The plasma protein vitronectin is thought to be an important regulator of extravascular plasminogen activation. In previous studies we have shown that a disulfide stabilized multimeric form of vitronectin is endocytosed and degraded by fibroblast cells (T.S. Panetti, P.J. McKeown-Longo, J. Biol. Chem. 268 (1993) 11988-11993; P.J. McKeown-Longo, T.S. Panetti, in: K.T. Preissner, S. Rosenblatt, C. Kost, J. Wegerhoff, D.F. Mosher (Eds.), Biology of Vitronectins and their Receptors, Elsevier Science Publishers, Amsterdam, 1993, pp. 111-118). The preparation of multimeric vitronectin used in these earlier studies was in the form of high molecular weight disulfide-bonded aggregates which were stable in sodium dodecyl sulfate (SDS). To address the question of whether vitronectin needed to be in the form of disulfide stabilized multimers in order to be endocytosed, a multimeric vitronectin, which was not disulfide stabilized, was prepared from vitronectin that had been treated with reducing agent and alkylated with iodoacetamide. The resulting protein migrated as a 65/75 kDa protein on SDS gels in the absence of reducing agent, confirming that this form of vitronectin was no longer stabilized into disulfide-bonded aggregates. However, the protein was still multimeric when analyzed by native gels and could be converted to SDS stable multimers by cross-linking agents. This result demonstrated that reduced and alkylated vitronectin aggregates into multimeric forms which are not stable in SDS. Similar to disulfide stabilized multimers, alkylated multimers of vitronectin bound to sulfated proteoglycans in the extracellular matrix and were endocytosed and degraded. Degradation of both forms of vitronectin was inhibited with arginine-glycine-aspartic acid peptides, an anti-alphavbeta5 antibody and heparin. Chloroquine and wortmannin were also able to inhibit degradation of both forms of vitronectin, indicating that both multimeric forms were following the same endocytic and degradative pathway. These results suggest that the organization of vitronectin into a multimeric form which will be recognized for endocytosis does not require disulfide bond stabilization. This study further suggests that recognition of vitronectin for endocytosis is dependent upon its conversion from a monomeric to a multivalent form (C.E. Wilkins-Port, P.J. McKeown-Longo, Mol. Biol. Cell 8:S:64A (1997).

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.

Surface functionalization strategies of synthetic materials for regenerative medicine applications comprise the development of microenvironments that recapitulate the physical and biochemical cues of physiological extracellular matrices. In this context, material-driven fibronectin (FN) nanonetworks obtained from the adsorption of the protein on poly(ethyl acrylate) provide a robust system to control cell behavior, particularly to enhance differentiation. This study aims at augmenting the complexity of these fibrillar matrices by introducing vitronectin, a lower-molecular-weight multifunctional glycoprotein and main adhesive component of serum. A cooperative effect during co-adsorption of the proteins is observed, as the addition of vitronectin leads to increased fibronectin adsorption, improved fibril formation, and enhanced vitronectin exposure. The mobility of the protein at the material interface increases, and this, in turn, facilitates the reorganization of the adsorbed FN by cells. Furthermore, the interplay between interface mobility and engagement of vitronectin receptors controls the level of cell fusion and the degree of cell differentiation. Ultimately, this work reveals that substrate-induced protein interfaces resulting from the cooperative adsorption of fibronectin and vitronectin fine-tune cell behavior, as vitronectin micromanages the local properties of the microenvironment and consequently short-term cell response to the protein interface and higher order cellular functions such as differentiation.

Vitronectin (VN) is a multi-functional protein involved in extracellular matrix (ECM)-cell binding through integrin receptors on the cell surface, which is an important environmental process for maintaining biological homeostasis. We investigated how VN affects the survival of endothelial cells after radiation damage. VN attenuated radiation-induced expression of p21, an inhibitor of cell cycle progression, and selectively inhibited Erk- and p38 MAPK-dependent p21 induction after radiation exposure through regulation of the activity of GSK-3β. VN also reduced the cleavage of caspase-3, thereby inhibiting radiation-induced apoptotic cell death. These results suggest that VN has important roles in cell survival after radiation damage.

Moraxella catarrhalis (Mx) is a common cause of otitis media and exacerbation of chronic obstructive pulmonary disease, an increasing worldwide problem. Surface proteins UspA1 and UspA2 of Mx bind to a number of human receptors and may function in pathogenesis. Genetic recombination events in the pathogen can generate hybrid proteins termed UspA2H. However, whether certain key functions (e.g. UspA1-specific CEACAM binding) can be exchanged between these adhesin families remains unknown. In this study, we have shown that Mx can incorporate the UspA1 CEACAM1-binding region not only into rare UspA1 proteins devoid of CEACAM-binding ability, but also into UspA2 which normally lack this capacity. Further, a screen of Mx isolates revealed the presence of novel UspA2 Variant proteins (UspA2V) in ∼14% of the CEACAM-binding population. We demonstrate that the expression of UspA2/2V with the CEACAM-binding domain enable Mx to bind both to cell surface CEACAMs and to integrins, the latter via vitronectin. Such properties of UspA2/2V have not been reported to date. The studies demonstrate that the UspA family is much more heterogeneous than previously believed and illustrate the in vivo potential for exchange of functional regions between UspA proteins which could convey novel adhesive functions whilst enhancing immune evasion.

Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206-217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M-CSF- and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.

Vitronectin (VTN) is a glycoprotein enriched in the blood and activates integrin receptors. VTN blood levels increase only in female mice 24 h after an ischemic stroke and exacerbate brain injury through IL-6-driven inflammation, but the VTN induction mechanism is unknown. Here, a 30 min middle cerebral artery occlusion (MCAO) in female mice induced VTN protein in the liver (normally the main source) in concert with plasma VTN. Male mice were excluded as VTN is not induced after stroke. MCAO also increased plasma VTN levels after de novo expression of VTN in the liver of VTN-/- female mice, using a hepatocyte-specific (SERPINA1) promoter. MCAO did not affect SERPINA1 or VTN mRNA in the liver, brain, or several peripheral organs, or platelet VTN, compared to sham mice. Thus, hepatocytes are the source of stroke-induced increases in plasma VTN, which is independent of transcription. The cholinergic innervation by the parasympathetic vagus nerve is a potential source of brain-liver signaling after stroke. Right-sided vagotomy at the cervical level led to increased plasma VTN levels, suggesting that VTN release is inhibited by vagal tone. Co-culture of hepatocytes with cholinergic neurons or treatment with acetylcholine, but not noradrenaline (sympathetic transmitter), suppressed VTN expression. Hepatocytes have muscarinic receptors and the M1/M3 agonist bethanechol decreased VTN mRNA and protein release in vitro via M1 receptors. Finally, systemic bethanechol treatment blocked stroke-induced plasma VTN. Thus, VTN translation and release are inhibited by muscarinic signaling from the vagus nerve and presents a novel target for lessening detrimental VTN expression.

As the primary resident immune cells, microglia play a central role in regulating inflammatory processes in the CNS. The extracellular matrix (ECM) protein vitronectin promotes microglial activation, switching microglia into an activated phenotype. We have shown previously that microglia express two vitronectin receptors, αvβ3 and αvβ5 integrins. As these integrins have well-defined roles in activation and phagocytic processes in other cell types, the purpose of the current study was to investigate the contribution of these two integrins in microglial activation.

We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5 mg/mL results for NPs. However, NPs in the range 0.01-0.30 mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1 μg/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation.

Vitronectin is a plasma protein which can deposit into the extracellular matrix where it supports integrin and uPA dependent cell migration. In earlier studies, we have shown that the plasma protein, vitronectin, stimulates focal adhesion remodeling by recruiting urokinase-type plasminogen activator (uPA) to focal adhesion sites [Wilcox-Adelman, S. A., Wilkins-Port, C. E., McKeown-Longo, P. J., 2000. Localization of urokinase-type plasminogen activator to focal adhesions requires ligation of vitronectin integrin receptors. Cell. Adhes. Commun.7, 477-490]. In the present study, we used a variety of vitronectin constructs to demonstrate that the localization of uPA to adhesion sites requires the binding of both vitronectin integrin receptors and the uPA receptor (uPAR) to vitronectin. A recombinant fragment of vitronectin containing the connecting sequence (VN(CS)) was able to support integrin-dependent adhesion, spreading and focal adhesion assembly by human microvessel endothelial cells. Cells adherent to this fragment were not able to localize uPA to focal adhesions. A second recombinant fragment containing both the amino-terminal SMB domain and the CS domain was able to restore the localization of uPA to adhesion sites. This fragment, which contains a uPAR binding site, also resulted in the localization of uPAR to adhesion sites. uPAR blocking antibodies as well as phospholipase C treatment of cells inhibited uPA localization to adhesion sites confirming a role for uPAR in this process. The SMB domain alone was unable to direct either uPAR or uPA to adhesion sites in the absence of the CS domain. Our results indicate that vitronectin-dependent localization of uPA to adhesion sites requires the sequential binding of vitronectin integrins and uPAR to vitronectin.

Pancreatic cancer is a malignant cancer common worldwide having poor prognosis, even when diagnosed at its early stage. Cell adhesion plays a critical role in cancer invasion and metastasis. Integrins are major mediators of cell adhesion and play an important role in invasion and metastatic growth of human pancreatic cancer cells. Snake disintegrins are the most potent ligands of several integrins and have potential therapeutic applications for cancers. We have previously cloned and expressed a new recombinant RGD-disintegrin from Crotalus adamanteus (r-Cam-dis). This recently published r-Cam-dis has an extra nine amino acids derived from the vector (SPGARGSEF) at the N-terminus end and has strong anti-platelet activity. However, this r-Cam-dis contains the contamination of the cleavage of the N-terminal end of the pET-43.1a cloning vector. In this study, we have cloned r-Cam-dis in a different cloning vector (pGEX-4T-1) showing five different amino acids (GSPEF) at the N-terminal part. This new r-Cam-dis was expressed and tested for inhibition of platelet aggregation, specific binding activity with seven different integrins, and inhibition of adhesion of three different pancreatic cancer cell lines on laminin-1 and vitronectin. The r-Cam-dis showed potent binding to αvβ3 integrin, but was moderate to weak with αvβ5, αvβ6, α2β1, and α6β1. Interestingly, the inhibition of r-Cam-dis on pancreatic cancer cell lines adhesion to laminin-1 was more effective than that to vitronectin. Based on our binding results to integrin receptors and previous adhesion studies using function-blocking monoclonal antibodies, it is suggested that r-Cam-dis could be inhibiting adhesion of pancreatic cancer cell lines through integrins α2β1, α6β1, αvβ5, and αvβ6.

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50-100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.

Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/β5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.

Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.

Integrin-mediated cell attachment modulates growth responses and growth factors regulate cell attachment. Moreover, both cell attachment to extracellular matrix and mitogenic signaling by growth factors are necessary for the proliferation of most types of normal cells, suggesting that integrin and growth factor receptor signaling pathways meet at some downstream point. We report here that a small, highly tyrosine-phosphorylated fraction of PDGFbeta and insulin receptors co-immunoprecipitates with the alphavbeta3 integrin from cells. The integrin association requires growth factor stimulation of the receptors. Several signaling molecules that are known to be associated with activated growth factor receptors were present in the alphavbeta3 integrin complexes. Mitogenicity and chemotaxis induced by PDGF-BB were enhanced in cells plated on the alphavbeta3 ligand vitronectin compared with cells plated on the beta1 integrin ligand collagen. Thus, the engagement of the alphavbeta3 integrin in cell-matrix interactions appears to coordinate an intense response to growth factors, helping to explain the importance of this integrin for tissue regeneration, angiogenesis and tumor metastasis.

During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR(88-92) chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR(S90P) exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR(S90P) cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR(S90E) cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR(S90P). In conclusion, our findings indicate that Ser(90) is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR(S90E) and uPAR(S90P) are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types. Following principles of developmental biology and translational applicability, here we developed an efficient stepwise differentiation method for peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest, followed by differentiation into sensory neurons. Detailed characterization, including ultrastructural analysis, confirmed that the hPSC-derived nociceptors displayed cellular and molecular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors relevant for pain research and axonal growth/regeneration studies (e.g., TRPV1, NAV1.7, NAV1.8, TAC1, CALCA, GAP43, DPYSL2, NMNAT2). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.

Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor alphavbeta3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.