Extracellular Matrix Protein Vitronectin Research Articles

Platycodin D Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Blocking the Activation of Mitogen-Activated Protein Kinases and the Production of Interleukin-8.

Platycodin D is a major constituent in the root of Platycodon grandiflorum and has diverse pharmacologic activities, including anti-inflammatory, anti-allergic, and antitumor activities. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are potent angiogenic factors and contribute to tumor angiogenesis by directly and indirectly promoting angiogenic processes, including the proliferation, adhesion, migration, and tube formation of endothelial cells. Here, we found that platycodin D at noncytotoxic concentrations inhibited VEGF-induced proliferation, adhesion to the extracellular matrix proteins fibronectin and vitronectin, chemotactic motility, and tube formation of human umbilical vein endothelial cells (HUVECs). Platycodin D reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) and the secretion of IL-8 in VEGF-stimulated HUVECs. Moreover, platycodin D inhibited tube formation and the phosphorylation of ERK and p38 in IL-8-stimulated HUVECs. The in vitro anti-angiogenic activity of platycodin D was confirmed by in vivo experimental models. Platycodin D inhibited the formation of new blood vessels into mouse Matrigel plugs with VEGF or IL-8. In mice injected with MDA-MB-231 human breast cancer cells, orally administered platycodin D inhibited tumor growth, the number of CD34 [Formula: see text]vessels, and the expression of VEGF and IL-8. Taken together, platycodin D directly and indirectly prevents VEGF-induced and IL-8-induced angiogenesis by blocking the activation of mitogen-activated protein kinases (MAPKs). Platycodin D may be beneficial for the prevention or treatment of tumor angiogenesis and angiogenesis-related human diseases.

The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin

Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16-18 nm Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvβ3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvβ3 integrin.

Small changes in PDMMLA structure influence the adsorption behavior of ECM proteins and syndecan-4 on PDMMLA derivative surfaces: Experimental validation by tensiometric surface force measurements

Cardiovascular diseases are the leading cause of death around the world according to the World Health Organization. In-stent restenosis is an inflammatory response of the immune system to endovascular stent implantation in atherosclerotic patients. Biocompatible and biodegradable polymers are of great interest in this field in order to limit the side effects of stent treatments. Poly([R,S]-3,3-dimethylmalic acid) (PDMMLA) is a new biodegradable statistical polyester which presents promising properties as a stent coating. In this work, we studied by dynamic tensiometry, the adhesion of extracellular matrix proteins (bovine serum albumin, fibronectin, fibrinogen, and vitronectin) and plasma membrane proteoglycan (syndecan-4) on three PDMMLA derivatives with different hydrophilicity levels. The results show that proteins have different adhesion profiles and affinity on these surfaces. They show similar behavior on the most hydrophilic surface, making hydrophilic, ionic and hydrogen type bonds. Then we compared each protein’s individual profile to that of a mixture of all studied proteins. The comparison shows that vitronectin and syndecan-4 are the quantitatively dominating proteins adsorbed by specific interactions. Based on the results from previous studies, this work allowed us to identify the most important PDMMLA surface as a promising biomaterial for bioactive stent-coating.

Vascular differentiation from pluripotent stem cells in 3-D auxetic scaffolds.

Auxetic scaffolds, that is, scaffolds that can display negative Poisson's ratio, have unique physical properties and can expand transversally when axially strained or contract under compression. Auxetic materials have been used for bioprostheses and artery stents due to the enhanced compressive strength and shear stiffness. In vascular tissue engineering, auxetic scaffolds allow the widening of blood vessels when blood flows through (creating compressive stress) to prevent the blockage. However, the influence of auxetic materials on the cellular fate decision in local environment is unclear. In this study, auxetic polyurethane foams were used to support vascular differentiation from pluripotent stem cells. The expression of alkaline phosphatase, Oct-4, and Nanog was lower after 4days of differentiation for the cells grown in auxetic scaffolds. Higher expression of vascular markers CD31 and VE-cadherin was observed for the cells from auxetic scaffolds compared with those from the scaffolds before auxetic conversion. Little influence on the expression of cardiac marker α-actinin was observed. The vascular cells secreted extracellular matrix proteins vitronectin and laminin and expressed membrane-bound matrix metalloproteinase 9. The examination of Yes-associated protein expression indicated more cytoplasmic retention in the cells from auxetic scaffolds compared with those from regular scaffolds, suggesting that the auxetic scaffolds may affect cellular contraction. This study demonstrates a novel 3-D culture based on auxetic scaffolds for vascular differentiation and provides a platform to study the influence of biophysical microenvironments on differentiation of pluripotent stem cells. The outcome of this study has implications for regenerative medicine and drug discovery.

Overexpression of the Vitronectin V10 Subunit in Patients with Nonalcoholic Steatohepatitis: Implications for Noninvasive Diagnosis of NASH.

Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM protein vitronectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosis.

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae.

The disintegrin tzabcanin inhibits adhesion and migration in melanoma and lung cancer cells

Integrins play an essential role in cancer survival and invasion, and they have been major targets in drug development and design. Disintegrins are small (4–16kDa) viperid snake venom proteins that exhibit a canonical integrin-binding site (often RGD). These non-enzymatic proteins inhibit integrin-mediated cell–cell and cell-extracellular matrix interactions, making them potential candidates as therapeutics in cancer and numerous other human disorders. The present study examined the cytotoxic, anti-adhesion, and anti-migration effects of a recently characterized disintegrin, tzabcanin, towards melanoma (A-375) and lung (A-549) cancer cell lines. Tzabcanin inhibits adhesion of both cells lines to vitronectin and exhibited very weak cytotoxicity towards A-375 cells; however, it had no effect on cell viability of A-549 cells. Further, tzabcanin significantly inhibited migration of both cell lines in cell scratch/wound healing assays. Flow cytometric analysis indicates that both A-375 and A-549 cell lines express integrin αvβ3, a critical integrin in tumor motility and invasion, and a major receptor of the extracellular matrix protein vitronectin. Flow cytometric analysis also identified αvβ3 as a binding site of tzabcanin. These results suggest that tzabcanin may have utility in the development of anticancer therapies, or may be used as a biomarker to detect neoplasms that over-express integrin αvβ3.

A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels.

Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.

The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization

Although it belongs to the group of coagulase-negative staphylococci, Staphylococcus lugdunensis has been known to cause aggressive courses of native and prosthetic valve infective endocarditis with high mortality similar to Staphylococcus aureus. In contrast to S. aureus, only little is known about the equipment of S. lugdunensis with virulence factors including adhesins and their role in mediating attachment to extracellular matrix and plasma proteins and host cells. In this study, we show that the multifunctional autolysin/adhesin AtlL of S. lugdunensis binds to the extracellular matrix and plasma proteins fibronectin, fibrinogen, and vitronectin as well as to human EA.hy926 endothelial cells. Furthermore, we demonstrate that AtlL also plays an important role in the internalization of S. lugdunensis by eukaryotic cells: The atlL-deficient mutant Mut17 adheres to and becomes internalized by eukaryotic cells to a lesser extent than the isogenic wild-type strain Sl253 and the complemented mutant Mut17 (pCUatlL) shows an increased internalization level in comparison to Mut17. Thus, surface localized AtlL that exhibits a broad binding spectrum also mediates the internalization of S. lugdunensis by eukaryotic cells. We therefore propose an internalization pathway for S. lugdunensis, in which AtlL plays a major role. Investigating the role of AtlL in biofilm formation of S. lugdunensis, Mut17 shows a significantly reduced ability for biofilm formation, which is restored in the complemented mutant. Thus, our data provide evidence for a significant role for AtlL in adherence and internalization processes as well as in biofilm formation of S. lugdunensis.

Abstract 3468: Integrin-β6 is required for transforming growth factor (TGF)-β-mediated epithelial-mesenchymal transition (EMT) and promotes the disease progression of intrahepatic cholangiocellular carcinoma

Abstract Background: Intrahepatic cholangiocellular carcinoma (ICC) is characterized as a poor prognostic disease represented by the highly invasiveness. Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer invasion and metastasis, and then TGF-β signaling is a potent inducible factor of EMT. Integrin-β6, which is an epithelial restricted trans-membrane protein, promotes binding to extracellular matrix proteins (fibronectin, vitronectin and tenascin) facilitating cell migration and activates TGFβ1 via binding to the latency associated peptide (LAP) of the TGF-β complex during embryogenesis and wound healing. In this study, we investigated the role of integrin-β6 during TGF-β-mediated EMT using ICC cell lines, and examined the clinic-pathological significance of Integrinβ6 expression in ICC patients. Material and Methods: We measured integrin-β6 mRNA expression by real time PCR using 7 ICC cell lines (Hucct1, HuH28, OZ, RBE, Ssp25, TFK1, Ysccc). To examine the role of integrin-β6 during TGF-β-mediated EMT in ICC cell lines, we downregulated integrin-β6 expression by RNAi during TGF-β-medaited EMT. Furthermore, using 55 resected ICC specimens, we evaluated integrin-β6 expression by immunohistochemical staining. Results: Of 7 ICC cell lines, Ysccc and OZ cell lines revealed high integrin-β6 expressions. Addition of TGF-β to Ysccc cell successfully induced EMT, characterized by the upregulated vimentin expression, downregulated E-cadherin expression, spindle-shaped morphology, and high invasiveness, with the integrin-β6 induction. The inhibition of integrin-β6 expression using RNAi abolished TGF-β-mediated EMT induction. In clinical setting, high integrin-β6 expression was significantly associated with Stage IV, lymph node metastasis, and blood vessel invasion, and then resulted in worse prognosis compared with low expression group. Conclusion : Integrin-β6 is required for TGF-β-mediated EMT and promotes the disease progression of ICC. Citation Format: Takaaki Higashi, Hiromitu Hayashi, Hideyuki Kuroki, Shigeki Nakagawa, Keita Sakamoto, Hidetoshi Nitta, Akira Chikamoto, Toru Toru, Hideo Baba. Integrin-β6 is required for transforming growth factor (TGF)-β-mediated epithelial-mesenchymal transition (EMT) and promotes the disease progression of intrahepatic cholangiocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3468. doi:10.1158/1538-7445.AM2014-3468

Acute and delayed deferoxamine treatment attenuates long-term sequelae after germinal matrix hemorrhage in neonatal rats.

This study investigated if acute and delayed deferoxamine treatment attenuates long-term sequelae after germinal matrix hemorrhage (GMH). Bacterial collagenase (0.3 U) was infused intraparenchymally into the right hemispheric ganglionic eminence in P7 rat pups to induce GMH. GMH animals received either deferoxamine or vehicle twice a day for 7 consecutive days. Deferoxamine administration was initiated at either 1 hour or 72 hours post-GMH. Long-term neurocognitive deficits and motor coordination were assessed using Morris water maze, rotarod, and foot fault tests between day 21 to 28 post-GMH. At 28 days post-GMH, brain morphology was assessed and extracellular matrix protein (fibronectin and vitronectin) expression was determined. Acute and delayed deferoxamine treatment improved long-term motor and cognitive function at 21 to 28 days post-GMH. Attenuated neurofunction was paralleled with improved overall brain morphology at 28 days post-GMH, reducing white matter loss, basal ganglia loss, posthemorrhagic ventricular dilation, and cortical loss. GMH resulted in significantly increased expression of fibronectin and vitronectin, which was reversed by acute and delayed deferoxamine treatment. Acute and delayed deferoxamine administration ameliorated long-term sequelae after GMH.

9-cis-Retinoic Acid and Troglitazone Impacts Cellular Adhesion, Proliferation, and Integrin Expression in K562 Cells

Retinoids are established pleiotropic regulators of both adaptive and innate immune responses. Recently, troglitazone, a PPAR gamma agonist, has been demonstrated to have anti-inflammatory effects. Separately, retinoids and troglitazone are implicated in immune related processes; however, their combinatory role in cellular adhesion and proliferation has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) and troglitazone on K562 cellular adhesion and proliferation was investigated. Troglitazone exposure decreased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin, FN-120, and vitronectin in a concentration and time-dependent manner. In the presence of troglitazone, 9-cis-retinoic acid restores cellular adhesion to levels comparable to vehicle treatment alone on fibronectin, FN-120, and vitronectin substrates within 72 hours. Due to the prominent role of integrins in attachment to extracellular matrix proteins, we evaluated the level of integrin α5 subunit expression. Troglitazone treatment results in decrease in α5 subunit expression on the cell surface. In the presence of both agonists, cell surface α5 subunit expression was restored to levels comparable to vehicle treatment alone. Additionally, troglitazone and 9-cis-RA mediated cell adhesion was decreased in the presence of a function blocking integrin alpha 5 inhibitor. Further, through retinoid metabolic profiling and HPLC analysis, our study demonstrates that troglitazone augments retinoid availability in K562 cells. Finally, we demonstrate that troglitazone and 9-cis-retinoic acid synergistically dampen cellular proliferation in K562 cells. Our study is the first to report that the combination of troglitazone and 9-cis-retinoic acid restores cellular adhesion, alters retinoid availability, impacts integrin expression, and dampens cellular proliferation in K562 cells.

Integrin alphavbeta5 identifies human corneal epithelial stem cells

AbstractPurpose Stem cell (SC) therapy is the main treatment modality to restore vision in patients with limbal stem cell deficiency. If corneolimbal epithelial stem cells (LESC) can be readily identified, isolated and maintained ex vivo, better quality grafts can be generated for patients with this debilitating disease. With prior knowledge that the extracellular matrix protein vitronectin (VN) is present within the LESC niche and that it supports SC in vitro, we postulated that the VN receptor αvβ5 is expressed by, and can be used to identify and isolate SC within the cornea.Methods Epithelial cells were isolate and expand from cadaveric human corneas and αvβ5 expression determined by flow cytometry and immunofluorescence. Integrin expressing cells were isolated by magnetic‐activated cell sorting then assessed by immunocytology, colony forming efficiency, RT‐PCR and microarray analysis.Results Integrin αvβ5+ cells co‐localized to N‐cadherin+/CK‐15+ putative LESC. αvβ5 was restricted to 3% of the total epithelial cells, which expressed more SC markers and formed more colonies compared to αvβ5‐ cells. Transcriptional profiling of αvβ5+/‐ cells by global gene array identified several interferon‐inducible genes, including the chemokine IP‐10, which localized to LESC. The role of IP‐10 in these cells is yet to be elucidated, but may be involved in immunoprotection by attracting mesenchymal SC into the corneal stroma.Conclusion Integrin αvβ5 is a candidate SC marker since its expression is restricted and αvβ5+ limbal epithelial cells have properties of LESC. Knowledge of the niche’s composition and the genes expressed by its SC will facilitate isolation and maintenance of these cells in culture for therapeutic purposes.

Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces

Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1μM) concentration of MeHgCl. β-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to μm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.

Vitronectin–αvβ3 Integrin Engagement Directs Hypoxia-Resistant mTOR Activity and Sustained Protein Synthesis Linked to Invasion by Breast Cancer Cells

The tumor microenvironment is a crucial player in the ability of cancer cells to acquire the ability to survive under the hypoxic environment and promote migration and invasion. Translational regulation is an essential part of cancer development and progression. Protein synthesis consumes considerable cellular metabolic energy and is therefore highly regulated, in turn controlling tumor cell proliferation and survival in extreme tumor-host conditions. Protein synthesis is typically downregulated by hypoxia, impairing cell proliferation and migration. Here, we show that breast cancer cells expressing integrin αvβ3, when engaging the extracellular matrix (ECM) protein vitronectin, strongly upregulate both mTOR activity and cap-dependent mRNA translation, which overrides their inhibition by hypoxia and facilitates tumor cell invasion. Interaction of vitronectin with integrin αvβ3 results in the continued activation of the kinase mTOR despite hypoxia through a mechanism that is dependent on integrin-linked kinase but is independent of focal adhesion kinase. Continuous activation of mTOR despite hypoxia involves release of translation initiation factor eIF4E from its repressor protein 4E-BP1, which is required for vitronectin-mediated tumor cell invasion. As integrin αvβ3 is associated with breast cancer cell invasion and metastasis to bone, we propose that the interaction with specific ECM proteins can influence cancer cell invasion, in part, by hyperactivation of mTOR, thereby promoting and sustaining protein synthesis under hypoxic conditions.

Lotus-Leaf-Like Topography Predominates over Adsorbed ECM Proteins in Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Surface/Cell Interactions

It is well-known that extracellular matrix (ECM) proteins mediate cell/surface interactions. However, introduction of a specific surface topography may disturb the correlation between ECM proteins adsorption and cells adhesion on a given surface. In present study, lotus-leaf-like topography was introduced on the surface of a biodegradable material, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Protein adsorption and cell interactions with this lotus-leaf-like surface (designated PHBHHx-L) were investigated. Water contact angle data indicated that the hydrophobicity of PHBHHx was enhanced by the introduction of lotus-leaf-like topography. The adsorption of extracellular matrix proteins (fibronectin and vitronectin) on PHBHHx-L was measured by enzyme linked immunosorbent assay (ELISA). Compared with flat PHBHHx, adsorption on the PHBHHx-L surface increased by ~260% for fibronectin and ~40% for vitronectin. In contrast, fibroblast and endothelial cell adhesion and proliferation were reduced on the PHBHHx-L compared to the flat polymer surface. These results suggest that the inhibition of cell adhesion and proliferation caused by the lotus-leaf-like topography dominates over the effect of the adsorbed adhesive proteins in promoting adhesion and proliferation. It can be concluded that the lotus-leaf-like topography plays a dominant role in cell/PHBHHx-L interactions. The present findings indicate the complexity of the interplay among surface topography, adsorbed proteins, and cell-surface interactions.

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

AbstractExtensive 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 (muPARWT), an uPAR mutant specifically deficient in VN binding (muPARW32A), and a truncation variant (muPARΔD1) deficient in both VN and uPA binding. In vitro cells expressing muPARWT 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 muPARWT, but not muPARW32A or muPARΔD1, 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.

Complex protein nanopatterns over large areas via colloidal lithography

The patterning of biomolecules at the nanoscale provides a powerful method to investigate cellular adhesion processes. A novel method for patterning is presented that is based on colloidal monolayer templating combined with multiple and angled deposition steps. Patterns of gold and SiO2 layers are used to generate complex protein nanopatterns over large areas. Simple circular patches or more complex ring structures are produced in addition to hierarchical patterns of smaller patches. The gold regions are modified through alkanethiol chemistry, which enables the preparation of extracellular matrix proteins (vitronectin) or cellular ligands (the extracellular domain of E-cadherin) in the nanopatterns, whereas the selective poly(l-lysine)–poly(ethylene glycol) functionalization of the SiO2 matrix renders it protein repellent. Cell studies, as a proof of principle, demonstrate the potential for using sets of systematically varied samples with simpler or more complex patterns for studies of cellular adhesive behavior and reveal that the local distribution of proteins within a simple patch critically influences cell adhesion.

The Unique Structure of Haemophilus influenzae Protein E Reveals Multiple Binding Sites for Host Factors

Haemophilus influenzae protein E (PE) is a multifunctional adhesin involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data at 1.8 Å. Here, we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened β-barrel. It is, however, not a true β-barrel, as there are differences in both the hydrogen-bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel β-sheet with a rigid α-helix at the C terminus tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41 to 68) is exposed, while the vitronectin binding region (residues 84 to 108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin to be present in many bacterial genera of the family Pasteurellaceae and also orthologues in other, unrelated species (Enterobacter cloacae and Listeria monocytogenes). Peptides corresponding to the surface-exposed regions PE 24 to 37, PE 74 to 89, and PE 134 to 156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognized PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor of H. influenzae simultaneously interacts with host vitronectin, laminin, or plasminogen, promoting bacterial pathogenesis.

The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG’s effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.