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.

Wound healing in the adult is commonly compromised by excessive scar formation. In contrast, fetal wound healing is a regenerative process characterised by the conspicuous absence of scarring. Available evidence suggests that phenotypic differences between fetal and adult fibroblasts are important determinants of these distinct modes of tissue repair. In this context, a number of groups (including our own) have documented differences between fetal and adult fibroblasts with respect to such potentially relevant characteristics as migratory activity, motogenic response to cytokines and the synthesis of motility factors, cytokines and matrix macromolecules. The oral mucosa appears to be a privileged site in the adult in that it continues to display a fetal‐like mode of wound healing. Data are presented in this review indicating that a subpopulation of gingival fibroblasts expresses several ‘fetal‐like’ phenotypic characteristics. These observations are discussed in terms of both the continued expression of a fetal‐like mode of wound healing in the oral mucosa and the possible differential involvement of distinct fibroblast subpopulations in the progression of periodontal disease.

We have previously shown that (i) human skin fibroblasts of fetal and adult origin display distinctive migratory phenotypes, (ii) this difference in cell behavior results from the production of a soluble "migration stimulating factor" (MSF) by fetal cells, and (iii) skin fibroblasts from breast cancer patients commonly resemble fetal fibroblasts both in migratory phenotype and in production of MSF. Data are now presented indicating that MSF present in the conditioned medium of fetal and cancer patient fibroblasts is precipitated at 10% saturation ammonium sulfate and binds to heparin and cation-exchange resins. Based on this information, we have devised a scheme for the purification of MSF involving the sequential application of ammonium sulfate precipitation, heparin affinity, gel filtration, and reverse-phase chromatography. Purified MSF has an estimated molecular mass of 70 kDa; amino acid analysis reveals a relatively high level of proline (13.34 residues per 100). Our results further suggest that skin fibroblasts from breast cancer patients produce an additional factor with migration stimulating activity; this factor is precipitated at higher concentrations of ammonium sulfate and binds to anion-exchange resins. We have previously discussed the possible direct involvement of fetal-like fibroblasts in cancer pathogenesis. The availability of MSF obtained from cancer patient fibroblasts provides a potential means with which to examine the complex cellular interactions contributing to this process as well as develop a screening regime for identifying individuals at elevated risk of developing cancer.

No abstract

No abstract

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 cellbased 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.Invasion of nonprofessional phagocytes is a common virulence mechanism among bacterial pathogens. It involves the subversion of cellular uptake mechanisms, cell signaling and cytoskeletal dynamics (1, 2). For Staphylococcus aureus and Streptococcus pyogenes, adhesion to, and internalization by, epithelial and endothelial cells is mediated by cell wall-attached fibronectin-binding proteins (FnBPs) 4 FnBPA and SfbI, respectively. FnBPs recruit fibronectin (FN) to the bacterial surface where it forms a molecular bridge between bacteria and host cell integrins (3, 4).FN occurs ubiquitously in vertebrate tissues either in its soluble form or as an insoluble component of fibrillar matrix networks. FN is a disulfide-linked homodimer consisting almost entirely of three types of domains or modules, FNI, FNII, and FNIII (Fig. 1A). FN interacts with other extracellular components and different integrin subfamilies, most notably ␣5␤1, ␣v␤3, and ␣4␤1 (5). The major integrin-binding site in FN, the Arg-Gly-Asp (RGD) motif, is located in the 10th FNIII module ( 10 FNIII). RGD may be cryptic in plasma FN and only exposed upon fibrillogenesis (6 -8). Understanding of FN activation is hampered by the lack of information on its in-solution conformation. Important progress has been made with the discovery of long range interactions between FNI modules and module 3 FNIII in an N-terminal 100-kDa FN fragment (FN100kDa) (Fig. 1A) (9). Although FN100kDa lacks the RGD motif, it is a soluble construct containing cryptic sites that are also observed in full-length FN. Neither FN nor FN100kDa stimulate fibroblast migration into collagen gels (9, 10), which depends on motogenic Ile-Gly-Asp (IGD) motifs (located on 7 FNI and 9 FNI) and ␣v␤3 integrins (11). In contrast, a truncated oncofetal FN isoform known as migrationstimulating factor and the very similar proteolytic N-terminal 70-kDa FN fragment (FN70kDa) (Fig. 1A) are efficient triggers of cell migration (10, 12). The IGD sites appear to be partially exposed in an FN100kDa mutant (FN100kDa-R222A). FN100kDa-R222A lacks a salt bridge between 4 FNI and 3 FNIII that stabilizes a closed (inactive) FN100kDa conformation and is proposed to be broken upon FN activati...

Background The objective of this study was to characterise the incidence and prognostic correlation of perineural invasion (PNI) in oral squamous cell carcinoma and determine whether nerve growth factor and its receptor tyrosine Kinase A expression could be used as biological markers for PNI. Methods A retrospective review of pathology reports of 430 patients with oral squamous cell carcinoma who were treated from 1992 to 2014 in Tayside, Scotland, was carried out. The expression of nerve growth factor and tyrosine kinase A was assessed with immunohistochemistry in 132 tissue sections of oral squamous cell carcinoma. Results Perineural invasion was identified in 17.4% of oral squamous cell carcinomas. High expression of nerve growth factor and tyrosine kinase A was seen in 84% and 92% of oral squamous cell carcinoma, respectively. Tumours with PNI expressed nerve growth factor and tyrosine kinase A with a greater frequency than tumours without PNI. PNI and high expression of nerve growth factor were significantly associated with pain. PNI was significantly associated with stage IV tumours and poor disease‐specific survival. Conclusions A higher level of expression of nerve growth factor and tyrosine kinase A may predict PNI and therefore may be considered as biological markers for PNI in oral squamous cell carcinoma. PNI and nerve growth factor overexpression may contribute to the pain generation in oral cancer patients. PNI and nerve growth factor expression can predict the aggressiveness and prognosis of oral squamous cell carcinoma patients.

Migration stimulating factor (MSF) is a truncated oncofetal fibronectin isoform expressed by fetal and tumor-associated cells. MSF mRNA is distinguished from other fibronectin isoforms by its size (2.1 kb) and the inclusion of a specific intronic sequence at its 3V end. Initial Northern blot analysis with a MSF-specific probe indicated the presence of this 2.1-kb transcript and an additional unexpected 5.9-kb RNA present in both MSF-secreting ( fetal) and nonsecreting (adult) fibroblasts. Our investigations into the nature of these transcripts and their relationship to MSF protein secretion revealed that the 5.9-kb mRNA is a second MSF-encoding transcript. Both these mRNAs have identical coding sequence and differ only in the length of their intron-derived 3V -untranslated region (UTR). The 5.9-kb MSF mRNA is retained in the nucleus whereas the 2.1-kb mRNA is not. MSF-secreting fetal fibroblasts have significantly lower nuclear levels of the 5.9-kb mRNA and correspondingly higher cytoplasmic levels of the 2.1-kb transcript than their nonsecreting adult counterparts. Adult fibroblasts induced to secrete MSF by treatment with transforming growth factor-B1 displayed similar changes in their respective levels of MSF mRNA, but not those of a control gene. When cloned downstream of a reporter gene, only the longer 3V -UTR retained coding sequence within the nucleus. We conclude that expression of MSF protein is regulated by 3V -UTR truncation of the 5.9-kb nuclear-sequestered ''precursor'' MSF mRNA and nuclear export of mature 2.1-kb message. Inducible 3V -UTR processing represents a novel regulatory mechanism involved in cancer pathogenesis that may open new avenues for therapeutic gene delivery. (Cancer Res 2005; 65(23): 10742-9)