: αvβ3 Selective Pro-Apoptotic Peptide as Potential Carrier for Drug Delivery into Melanoma Metastatic Cells

αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.     

Release of cAMP Gating by the α6β4 Integrin Stimulates Lamellae Formation and the Chemotactic Migration of Invasive Carcinoma Cells

The α6β4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949–960). We demonstrate here using MDA-MB-435 breast carcinoma cells that α6β4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by α6β4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon α6β4 expression. Both lamellae formation and chemotactic migration are inhibited or “gated” by cAMP and our results reveal that a critical function of α6β4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that α6β4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.     

Impact of α-Targeted Radiation Therapy on Gene Expression in a Pre-Clinical Model for Disseminated Peritoneal Disease when Combined with Paclitaxel

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.     

Distribution of Laminin Variants and Their Integrin Receptors in Human Secondary Lymphoid Tissue: Colocalization suggests that the α6β4-integrin is a receptor for laminin-5 in lymphoid follicles

Laminins are a family of multifunctional basement membrane glycoproteins. Over the last years, many laminin isoforms have been characterized, which were shown to be composed of distinct combinations of variant α β and γ chains. Some of these isoforms show remark-able tissue specificity, which suggests functional involvement in local processes. In this study the previously described mAb 4C7. which recognize epithelial basement membranes as well as endothelial basement membranes in lymphoid follicles, was identified as an anti-laminin-5 antibody. Using a set of mAbs against various variant laminin chains we established that specifically the γ₂ chain of laminin-5 was confined to the endothelial basement membranes of vessels in lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid tissue. Additionally. the expression of the known integrin receptors of laminin-5 was also examined. The α6β4 integrin-receptor for laminin was found to be colocalized with the laminin-5 γ₂ chain on the abluminal surface of endothelial cells, whereas the α3 integrin chain could not be detected in lymphoid follicles. This finding suggests that the α6β4 integrin (and not the α3β1 integrin) serves as a laminin-5 receptor on endothelial cells in the follicular compartment of lymphoid tissue. Furthermore, α6β4 was also found in the same punctuated pattern on FDCs as laminin-5. The function of the laminin-α6β4 complex in this particular localisation is still obscure, but a role in the maintainance of the follicular compartment via hemidesmosome-like attachment sites is postulated.     

Integrin α4β1 controls G9a activity that regulates epigenetic changes and nuclear properties required for lymphocyte migration

The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4β1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4β1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4β1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.     

Integrin α6β4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes

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Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival

Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.     

Strain-induced Differentiation of Fetal Type II Epithelial Cells Is Mediated via the Integrin α6β1-ADAM17/Tumor Necrosis Factor-α-converting Enzyme (TACE) Signaling Pathway

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α₆ and β₁ to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α₆ and β₁ antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α₆β₁ integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.     

A Novel Aminothiazole KY-05009 with Potential to Inhibit Traf2- and Nck-Interacting Kinase (TNIK) Attenuates TGF-β1-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Adenocarcinoma A549 Cells

Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (K _i, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.     

Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex

The human adenovirus E4orf6 and E1B55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains elongins B and C and cullin family member Cul5. E1B55K associates with this complex, where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. In addition to p53, its first known substrate, the E4orf6/E1B 55-kDa complex (E4orf6/E1B55K) was shown to promote the degradation of Mre11 and DNA ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seems likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and blockage of export of the bulk cellular mRNAs from the nucleus. In an attempt to identify new E4orf6/E1B55K substrates, we undertook a proteomic screen using human p53-null, non-small-cell lung carcinoma H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection by appropriate adenovirus vectors. One cellular protein that appeared to be degraded by E1B55K in combination with the E4orf6 protein was a species of molecular mass ∼130 kDa that was identified as the integrin α3 subunit (i.e., very late activation antigen 3 alpha subunit). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions.Viruses are well known to promote replication by inhibiting or enhancing endogenous cellular machinery or, in some cases, by reprogramming key cellular pathways. Human adenoviruses have developed effective ways to modulate the immune response, apoptosis, double-strand break repair, mRNA export, and translation to optimize virus replication and the spreading of progeny virions. The expression of adenovirus E1A proteins stabilizes p53 and induces apoptosis (8, 33); however, this effect is reversed in infected cells by the action of two early products: the E1B 55-kDa (E1B55K) and E4orf6 proteins (35, 36). We and others have shown that these proteins act through the formation of an E3 ubiquitin ligase complex analogous to the SCF and VBC complexes but which contains, in addition to elongins B and C and the RING protein Rbx1, the cullin family member Cul5 (18, 41, 43). This E4orf6-mediated E3 ligase complex blocks p53-induced apoptosis (35, 36) by promoting the ubiquitination of p53, followed by its degradation by proteasomes (41, 43). E4orf6 protein mediates the assembly of the complex by its interaction with elongin C through its three BC boxes (11, 41, 43). E1B55K, which appears to associate with the E4orf6 protein only when present in the ligase complex (4), is thought to function as a substrate recognition factor that brings substrates to the complex because, although both E4orf6 and E1B55K bind p53 independently, interaction of E1B55K with p53 is essential for the efficient degradation of p53 (41, 48). In addition to protecting infected cells from early lysis via p53-induced apoptosis, the E4orf6/E1B55K ligase complex performs other functions essential for virus replication. Two other substrates of the complex have been identified: a member of the MRN DNA repair complex, Mre11, and the central component of the nonhomologous end-joining DNA repair system, DNA ligase IV (2, 56). Degradation of both of these proteins prevents viral genome concatenation, which interferes with the packaging of viral DNA into virions (2, 56). E1B55K binds to p53, Mre11, and DNA ligase IV and has been demonstrated to colocalize with p53 and Mre11 in perinuclear cytoplasmic bodies termed aggresomes (1, 2, 32). More recently, we and others have obtained results that suggest that the E4orf6-associated E3 ligase complex regulates viral and cellular mRNA export (5, 66). The Cul5-based ligase activity was shown to be essential for selective viral mRNA export and the block of cellular mRNA export from the nucleus (66), thus contributing to the shutoff of cellular protein synthesis initiated by L4-100K (20). The actual substrates of the complex responsible for regulating mRNA export are currently unknown.As discussed in detail below, our efforts to identify substrates of the E4orf6/E1B55K complex led us to consider a member of the integrin family as a potential substrate. Integrins are members of a family of surface receptors that function in several ways through the formation of cell-extracellular matrices and cell-cell interactions (reviewed in references 21, 26, and 63). Integrins are typically composed of two transmembrane glycoproteins forming heterodimers of α and β subunits each of approximately 80 to 150 kDa. There are at least 18 α subunits and 8 β subunits in mammals that can dimerize in limited combinations to form more than 20 functionally distinct integrins with different ligand specificities. Integrin heterodimers function as transmembrane receptors that link external factors to intracellular signaling pathways. In addition to roles in cell adhesion, these communication events are implicated in a large range of cellular processes, including proliferation, differentiation, translation, migration, and apoptosis. Some of these processes depend on the intracellular trafficking pathways of the integrins (reviewed in references 9, 24, 40, and 44), including the long-loop recycling pathway in which integrins present in clathrin-coated endosomes move first to the perinuclear recycling center, where some accumulate, including the β1 integrin subunit (31), before returning to the plasma membrane. The integrin α3β1 is a member of the β1 integrin subfamily in which the α3 subunit (VLA-3a) is coupled to the β1 subunit to form the very late activation antigen (VLA-3 or CD49c) (21, 59, 60). α3β1 is expressed in a wide range of tissues in which it binds a variety of extracellular matrix substrates, including fibronectin, collagen, thrombospondin 1, and laminins 1, 5, 8, 10, and 11 (13). These associations allow the integrin α3β1 to fill its primary role in cell adhesion. α3β1 also participates in intercellular adhesion through several protein-protein interactions (10, 27, 53, 55, 58), making it a major contributor in the regulation of cellular adhesion.Human adenovirus type 5 (Ad5) particles interact with cell surface receptors to facilitate internalization into target cells. In the high-affinity interacting model (reviewed in reference 29), the viral fiber knob polypeptide binds the coxsackie adenovirus receptor (CAR) protein on the surface of cells as the primary cell binding event (primary receptor). The penton base polypeptide then binds a cell surface integrin (secondary receptor), leading to entry of the capsid into the cell by a process termed receptor-mediated endocytosis or clathrin-mediated endocytosis. Several types of integrins have been identified as being used by Ad5 to mediate virus internalization: αMβ1, αMβ2, αVβ1, αVβ3, αVβ5, and α5β1 (22, 30, 49, 65). Salone et al. have shown that α3β1 serves as an alternative cellular receptor for adenovirus serotype 5 (49). It promotes entry of the virus into cells, transduction of DNA, and mediates adenovirus infection in both CAR-positive and CAR-negative cell lines. Thus, in addition to functions related to cell adhesion, integrin α3β1 plays an important role in the adenovirus infection cycle.To identify new targets for degradation by the E4orf6/E1B55K ubiquitin ligase, we used a proteomic screen covering most cellular proteins to look for any polypeptide that exhibited a significant decrease in amount following the coexpression from appropriate adenovirus vectors of the E4orf6 protein and E1B55K. This screen revealed several interesting candidates, including integrin α3, a species of 130 kDa that also was found to be reduced in wild-type (wt) virus infection. The degradation of α3 was seen to be dependent on the Cul5-based ligase complex driven by E4orf6 and E1B55K. We also found evidence that the E4orf6/E1B55K ligase complex appears to be involved in cell detachment from the extracellular matrix, a function that could play a role in virus spread.     

α-Mangostin Suppresses Phorbol 12-myristate 13-acetate-Induced MMP-2/MMP-9 Expressions via αvβ3 Integrin/FAK/ERK and NF-κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

The purpose of this study is to investigate the anti-metastatic effect of α-mangostin on phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions in A549 human lung adenocarcinoma cells. Firstly, α-mangostin could inhibit PMA-induced abilities of the adhesion, invasion, and migration. Data also showed α-mangostin could inhibit the activation of αvβ3 integrin, focal adhesion kinase (FAK), and extracellular signal-regulated kinase1/2 (ERK1/2) involved in the downregulation the enzyme activities, protein and messenger RNA levels of MMP-2 and MMP-9 induced by PMA. Next, α-mangostin also strongly inhibited PMA-induced degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding abilities of NF-κB by α-mangostin treatment was further observed. Furthermore, reduction of FAK or ERK1/2 phosphorylation by FAK small interfering RNA (FAK siRNA) potentiated the effect of α-mangostin. Finally, the transient transfection of ERK siRNA significantly down-regulated the expressions of MMP-2 and MMP-9 concomitantly with a marked inhibition on cell invasion and migration. Presented results indicated α-mangostin is a novel, effect, anti-metastatic agent that functions by downregulating MMP-2 and MMP-9 gene expressions.