A mathematical model of integrin-mediated haptotactic cell migration

Haptotactic cell migration, a directed response to gradients of cell—extracellular matrix adhesion, is an important process in a number of biological phenomena such as wound healing and tumour cell invasion. Previously, mathematical models of haptotaxis have been developed on the premise that cells migrate in response to gradients in the density of the extracellular matrix. In this paper, we develop a novel mathematical model of haptotaxis which includes the adhesion receptors known as integrins and a description of their functional activation, local recruitment and protrusion as part of lamellipodia. Through the inclusion of integrins, the modelled cell matter is able to respond to a true gradient of cell–matrix adhesion, represented by functionally active integrins. We also show that previous matrix-mediated models are in fact a subset of the novel integrin-mediated models, characterised by specific choices of diffusion and haptotaxis coefficients in their model equations. Numerical solutions suggest the existence of travelling waves of cell migration that are confirmed via a phase plane analysis of a simplified model.

Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding

We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 3 1, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3 beta. We also present evidence that PV signaling is activated via the alpha 6 beta 4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection. (c) 2006 Elsevier Inc. All rights reserved

Palmitate induces insulin resistance in monocytes and increases expression of the integrin CD11b

Obesity and insulin resistance are important risk factors for atherosclerosis, and elevated level of plasma NEFA is a common feature in individuals with obesity and insulin resistance. Palmitate, one of the most abundant non-esterified SFA in plasma, has been reported to induce insulin resistance in adipose tissues and skeletal muscles and to cause an increased inflammatory response in monocytes. The present study investigated whether palmitate can induce insulin resistance in monocytes and its effect on monocyte adhesion molecular expression (CD11b). Insulin resistance was measured by in vitro uptake of insulin-stimulated 3H-labelled 2-deoxy-D-glucose into THP-1 cells, cell surface CD11b expression was measured by flow cytometry. The data showed that palmitate-induced insulin resistance in THP-1 monocytes was concentration and time dependent (Figure 1). The insulin-stimulated glucose uptake was significantly decreased in cells treated with 300 mM-palmitate compared with control cells (P<0.001) and was observed within 6 h, but was not a result of palmitate toxicity. There was no significant increase in caspase 3 activation (P>0.05). Treatment with 300 mM-palmitate for 24 h also caused a significant increase in surface CD11b expression in both U937 and THP-1 monocytic cell lines and human primary monocytes compared with the control (P<0.001). Both these effects were inhibited by co-incubation with Fumonisin B1, an inhibitor of de novo ceramide synthesis. In conclusion, these data show that palmitate, at physiological concentrations, can cause insulin resistance in monocytes and increase monocyte surface integrin CD11b expression, which is in part the result of the synthesis of ceramide.

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and α5β1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ß1 integrin co-signaling pathway. By using a5 null cells, ß1 integrin functional blocking antibody, and a a5ß1 integrin targeting peptide A5-1, we demonstrate that the a5 and ß1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCa is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.

Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFa production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the I?Ba. Low basal levels of I?Ba explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-?B. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

Fibronectin-tissue transglutaminase matrix rescues RGD-impaired celladhesion through syndecan-4 and β integrin co-signaling

Heterotropic association of tissue transglutaminase (TG2) with extracellular matrix-associated fibronectin (FN) can restore the adhesion of fibroblasts when the integrin-mediated direct binding to FN is impaired using RGD-containing peptide. We demonstrate that the compensatory effect of the TG-FN complex in the presence of RGD-containing peptides is mediated by TG2 binding to the heparan sulfate chains of the syndecan-4 cell surface receptor. This binding mediates activation of protein kinase Ca (PKCa) and its subsequent interaction with ß1 integrin since disruption of PKCa binding to ß1 integrins with a cell-permeant competitive peptide inhibits cell adhesion and the associated actin stress fiber formation. Cell signaling by this process leads to the activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases. Fibroblasts deficient in Raf-1 do not respond fully to the TG-FN complex unless either the full-length kinase competent Raf-1 or the kinase-inactive domain of Raf-1 is reintroduced, indicating the involvement of the Raf-1 protein in the signaling mechanism. We propose a model for a novel RGD-independent cell adhesion process that could be important during tissue injury and/or remodeling whereby TG-FN binding to syndecan-4 activates PKCa leading to its association with ß1 integrin, reinforcement of actin-stress fiber organization, and MAPK pathway activation.

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons.

Angiopoietin-1 induces migration of monocytes in a tie-2 and integrin-independent manner

Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whether Ang-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in the progression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that is independent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1 promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulated transendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly to monocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primary monocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1 binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogated Ang-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. In summary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase and heparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role in inflammatory angiogenesis and may promote atherosclerosis.

Mathematical modelling of integrin-like receptors systems

Nel presente lavoro, ho studiato e trovato le soluzioni esatte di un modello matematico applicato ai recettori cellulari della famiglia delle integrine. Nel modello le integrine sono considerate come un sistema a due livelli, attivo e non attivo. Quando le integrine si trovano nello stato inattivo possono diffondere nella membrana, mentre quando si trovano nello stato attivo risultano cristallizzate nella membrana, incapaci di diffondere. La variazione di concentrazione nella superficie cellulare di una sostanza chiamata attivatore dà luogo all’attivazione delle integrine. Inoltre, questi eterodimeri possono legare una molecola inibitrice con funzioni di controllo e regolazione, che chiameremo v, la quale, legandosi al recettore, fa aumentare la produzione della sostanza attizzatrice, che chiameremo u. In questo modo si innesca un meccanismo di retroazione positiva. L’inibitore v regola il meccanismo di produzione di u, ed assume, pertanto, il ruolo di modulatore. Infatti, grazie a questo sistema di fine regolazione il meccanismo di feedback positivo è in grado di autolimitarsi. Si costruisce poi un modello di equazioni differenziali partendo dalle semplici reazioni chimiche coinvolte. Una volta che il sistema di equazioni è impostato, si possono desumere le soluzioni per le concentrazioni dell’inibitore e dell’attivatore per un caso particolare dei parametri. Infine, si può eseguire un test per vedere cosa predice il modello in termini di integrine. Per farlo, ho utilizzato un’attivazione del tipo funzione gradino e l’ho inserita nel sistema, valutando la dinamica dei recettori. Si ottiene in questo modo un risultato in accordo con le previsioni: le integrine legate si trovano soprattutto ai limiti della zona attivata, mentre le integrine libere vengono a mancare nella zona attivata.

Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4β7 Integrin CD4+ T cells : Implications for HIV Microbicides

This work was supported by the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No 305316 as part of the MOTIF (Microbicides Optimisation Through Innovative Formulation for Vaginal and Rectal Delivery) project. We would like to extend our thanks to all the study participants for their invaluable contribution and to Grampian Biorepository staff for help with collection of fresh colorectal resection tissue.

Integrin traffic and signalling – from plasma membrane to endosomes

Integrins are the main cell surface receptors by which cells adhere to the surrounding extracellular matrix (ECM). Cells regulate integrin-mediated adhesions by integrin endo/exocytic trafficking or by altering the integrin activation status. Integrin binding to ECM-components induces several intracellular signalling cascades, which regulate almost every aspect of cell behaviour from cell motility to survival, and dysregulation of integrin traffic or signalling is associated with cancer progression. Upon detachment, normal cells undergo a specialised form of programmed cell death namely anoikis and the ECM-integrin -mediated activation of focal adhesion kinase (FAK) signalling at the cell surface has been considered critical for anoikis suppression. Integrins are also constantly endocytosed and recycled back to the plasma membrane, and so far the role of integrin traffic in cancer has been linked to increased adhesion site turnover and cell migration. However, different growth factor receptors are known to signal also from endosomes, but the ability of integrins to signal from endosomes has not been previously studied. In this thesis, I demonstrate for the first time that integrins are signalling also from endosomes. In contrast to previous believes, integrin-induced focal adhesion kinase (FAK) signalling occurs also on endosomes, and the endosomal FAK signalling is critical for anoikis suppression and for cancer related processes such as anchorage-independent growth and metastasis. Moreover, we have set up a new integrin trafficking assay and demonstrate for the first time in a comprehensive manner that active and inactive integrins undergo distinct trafficking routes. Together these results open up new horizons in our understanding of integrins and highlight the fundamental connection between integrin traffic and signalling.

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.

Peptidomimetic ligands for tuning integrin-mediated signalling: rational design, synthesis and theranostic applications

Integrins are α/β-heterodimeric transmembrane adhesion receptors that mediate cell-cell and cell-ECM interactions. Integrins are bidirectional signalling receptors that respond to external signals (“outside-in” signalling) and in parallel, transduce internal signals to the matrix (“inside-out” signalling), to regulate vital cellular functions including migration, survival, growth and differentiation. Therefore, dysregulation of these tightly regulated processes often results in uncontrolled integrin activation and abnormal tissue expression that is responsible for many diseases. Because of their important roles in physiological and pathological events, they represent a validated target for therapeutic and diagnostic purposes. The aim of the present Thesis was focused on the development of peptidic ligands for α4β1 and αvβ3 integrin subtypes, involved in inflammatory responses (leukocytes recruitment and extravasation) and cancer progression (angiogenesis, tumor growth, metastasis), respectively. Following the peptidomimetic strategy, we designed and synthesized a small library of linear and cyclic hybrid α/β-peptidomimetics based on the phenylureido-LDV scaffolds for the treatment of chronic inflammatory autoimmune diseases. In order to implement a fast and non-invasive diagnostic method for monitoring the course of the inflammatory processes, a flat glass-surface of dye-loaded Zeolite L-crystal nanoparticles was coated with bioactive α4β1-peptidomimetics to detect specific integrin-expressing cells as biomarkers of inflammatory diseases. Targeted drug delivery has been considered a promising alternative to overcome the pharmacokinetic limitations of conventional anticancer drugs. Thus, a novel Small-Molecule Drug Conjugate was synthesized by connecting the highly cytotoxic Cryptophycin to the tumor-targeting RGDfK-peptide through a protease-cleavable linker. Finally, in view to making the peptide synthesis more sustainable and greener, we developed an alternative method for peptide bonds formation employing solvent-free mechanochemistry and ultra-mild minimal solvent-grinding conditions in common, inexpensive laboratory equipment. To this purpose, standard amino acids, coupling agents and organic-green solvents were used in the presence of nanocrystalline hydroxyapatite as a reusable, bio-compatible inorganic basic catalyst.

Disarray Of Glomerular And Tubular Cell Adhesion Molecules In The Course Of Experimental Bothrops Moojeni Envenomation.

In this study, we show that administration of Bothrops moojeni venom in rats induces a general disturbance in the distribution and content of the tight junctional protein ZO-1, the cell-matrix receptor beta 1 integrin, the cytoskeletal proteins, vinculin and F-actin, and of the extracellular matrix component laminin in renal corpuscles and cortical nephron tubules. These findings suggest that cell-cell and cell-matrix adhesion proteins may be molecular targets in the B. moojeni-induced kidney injury.