A cryptic fragment from fibronectin's III1 module localizes to lipid rafts and stimulates cell growth and contractility

The interaction of cells with the extracellular matrix (ECM) form of fibronectin (FN) triggers changes in growth, migration, and cytoskeletal organization that differ from those generated by soluble FN. As cells deposit and remodel their FN matrix, the exposure of new epitopes may serve to initiate responses unique to matrix FN. To determine whether a matricryptic site within the III1 module of FN modulates cell growth or cytoskeletal organization, a recombinant FN with properties of matrix FN was constructed by directly linking the cryptic, heparin-binding COOH-terminal fragment of III1 (III1H) to the integrin-binding III8–10 modules (glutathione-S-transferase [GST]–III1H,8–10). GST–III1H,8–10 specifically stimulated increases in cell growth and contractility; integrin ligation alone was ineffective. A construct lacking the integrin-binding domain (GST–III1H,2–4) retained the ability to stimulate cell contraction, but was unable to stimulate cell growth. Both GST–III1H,2–4 and matrix FN colocalized with caveolin and fractionated with low-density membrane complexes by a mechanism that required heparan sulfate proteoglycans. Disruption of caveolae inhibited the FN- and III1H-mediated increases in cell contraction and growth. These data suggest that a portion of ECM FN partitions into lipid rafts and differentially regulates cytoskeletal organization and growth, in part, through the exposure of a neoepitope within the conformationally labile III1 module

Identification of the heparin-binding determinants within fibronectin repeat III1: Role in cell spreading and growth

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III1 repeat (FNIII 1). In this study, we localized the heparin-binding activity of FNIII1 to a cluster of basic amino acids, Arg613, Trp 614, Arg615, and Lys617. Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin- and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII1. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin\u27s heparin-binding domain inhibited both β1 and non-β1 integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin- associated proteins α-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton

Lack of Chemokine Signaling through CXCR5 Causes Increased Mortality, Ventricular Dilatation and Deranged Matrix during Cardiac Pressure Overload

RATIONALE: Inflammatory mechanisms have been suggested to play a role in the development of heart failure (HF), but a role for chemokines is largely unknown. Based on their role in inflammation and matrix remodeling in other tissues, we hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling during HF. OBJECTIVE: We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5 in cardiac pathophysiology leading to HF. METHODS AND RESULTS: Mice harboring a systemic knockout of the CXCR5 (CXCR5(-/-)) displayed increased mortality during a follow-up of 80 days after aortic banding (AB). Following three weeks of AB, CXCR5(-/-) developed significant left ventricular (LV) dilatation compared to wild type (WT) mice. Microarray analysis revealed altered expression of several small leucine-rich proteoglycans (SLRPs) that bind to collagen and modulate fibril assembly. Protein levels of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced in AB CXCR5(-/-) compared to AB WT mice. Electron microscopy revealed loosely packed extracellular matrix with individual collagen fibers and small networks of proteoglycans in AB CXCR5(-/-) mice. Addition of CXCL13 to cultured cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF, we observed increased myocardial levels of CXCR5 and SLRPs, which was reversed following LV assist device treatment. CONCLUSIONS: Lack of CXCR5 leads to LV dilatation and increased mortality during pressure overload, possibly via lack of an increase in SLRPs. This study demonstrates a critical role of the chemokine CXCL13 and CXCR5 in survival and maintaining of cardiac structure upon pressure overload, by regulating proteoglycans essential for correct collagen assembly

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Fibronectin Polymerization Regulates the Composition and Stability of Extracellular Matrix Fibrils and Cell-Matrix Adhesions

Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell–matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither α5β1 integrin nor tensin localize to fibrillar cell–matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell–matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation

The influence of extracellular matrix fibronectin on platelet-derived growth factor signaling

Thesis (Ph. D.)--University of Rochester. Department of Biomedical Engineering, 2017.Cell signaling in response to platelet-derived growth factor (PDGF) has been found to be either up- or downregulated in a variety of pathological conditions, making PDGF-induced signaling pathways important and attractive therapeutic targets. The difficulty in translating PDGF signaling to therapeutic interventions arises from the complexity of the cellular responses to PDGF, wherein broadly up- or downregulating PDGF signaling often has off-target effects due to the multitude of PDGF-induced signaling pathways and cellular responses to PDGF. The limited success of PDGF in tissue engineering and regenerative medicine applications may be overcome with a clearer understanding of how cells integrate signals from PDGF and the extracellular matrix (ECM). Fibronectin is a principal component of the ECM that is produced in a soluble, protomeric form and then assembled by cells into an insoluble, fibrillar matrix. ECM fibronectin is known to influence ECM composition, as well as cell and tissue function. Understanding how ECM fibronectin directs cell and tissue responses to PDGF is critical to designing targeted therapies for pathologies characterized by both aberrant ECM assembly and PDGF signaling, such as asthma, liver cirrhosis, atherosclerosis, pulmonary fibrosis, cancer and chronic wounds. In this thesis, the effect of cell-assembled ECM fibronectin on cellular responsiveness to PDGF was assessed using intracellular calcium release as a measure of cellular responsiveness to PDGF. Results of this work demonstrate that ECM fibronectin attenuates the PDGF-PI3K-calcium signaling axis at the level of PI3K activation. ECM fibronectin did not have an effect on other intracellular signals activated by PDGF, including activation of PDGF receptor β, AKT, phospholipase Cγ1, or ERK1/2. The bioactive effects of fibronectin were localized to the α5β1 integrinbinding FNIII9-10 region. Finally, a cell-binding fibronectin fragment with a truncated FNIII module, FNIII8c-10, attenuated PDGF-induced intracellular calcium release and PI3K activation. Thus, this newly developed protein has the potential to be employed as a therapeutic to specifically attenuate the PDGF-induced intracellular calcium release cascade while leaving intact other PDGF signaling pathways

Using fibronectin matrix mimetics to stimulate 3D cellular self-assembly

Thesis (Ph. D.)--University of Rochester. Department of Biomedical Engineering, 2015.Cellular self-assembly is a process that occurs during embryonic development in which cells and extracellular matrix (ECM) spontaneously organize into three-dimensional (3D) tissues in the absence of external forces. Cellular self-assembly can also be initiated in vitro, and represents a potential tool for tissue engineers to organize cells and ECM in an in vivo-like manner. Understanding the cell-ECM interactions that lead to the formation of 3D cellular structures will better allow tissue engineers to develop biological constructs in vitro for regenerative medicine purposes. Fibronectin is an ECM protein that plays an important role in tissue formation during embryonic development as well as 3D cellular self-assembly in vitro. Our lab has developed a series of fibronectin matrix mimetic proteins that mimic many of the cellular effects of the bioactive, ECM form of fibronectin. The goal of this work was use fibronectin matrix mimetics to identify the cell-fibronectin interactions that lead to cellular self-assembly. In this work, we showed that fibronectin matrix mimetic substrates are able to stimulate fibronectin-dependent cellular self-assembly. The fibronectin matrix mimetic substrate conditions that permit cellular self-assembly are dependent on the cell-substrate binding strength. We have identified roles for fibronectin matrix assembly and Rac GTPase in mediating cell-substrate binding strength and subsequently cellular self-assembly. These findings were then applied to tissue engineering applications. We showed that fibronectin matrix mimetics are also able to control cellular self-assembly of mesenchymal stem cells (MSCs) and stimulate early osteogenic differentiation based on substrate coating concentration. The studies presented herein suggest that fibronectin matrix mimetics provide a model for studying the role that fibronectin matrix assembly and cell-substrate binding strength play in stimulating tissue development