TGF-? triggers rapid fibrillogenesis via a novel T?RII-dependent fibronectin-trafficking mechanism.
ABSTRACT: Fibronectin (FN) is a critical regulator of extracellular matrix (ECM) remodeling through its availability and stepwise polymerization for fibrillogenesis. Availability of FN is regulated by its synthesis and turnover, and fibrillogenesis is a multistep, integrin-dependent process essential for cell migration, proliferation, and tissue function. Transforming growth factor ? (TGF-?) is an established regulator of ECM remodeling via transcriptional control of ECM proteins. Here we show that TGF-?, through increased FN trafficking in a transcription- and SMAD-independent manner, is a direct and rapid inducer of the fibrillogenesis required for TGF-?-induced cell migration. Whereas TGF-? signaling is dispensable for rapid fibrillogenesis, stable interactions between the cytoplasmic domain of the type II TGF-? receptor (T?RII) and the FN receptor (?5?1 integrin) are required. We find that, in response to TGF-?, cell surface-internalized FN is not degraded by the lysosome but instead undergoes recycling and incorporation into fibrils, a process dependent on T?RII. These findings are the first to show direct use of trafficked and recycled FN for fibrillogenesis, with a striking role for TGF-? in this process. Given the significant physiological consequences associated with FN availability and polymerization, our findings provide new insights into the regulation of fibrillogenesis for cellular homeostasis.
Project description:BACKGROUND:Fibronectin (FN) assembly into an insoluble fibrillar matrix is a crucial step in many cell responses to extracellular matrix (ECM) properties, especially with regards to the integrin-related mechanosensitive signaling pathway. We have previously reported that the silencing of expression of integrin-linked kinase (ILK) in human intestinal epithelial crypt (HIEC) cells causes significant reductions in proliferation and spreading through concomitantly acquired impairment of soluble FN deposition. These defects in ILK-depleted cells are rescued by growth on exogenous FN. In the present study we investigated the contribution of ILK in the fibrillogenesis of FN and its relation to integrin-actin axis signaling and organization. RESULTS:We show that de novo fibrillogenesis of endogenous soluble FN is ILK-dependent. This function seemingly induces the assembly of an ECM that supports increased cytoskeletal tension and the development of a fully spread contractile cell phenotype. We observed that HIEC cell adhesion to exogenous FN or collagen-I (Col-I) is sufficient to restore fibrillogenesis of endogenous FN in ILK-depleted cells. We also found that optimal engagement of the Ras homolog gene family member A (RhoA) GTPase/Rho-associated kinase (ROCK-1, ROCK-2)/myosin light chain (MLC) pathway, actin ventral stress fiber formation, and integrin adhesion complex (IAC) maturation rely primarily upon the cell's capacity to execute FN fibrillogenesis, independent of any significant ILK input. Lastly, we confirm the integrin ?5?1 as the main integrin responsible for FN assembly, although in ILK-depleted cells ?V-class integrins expression is needed to allow the rescue of FN fibrillogenesis on exogenous substrate. CONCLUSION:Our study demonstrates that ILK specifically induces the initiation of FN fibrillogenesis during cell spreading, which promotes RhoA/ROCK-dependent cell contractility and maturation of the integrin-actin axis structures. However, the fibrillogenesis process and its downstream effect on RhoA signaling, cell contractility and spreading are ILK-independent in human intestinal epithelial crypt cells.
Project description:Basolateral polymerization of cellular fibronectin (FN) into a meshwork drives endothelial cell (EC) polarity and vascular remodelling. However, mechanisms coordinating ?5?1 integrin-mediated extracellular FN endocytosis and exocytosis of newly synthesized FN remain elusive. Here we show that, on Rab21-elicited internalization, FN-bound/active ?5?1 is recycled to the EC surface. We identify a pathway, comprising the regulators of post-Golgi carrier formation PI4KB and AP-1A, the small GTPase Rab11B, the surface tyrosine phosphatase receptor PTPRF and its adaptor PPFIA1, which we propose acts as a funnel combining FN secretion and recycling of active ?5?1 integrin from the trans-Golgi network (TGN) to the EC surface, thus allowing FN fibrillogenesis. In this framework, PPFIA1 interacts with active ?5?1 integrin and localizes close to EC adhesions where post-Golgi carriers are targeted. We show that PPFIA1 is required for FN polymerization-dependent vascular morphogenesis, both in vitro and in the developing zebrafish embryo.
Project description:TGF-? and myofibroblasts play a key role in fibrosis, characterized by aberrant synthesis and deposition of extracellular matrix (ECM) proteins, such as fibronectin (Fn) and collagen type I. There are two major roles played by integrins in the fibrotic pathology: (i) Fn-integrin interaction, coupled with cytokines like TGF-?, facilitates the self-polymerization of Fn and regulates cell-matrix fibrillar adhesions, thereby promoting fibrillogenesis; (ii) Integrin interaction with an RGD (arginine-glycine-aspartic) consensus sequence in the latent TGF-?, resulting in its activation. This study describes an anti-fibrotic strategy using a combination of two antibodies: Fn52 (targeted against the N-terminal 30?kDa region of fibronectin, a major site for Fn self-association), and its engineered form, Fn52RGDS (which binds to integrins). Interestingly, a synergistic effect of the cocktail in causing a decline in fibrotic features was confirmed in the context of fibrotic posterior capsular opacification (PCO), mediated by the lens epithelial cells (left behind after cataract surgery). Inclusion of Fn52RGDS to Fn52 aids in better diffusion of the antibodies; such combination therapies could be useful in the context of pathologies involving extensive remodeling of the fibronectin matrix, where the thick ECM offers a major challenge for efficient drug delivery.
Project description:Fibronectin (FN) assembly into extracellular matrix is tightly regulated and essential to embryogenesis and wound healing. FN fibrillogenesis is initiated by cytoskeleton-derived tensional forces transmitted across transmembrane integrins onto RGD binding sequences within the tenth FN type III (10FNIII) domains. These forces unfold 10FNIII to expose cryptic FN assembly sites; however, a specific sequence has not been identified in 10FNIII. Our past steered molecular dynamics simulations modeling 10FNIII unfolding by force at its RGD loop predicted a mechanical intermediate with a solvent-exposed N terminus spanning the A and B ?-strands. Here, we experimentally confirm that the predicted 23-residue cryptic peptide 1 (CP1) initiates FN multimerization, which is mediated by interactions with 10FNIII that expose hydrophobic surfaces that support 8-anilino-1-napthalenesulfonic acid binding. Localization of multimerization activity to the C terminus led to the discovery of a minimal 7-amino acid "multimerization sequence" (SLLISWD), which induces polymerization of FN and the clotting protein fibrinogen in addition to enhancing FN fibrillogenesis in fibroblasts. A point mutation at Trp-6 that reduces exposure of hydrophobic sites for 8-anilino-1-napthalenesulfonic acid binding and ?-structure formation inhibits FN multimerization and prevents physiological cell-based FN assembly in culture. We propose a model for cell-mediated fibrillogenesis whereby cell traction force initiates a cascade of intermolecular exchange starting with the unfolding of 10FNIII to expose the multimerization sequence, which interacts with strand B of another 10FNIII domain via a Trp-mediated ?-strand exchange to stabilize a partially unfolded intermediate that propagates FN self-assembly.
Project description:Protein remodeling at the cell-material interface is an important phenomenon that should be incorporated into the design of advanced biomaterials for tissue engineering. In this work, we address the relationship between fibronectin (FN) activity at the material interface and remodeling, including proteolytic cascades. To do so, we studied FN adsorption on two chemically similar substrates, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), which resulted in different distribution and conformation of the protein at the material interface: FN organized spontaneously upon adsorption on PEA into physiological-like fibrils, through a process called material-driven FN fibrillogenesis. The amount of adsorbed FN and its conformation were investigated in two different coating concentrations (2 and 20??g/mL). Since FN activity at the material interface determines the initial cellular response, we followed the formation of focal adhesions (vinculin) and subsequent cell signaling by focal adhesion kinase (FAK) expression and its phosphorylation (pFAK). More detailed studies were performed to get further insights into integrin binding by crosslinking and extraction followed by immunofluorescence, as well as protein and gene expression for ?5 and ?v. To correlate cell adhesion with matrix degradation, gene expression and activity (zymography) of matrix metalloproteinases (MMPs) were measured. Overall, we demonstrated that the material-driven FN fibrillogenesis triggers proteolytic activity: MMP activity was higher on the material-driven FN fibrils, as a compensatory mechanism to the inability of cells to reorganize this FN network.
Project description:Hydroxysafflor yellow A (HSYA) is an active ingredient of Carthamus tinctorius L.. This study aimed to evaluate the effects of HSYA on transforming growth factor-?1 (TGF-?1)-induced changes in proliferation, migration, differentiation, and extracellular matrix accumulation and degradation in human fetal lung fibroblasts (MRC-5), to explore the mechanisms whereby HSYA may alleviate pulmonary fibrosis. MRC-5 cells were incubated with various doses of HSYA and/or the TGF-? receptor type I kinase inhibitor SB431542 and then stimulated with TGF-?1. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt assay. Cell migration was detected by wound-healing assay. Protein levels of ?-smooth muscle actin (?-SMA), collagen I ? 1 (COL1A1), and fibronectin (FN) were measured by immunofluorescence. Protein levels of matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-2, TGF-? type II receptor (T?RII), and TGF-? type I receptor were detected by western blotting. T?RII knockdown with siRNA interfered with the inhibitory effect of HSYA on ?-SMA, COL1A1, and FN expression, and TGF-?1-induced Sma and Mad protein (Smad), and extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway activation. The antagonistic effect of HSYA on the binding of fluorescein isothiocyanate-TGF-?1 to MRC-5 cell cytoplasmic receptors was measured by flow cytometry. HSYA significantly suppressed TGF-?1-induced cell proliferation and migration. HSYA could antagonize the binding of FITC-TGF-?1 to MRC-5 cell cytoplasmic receptors. Also HSYA inhibited TGF-?1-activated cell expression of ?-SMA, COL1A1, and FN and phosphorylation level of Smad2, Smad3, and ERK by targeting T?RII in MRC-5 cells. These findings suggest that T?RII might be the target responsible for the inhibitory effects of HSYA on TGF-?1-induced pathological changes in pulmonary fibrosis.
Project description:Objective- Alterations in extracellular matrix quantity and composition contribute to atherosclerosis, with remodeling of the subendothelial basement membrane to an FN (fibronectin)-rich matrix preceding lesion development. Endothelial cell interactions with FN prime inflammatory responses to a variety of atherogenic stimuli; however, the mechanisms regulating early atherogenic FN accumulation remain unknown. We previously demonstrated that oxLDL (oxidized low-density lipoprotein) promotes endothelial proinflammatory gene expression by activating the integrin ?5?1, a classic mediator of FN fibrillogenesis. Approach and Results- We now show that oxLDL drives robust endothelial FN deposition and inhibiting ?5?1 (blocking antibodies, ?5 knockout cells) completely inhibits oxLDL-induced FN deposition. Consistent with this, inducible endothelial-specific ?5 integrin deletion in ApoE knockout mice significantly reduces atherosclerotic plaque formation, associated with reduced early atherogenic inflammation. Unlike TGF? (transforming growth factor ?)-induced FN deposition, oxLDL does not induce FN expression (mRNA, protein) or the endothelial-to-mesenchymal transition phenotype. In addition, we show that cell-derived and plasma-derived FN differentially affect endothelial function, with only cell-derived FN capable of supporting oxLDL-induced VCAM-1 (vascular cell adhesion molecule 1) expression, despite plasma FN deposition by oxLDL. The inclusion of alternative exon EIIIA (EDA) of FN (EIIIA) and alternative exon EIIIB (EDB) of FN (EIIIB) domains in cell-derived FN mediates this effect, as EIIIA/EIIIB knockout endothelial cells show diminished oxLDL-induced inflammation. Furthermore, our data suggest that EIIIA/EIIIB-positive cellular FN is required for maximal ?5?1 recruitment to focal adhesions and FN fibrillogenesis. Conclusions- Taken together, our data demonstrate that endothelial ?5 integrins drive oxLDL-induced FN deposition and early atherogenic inflammation. Additionally, we show that ?5?1-dependent endothelial FN deposition mediates oxLDL-dependent endothelial inflammation and FN fibrillogenesis.
Project description:During embryonic development and tissue homeostasis, cells produce and remodel the extracellular matrix (ECM). The ECM maintains tissue integrity and can serve as a substrate for cell migration. Integrin ?5 (Itg?5) and ?V (Itg?V) are the ? subunits of the integrins most responsible for both cell adhesion to the ECM protein fibronectin (FN) and FN matrix fibrillogenesis. We perform a systems-level analysis of cell motion in the zebrafish tail bud during trunk elongation in the presence and absence of normal cell-FN interactions. Itg?5 and Itg?V have well-described roles in cell migration in vitro. However, we find that concomitant loss of itg?5 and itg?V leads to a trunk elongation defect without substantive alteration of cell migration. Tissue-specific transgenic rescue experiments suggest that the FN matrix on the surface of the paraxial mesoderm is required for body elongation via its role in defining tissue mechanics and intertissue adhesion.
Project description:Epithelial-Mesenchymal Transition (EMT) is a dynamic process through which epithelial cells transdifferentiate from an epithelial phenotype into a mesenchymal phenotype. Previous studies have demonstrated that both mechanical signaling and soluble growth factor signaling facilitate this process. One possible point of integration for mechanical and growth factor signaling is the extracellular matrix. Here we investigate the role of the extracellular matrix (ECM) protein fibronectin (FN) in this process. We demonstrate that inhibition of FN fibrillogenesis blocks activation of the Transforming Growth Factor-Beta (TGF-?) signaling pathway via Smad2 signaling, decreases cell migration and ultimately leads to inhibition of EMT. Results show that soluble FN, FN fibrils, or increased contractile forces are insufficient to independently induce EMT. We further demonstrate that inhibition of latent TGF-?1 binding to FN fibrils via either a monoclonal blocking antibody against the growth factor binding domain of FN or through use of a FN deletion mutant that lacks the growth factor binding domains of FN blocks EMT progression, indicating a novel role for FN in EMT in which the assembly of FN fibrils serves to localize TGF-?1 signaling to drive EMT.
Project description:The process by which fibronectin (FN), a soluble multidomain protein found in tissue fluids, forms insoluble fibrillar networks in the extracellular matrix is poorly understood. Cryptic sites found in FN type III domains have been hypothesized to function as nucleation points, thereby initiating fibrillogenesis. Exposure of these sites could occur upon tension-mediated mechanical rearrangement of type III domains. Here, we present the solution structures of the second type III domain of human FN ((2)FNIII), and that of an interaction complex between the first two type III domains ((1-2)FNIII). The two domains are connected through a long linker, flexible in solution. A weak but specific interdomain interaction maintains (1-2)FNIII in a closed conformation that associates weakly with the FN N-terminal 30 kDa fragment (FN30 kDa). Disruption of the interdomain interaction by amino-acid substitutions dramatically enhances association with FN30 kDa. Truncation analysis of (1-2)FNIII reveals that the interdomain linker is necessary for robust (1-2)FNIII-FN30 kDa interaction. We speculate on the importance of this interaction for FN function and present a possible mechanism by which tension could initiate fibrillogenesis.