Ian R. Ellis

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains 4–5FnI and 3FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes 3FnIII and disrupts the observed 4–5FnI-3FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.

Cooperative binding and activation of fibronectin by a bacterial surface protein.

Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens.

The Role of the Fibronectin IGD Motif in Stimulating Fibroblast Migration

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 106 times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.

Differential involvement of TGF-β1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1-10 microg/ml) and inhibition at high concentrations (25-100 microg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5-25 microg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to alphavbeta3 integrin. TGF-beta1 (0.1-200 ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-beta1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-beta1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-beta1, whereas the effects on fibroblast and tumour cell migration are TGF-beta1-independent.

Collagen Gel Assay for Angiogenesis

The inner lining of blood vessels, the endothelium, consists of a monolayer of endothelial cells (ECs), that present a free luminal surface and attach on their abluminal side to the underlying basement membrane (apart from a minimal amount of cell-cell overlap). A great deal of heterogeneity exists in the morphology of the endothelium and in the phenotype displayed by individual ECs. In spite of this, all ECs may be defined by two general criteria: anatomical location (i.e., luminal wall of blood vessels) and functionality (e.g., provision of a nonthrombogenic surface). In a mature resting vessel, the functionality and integrity of the endothelium is maintained under steady state conditions by the biosynthetic activity of the ECs, in conjunction with low levels of cell proliferation and motility. Significant changes in the motility of the endothelial cells, often accompanied by cell proliferation, occur during angiogenesis and in response to vessel injury.

Is there a pAkt between VEGF and oral cancer cell migration

The PI3K-Akt signalling pathway is a well-established driver of cancer progression. One key process promoted by Akt phosphorylation is tumour cell motility; however the mechanism of VEGF-induced Akt phosphorylation leading to motility remains poorly understood. Previously, we have shown that Akt phosphorylation induced by different factors causes both stimulation and inhibition of motility in different cell types. However, differential phosphorylation of Akt at T308 and S473 residues by VEGF and its role in head and neck cancer cell motility and progression is unknown. The cell lines investigated in this study exhibited a change in phosphorylation of Akt in response to VEGF. However, in terms of motility, VEGF stimulated oral cancer and its associated cell lines, but not normal keratinocytes or oral mucosal fibroblasts. The addition of a PI3 kinase and mTOR inhibitor, inhibited the phosphorylation of Akt and also effectively blocked VEGF-induced oral cancer cell motility, whereas only the PI3 kinase inhibitor blocked oral cancer associated fibroblast cell motility. This study therefore discloses that two different mechanisms of Akt phosphorylation control the motility potential of different cell lines. Akt phosphorylated at both residues controls oral cancer cell motility. Furthermore, immunohistochemical analysis of VEGF positive human head and neck tumour tissues showed a significant increase in Akt phosphorylation at the T308 residue, suggesting that pAkt T308 may be associated with tumour progression in vivo.

The control and importance of hyaluronan synthase expression in palatogenesis

Development of the lip and palate involves a complex series of events that requires the close co-ordination of cell migration, growth, differentiation, and apoptosis. Palatal shelf elevation is considered to be driven by regional accumulation and hydration of glycosoaminoglycans, principally hyaluronan (HA), which provides an intrinsic shelf force, directed by components of the extracellular matrix (ECM). During embryogenesis, the extracellular and pericellular matrix surrounding migrating and proliferating cells is rich in HA. This would suggest that HA may be important in both shelf growth and fusion. TGFβ3 plays an important role in palatogenesis and the corresponding homozygous null (TGFβ3−/−) mouse, exhibits a defect in the fusion of the palatal shelves resulting in clefting of the secondary palate. TGFβ3 is expressed at the future medial edge epithelium (MEE) and at the actual edge epithelium during E14.5, suggesting a role for TGFβ3 in fusion. This is substantiated by experiments showing that addition of exogenous TGFβ3 can “rescue” the cleft palate phenotype in the null mouse. In addition, TGFβ1 and TGFβ2 can rescue the null mouse palate (in vitro) to near normal fusion. In vivo a TGFβ1 knock-in mouse, where the coding region of the TGFβ3 gene was replaced with the full-length TGFβ1 cDNA, displayed complete fusion at the mid portion of the secondary palate, whereas the anterior and posterior regions failed to fuse appropriately. We present experimental data indicating that the three HA synthase (Has) enzymes are differentially expressed during palatogenesis. Using immunohistochemistry (IHC) and embryo sections from the TGFβ3 null mouse at days E13.5 and E14.5, it was established that there was a decrease in expression of Has2 in the mesenchyme and an increase in expression of Has3 in comparison to the wild-type mouse. In vitro data indicate that HA synthesis is affected by addition of exogenous TGFβ3. Preliminary data suggests that this increase in HA synthesis, in response to TGFβ3, is under the control of the PI3kinase/Akt pathway.

Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor)

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as (3)FnI, (5)FnI, (7)FnI and (9)FnI, respectively. We have previously reported that mutation of IGD motifs in modules (7)FnI and (9)FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in (3)FnI and (5)FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within (1-5)FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in (7)FnI and (9)FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.

Migration Stimulating Factor (MSF) promotes fibroblast migration by inhibiting AKT

The protein kinase AKT is activated strongly by many motogenic growth factors, yet has recently been shown capable of inhibiting migration in several cell types. Here we report that treatment with Migration Stimulating Factor (MSF), a truncated form of fibronectin that promotes the migration of many cell types, inhibits AKT activity in human fibroblasts and endothelial cells. In fibroblasts, treatment with either MSF or the AKT inhibitor, Akti-1/2, stimulated migration into 3D collagen gels to a similar extent and the effects of Akti-1/2 on migration could be blocked by the expression of an inhibitor-resistant mutant, AKT1 W80A. These data indicate that MSF promotes fibroblast migration, at least in part, by inhibiting the activity of AKT.

Bistable switch in migration stimulating factor expression: regulation by the concerted signalling of transforming growth factor-β1 and the extracellular matrix.

Migration stimulating factor (MSF) is an oncofetal motogenic/angiogenic cytokine constitutively expressed by epithelial and stromal cells in fetal and neoplastic tissues. Fibroblasts derived from healthy adult skin do not express MSF but can be induced to do so by treatment with transforming growth factor‐β1 (TGF‐β1). As the bioactivities of both MSF and TGF‐β1 are modulated by the extracellular matrix, we investigated whether the induction of MSF expression by TGF‐β1 is also matrix dependent. We now report that adult fibroblasts are induced to express MSF by a transient treatment with TGF‐β1 (as short as 2 hr) but only when the cells are adherent to a “wound” matrix, such as denatured type I collagen, fibrin or plastic tissue culture dishes. Unexpectedly, this induction of MSF expression persists unabated for the entire subsequent lifespan of the treated cells in the absence of further TGF‐β1 and irrespective of the substratum. Such “activated” MSF expression may, however, be persistently switched off again by a second transient exposure to TGF‐β1 but this time only when the cells are adherent to a “healthy” matrix of native type I collagen. Significantly, the constitutive expression of MSF by fetal and cancer patient fibroblasts could also be persistently switched off by this means. We conclude that TGF‐β1 may both switch on and switch off MSF expression in a manner critically determined by the nature of the matrix substratum and suggest that this may be a possible mechanism underlying the observed dual functionality of TGF‐β1 as both a tumour suppressor and tumour promoter.