Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases.

Changes in the organization and structure of the fibronectin matrix are believed to contribute to dysregulated wound healing and subsequent tissue inflammation and tissue fibrosis. These changes include an increase in the EDA isoform of fibronectin as well as the mechanical unfolding of fibronectin type III domains. In previous studies using embryonic foreskin fibroblasts, we have shown that fibronectin's EDA domain (FnEDA) and the partially unfolded first Type III domain (FnIII-1c) function as Damage Associated Molecular Pattern (DAMP) molecules to stimulate the induction of inflammatory cytokines by serving as agonists for Toll-Like Receptor-4 (TLR4). However, the role of signaling molecules downstream of TLR-4 such as TGF-ß Activated Kinase 1 (TAK1) and Mitogen activated protein kinases (MAPK) in regulating the expression of fibronectin DAMP induced inflammatory genes in specific cell types is not known. In the current study, we evaluate the molecular steps regulating the fibronectin driven induction of inflammatory genes in three human fibroblast cell lines: embryonic foreskin, adult dermal, and adult kidney. The fibronectin derived DAMPs each induce the phosphorylation and activation of TAK1 which results in the activation of two downstream signaling arms, IKK/NF-κB and MAPK. Using the specific inhibitor 5Z-(7)-Oxozeanol as well as siRNA, we show TAK1 to be a crucial signaling mediator in the release of cytokines in response to fibronectin DAMPs in all three cell types. Finally, we show that FnEDA and FnIII-1c induce several pro-inflammatory cytokines whose expression is dependent on both TAK1 and JNK MAPK and highlight cell-type specific differences in the gene-expression profiles of the fibroblast cell-lines.

Fibronectin Functions as a Selective Agonist for Distinct Toll-like Receptors in Triple-Negative Breast Cancer.

The microenvironment of tumors is characterized by structural changes in the fibronectin matrix, which include increased deposition of the EDA isoform of fibronectin and the unfolding of the fibronectin Type III domains. The impact of these structural changes on tumor progression is not well understood. The fibronectin EDA (FnEDA) domain and the partially unfolded first Type III domain of fibronectin (FnIII-1c) have been identified as endogenous damage-associated molecular pattern molecules (DAMPs), which induce innate immune responses by serving as agonists for Toll-Like Receptors (TLRs). Using two triple-negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231, we show that FnEDA and FnIII-1c induce the pro-tumorigenic cytokine, IL-8, by serving as agonists for TLR5 and TLR2, the canonical receptors for bacterial flagellin and lipoprotein, respectively. We also find that FnIII-1c is not recognized by MDA-MB-468 cells but is recognized by MDA-MB-231 cells, suggesting a cell type rather than ligand specific utilization of TLRs. As IL-8 plays a major role in the progression of TNBC, these studies suggest that tumor-induced structural changes in the fibronectin matrix promote an inflammatory microenvironment conducive to metastatic progression.

Hyaluronan, Transforming Growth Factor ß, and Extra Domain A-Fibronectin: A Fibrotic Triad.

Significance: Inflammation is a critical aspect of injury repair. Nonresolving inflammation, however, is perpetuated by the local generation of extracellular matrix-derived damage-associated molecular pattern molecules (DAMPs), such as the extra domain A (EDA) isoform of fibronectin and hyaluronic acid (HA) that promote the eventual acquisition of a fibrotic response. DAMPs contribute to the inflammatory environment by engaging Toll-like, integrin, and CD44 receptors while stimulating transforming growth factor (TGF)-ß signaling to activate a fibroinflammatory genomic program leading to the development of chronic disease. Recent Advances: Signaling through TLR4, CD44, and the TGF-ß pathways impact the amplitude and duration of the innate immune response to endogenous DAMPs synthesized in the context of tissue injury. New evidence indicates that crosstalk among these three networks regulates phase transitions as well as the repertoire of expressed genes in the wound healing program determining, thereby, repair outcomes. Clarifying the molecular mechanisms underlying pathway integration is necessary for the development of novel therapeutics to address the spectrum of fibroproliferative diseases that result from maladaptive tissue repair. Critical Issues: There is an increasing appreciation for the role of DAMPs as causative factors in human fibroinflammatory disease regardless of organ site. Defining the involved intermediates essential for the development of targeted therapies is a daunting effort, however, since various classes of DAMPs activate different direct and indirect signaling pathways. Cooperation between two matrix-derived DAMPs, HA, and the EDA isoform of fibronectin, is discussed in this review as is their synergy with the TGF-ß network. This information may identify nodes of signal intersection amenable to therapeutic intervention. Future Directions: Clarifying mechanisms underlying the DAMP/growth factor signaling nexus may provide opportunities to engineer the fibroinflammatory response to injury and, thereby, wound healing outcomes. The identification of shared and unique DAMP/growth factor-activated pathways is critical to the design of optimized tissue repair therapies while preserving the host response to bacterial pathogens.

Fibronectin in the Tumor Microenvironment Activates a TLR4-dependent Inflammatory Response in Lung Cancer Cells.

The microenvironment of solid tumors plays an essential role in tumor progression. In lung cancer, the stromal cells produce a fibronectin rich extracellular matrix which is known to contribute to both tumor metastasis and drug resistance. Due to its conformational lability, fibronectin is considerably remodeled by the contractile forces of the fibrotic microenvironment within the tumor stroma. As a result, the secondary structure of fibronectin's Type III domains is disrupted and the molecule becomes highly stretched. The contribution/impact of these strained forms of fibronectin on tumor growth and metastasis is not known. In the current study we show that the partially unfolded first Type III domain of fibronectin, III-1c, activates a toll-receptor/NF-κB pathway leading to an increase in the expression of IL-8. Using a 3-D model of tumor-associated extracellular matrix, we demonstrate that lung cancer cells seeded onto this matrix activate a TLR4/NF-κB signaling pathway leading to a robust increase in the release of IL-8. Cytokine release by these cells is completely dependent on the presence of fibronectin in the extracellular matrix. These findings suggest that paracrine signaling between the tumor and the stromal myofibroblasts causes a remodeling of the matrix fibronectin into a strained conformation which supports the activation of a TLR4/NF-κB signaling pathway resulting in the upregulation of fibro-inflammatory cytokines.

Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin.

Chronic inflammation and subsequent tissue fibrosis are associated with a biochemical and mechanical remodeling of the fibronectin matrix. Due to its conformational lability, fibronectin is considerably stretched by the contractile forces of the fibrotic microenvironment, resulting in the unfolding of its Type III domains. In earlier studies, we have shown that a peptide mimetic of a partially unfolded fibronectin Type III domain, FnIII-1c, functions as a Damage Associated Molecular Pattern (DAMP) molecule to induce activation of a toll-like receptor 4 (TLR4)/NF-B pathway and the subsequent release of fibro-inflammatory cytokines from human dermal fibroblasts. In the current study, we evaluated the requirement of the canonical TLR4/MD2/CD14 receptor complex in the regulation of FnIII-1c induced cytokine release. Using dermal fibroblasts and human embryonic kidney (HEK) cells, we found that all the components of the TLR4/MD2/CD14 complex were required for the release of the fibro-inflammatory cytokine, interleukin 8 (IL-8) in response to both FnIII-1c and the canonical TLR4 ligand, lipopolysaccharide (LPS). However, FnIII-1c mediated IL-8 release was strictly dependent on membrane-associated CD14, while LPS could use soluble CD14. These findings demonstrate that LPS and FnIII-1c share a similar but not identical mechanism of TLR4 activation in human dermal fibroblasts.

TLR4 Ligands Selectively Synergize to Induce Expression of IL-8.

Objective: Dysfunctional remodeling of the extracellular matrix contributes to the formation of TLR-dependent feed forward loops that drive chronic inflammation. We have previously shown that two Type III domains of Fibronectin, FnEDA and FnIII-1c, cooperate to induce the synergistic release of interleukin 8 (IL-8) from dermal fibroblasts. We now identify steps in the TLR4 pathway where synergy can be demonstrated as well as additional kinases functioning in fibronectin activation of TLR4 signaling. We also evaluate the ligand and cell-type specificity of this synergistic response. Approach: FnEDA, FnIII-1c, and lipopolysaccharide (LPS)-induced genes in fibroblasts were analyzed by a quantitative reverse transcription-polymerase chain reaction (qPCR) and protein was measured by an enzyme-linked immunosorbent assay (ELISA). Kinases functioning in gene expression were identified by using specific inhibitors. Activated TLR4-dependent effector molecules were identified by cell fractionation and Western blot and quantified by image analysis. Results: The addition of FnEDA and FnIII-1c to dermal fibroblasts resulted in a synergistic increase in the expression of IL-8, tumor necrosis factor alpha (TNF-α), and vascular cell adhesion molecule (VCAM-1). Synergy between these domains was detected at the level of nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) and inhibitor of kappa B kinase (IKK) activation. Induction of IL-8 by fibronectin ligands was partially attenuated in the presence of inhibitors to either epidermal growth factor receptor or Src kinases. FnIII-1c also synergized with LPS to induce IL-8 in dermal fibroblasts, whereas the combined effect of FnEDA and LPS on IL-8 synthesis was additive. In contrast, synergistic responses to these ligands were not observed in THP-1 monocytic cells. Innovation: The data suggest that chronic inflammation may be driven by matrix- and pathogen-derived TLR4 ligands that work in synergy to promote an exuberant innate response. Conclusion: The data suggest that the molecular mechanism underlying synergistic responses to TLR4 ligands lies upstream of IKK activation, likely in the molecular composition of the TLR4 receptor complex that assembles in response to each ligand. In addition, synergistic responses to TLR4 activation may be both cell-type and ligand specific.

Integration of Canonical and Noncanonical Pathways in TLR4 Signaling: Complex Regulation of the Wound Repair Program.

Significance: Chronic inflammation and maladaptive repair contribute to the development of fibrosis that negatively impacts quality of life and organ function. The toll-like receptor (TLR) system is a critical node in the tissue response to both exogenous (pathogen-associated) and endogenous (damage-associated) molecular pattern factors (PAMPs and DAMPs, respectively). The development of novel TLR ligand-, pathway-, and/or target gene-specific therapeutics may have clinical utility in the management of the exuberant inflammatory/fibrotic tissue response to injury without compromising the host defense to pathogens. Recent Advances: DAMP ligands, released upon wounding, and microbial-derived PAMPs interact with several TLRs, and their various coreceptor partners, engaging downstream pathways that include Src family kinases, the epidermal growth factor receptor, integrins and the tumor suppressor phosphatase and tensin homolog (PTEN). Toll-like receptor 4 (TLR4) activation enhances cellular responses to the potent profibrotic cytokine transforming growth factor-ß1 (TGF-ß1) by attenuating the expression of receptors that inhibit TGF-ß1 signaling. Critical Issues: Common as well as unique pathways may be activated by PAMP and DAMP ligands that bind to the repertoire of TLRs on various cell types. Dissecting mechanisms underlying ligand-dependent engagement of this complex, highly interactive, network will provide for adaptation of new and focused therapies directed to the regulation of pathologically significant profibrotic genes. Inherent in this diversity are therapeutic opportunities to modulate the pathophysiologic consequences of persistent TLR signaling. The recently identified involvement of receptor and nonreceptor kinase pathways in TLR signaling may present novel opportunities for pharmacologic intervention. Future Directions: Clarifying the identity and function of DAMP-activated TLR complexes or ligand-binding partners, as well as their engaged downstream effectors and target genes, are key factors in the eventual design of pathway-specific treatment modalities. Such approaches may be tailored to address the spectrum of TLR-initiated pathologies (including localized and persistent inflammation, maladaptive repair/fibrosis) and, perhaps, even titrated to achieve patient-unique beneficial clinical outcomes.

Integrin α4ß1 and TLR4 Cooperate to Induce Fibrotic Gene Expression in Response to Fibronectin's EDA Domain.

Alternative splicing of fibronectin increases expression of the EDA+ isoform of fibronectin (EDA+Fn), a damage-associated molecular pattern molecule, which promotes fibro-inflammatory disease through the activation of toll-like receptors. Our studies indicate that the fibronectin EDA domain drives two waves of gene expression in human dermal fibroblasts. The first wave, seen at 2 hours, consisted of inflammatory genes, VCAM1, and tumor necrosis factor. The second wave, evaluated at 24 hours, was composed of the fibrosis-associated cytokines IL-10 and IL-13 and extracellular matrix genes fibronectin and osteopontin. Gene expression was coordinately regulated by the α4ß1 integrin and the innate immune receptor toll-like receptor 4. Additionally, we found a significant toll-like receptor 4/α4ß1-dependent enrichment in the ratio of EDA+Fn to total fibronectin in response to EDA, consistent with EDA+Fn initiating further production of EDA+Fn. Our data also suggest that the EDA/α4ß1 integrin interaction primes the cell for an enhanced response to toll-like receptor 4 ligands. Our studies provide evidence that remodeling of the fibronectin matrix in injured or diseased tissue elicits an EDA-dependent fibro-inflammatory response in dermal fibroblasts. The data suggest a paradigm of damage-associated molecular pattern-based signaling whereby damage-associated molecular pattern binding integrins cooperate with innate immune receptors to stimulate inflammation and fibrosis.

The fibronectin III-1 domain activates a PI3-Kinase/Akt signaling pathway leading to αvß5 integrin activation and TRAIL resistance in human lung cancer cells.

BACKGROUND: Fibronectin is a mechanically sensitive protein which is organized in the extracellular matrix as a network of interacting fibrils. The lung tumor stroma is enriched for fibronectin which is thought to contribute to metastasis and drug resistance. Fibronectin is an elastic, multi-modular protein made up of individually folded domains, some of which can stretch in response to increased mechanical tension. Very little is known about the relationship of fibronectin's unfolded domains to lung cancer resistance to chemotherapy. In the present study, we evaluated the impact of unfolding the first Type III domain of fibronectin (FnIII-1c) on TNF-related apoptosis inducing ligand (TRAIL) resistance. METHODS: NCI-H460 non-small cell lung cancer cells were treated with FnIII-1c then assessed for TRAIL-induced apoptosis. Subsequent analysis of FnIII-1c-mediated signaling pathways was also completed. Human non-small cell lung cancer tissue sections were assessed for the expression of vitronectin by immunohistochemistry. RESULTS: FnIII-1c inhibited TRAIL-induced activation of caspase 8 and subsequent apoptosis in NCI-H460 lung cancer cells. FnIII-1c treatment was associated with the activation of the phosphatidylinositol-3-kinase/alpha serine/threonine kinase (PI3K/Akt) pathway and the αvß5 integrin receptor for vitronectin, both of which were required for TRAIL resistance. Immunohistochemical staining of sections from non-small cell lung cancers showed that vitronectin was localized around blood vessels and in the tumor-stroma interface. CONCLUSIONS: Unfolding of Type III domains within the fibronectin matrix may promote TRAIL resistance through the activation of a PI3K/Akt/αvß5 signaling axis and point to a novel mechanism by which changes in secondary structure of fibronectin contribute to cancer cell resistance to apoptosis.

Role of EGFR expression levels in the regulation of integrin function by EGF.

Activation of ß1 integrins in dormant tumor cells has been linked to metastatic progression, suggesting that therapies designed to maintain ß1 integrins in an inactive state may be useful in the prevention of metastatic disease. Our earlier studies have demonstrated that EGF regulates the activation state of the α5ß1 integrin in EGFR overexpressing tumor cells through an ERK/p90RSK signaling pathway. Activation of this pathway by EGF resulted in the filamin A dependent inactivation of the α5ß1 integrin receptor for fibronectin. The current study was designed to address the role of EGFR overexpression in the regulation of α5ß1 integrin activation state by EGF. Lentiviral knockdown of EGFR coupled with limited dilution cloning was used to develop A431 squamous carcinoma cell lines expressing high, moderate, and low levels of EGFR. Inactivation of α5ß1 integrin by EGF was shown to correlate with both the level of EGFR expression and the extent of p90RSK phosphorylation, but not with the level of ERK phosphorylation, suggesting that high levels of EGFR promote α5ß1 integrin inactivation through sustained activation of p90RSK. Treatment of cells with EGFR kinase inhibitor resulted in a reactivation of the integrin which could be reversed with the phosphatase inhibitor, menadione. Taken together, these findings indicate that p90RSK may function to maintain dormancy in tumor cells expressing high levels of EGFR. © 2015 Wiley Periodicals, Inc.

EDA Fibronectin in Keloids Create a Vicious Cycle of Fibrotic Tumor Formation.

During the early phase of wound healing, first plasma fibronectin (FN) and then in situ FN are deposited at the site of injury. In situ FN--FN made by tissue cells at the injury site--often contains an extra domain A (EDA) insert. Multiple wound-related signal transduction pathways control the deposition of EDA FN, and the EDA insert can in turn trigger pathways that induce inflammation, increased extracellular matrix molecule deposition including FN and collagen, and activation of fibroblasts. Together these pathways can create a vicious cycle that leads to fibrosis or keloid formation.

The α4ß1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts.

Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C-C' loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4ß1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition.

Cryptic activity within the Type III1 domain of fibronectin regulates tissue inflammation and angiogenesis.

The fibronectin matrix provides mechanical and biochemical information to regulate homeostatic and pathological processes within tissues. Fibronectin consists of independently-folded modules termed Types I, II and III. In response to cellular contractile force, Type III domains unfold to initiate a series of homophilic binding events which result in the assembly of a complex network of intertwining fibrils. The unfolding of Type III modules provides elasticity to the assembled fibronectin matrix allowing it to function as a dynamic scaffold which provides binding sites for cellular receptors, growth factors and other matrix molecules. Access to bioactive sites within the fibronectin matrix is under complex regulation and controlled through a combination of mechanical and proteolytic activity. Mechanical unfolding of Type III modules and limited proteolysis can alter the topographical display of bioactive sites within the fibronectin fibrils by exposing previously cryptic sites and disrupting functional sites. In this review we will discuss cryptic activity found within the first Type III module of fibronectin and its impact on tissue angiogenesis and inflammation.

Regulation of the innate immune response by fibronectin: synergism between the III-1 and EDA domains.

Fibronectin is a critical component of the extracellular matrix and alterations to its structure will influence cellular behavior. Matrix fibronectin is subjected to both mechanical and biochemical regulation. The Type III domains of fibronectin can be unfolded in response to increased cellular contractility, included or excluded from the molecule by alternative splicing mechanisms, or released from the matrix by proteolysis. Using Inflammatory Cytokine microarrays we found that the alternatively spliced fibronectin Type III domain, FnEDA, and the partially unfolded III-1 domain, FnIII-1c, induced the expression of a multitude of pro-inflammatory cytokines in human dermal fibroblasts, most notably CXCL1-3, IL-8 and TNF-α. FnIII-1c, a peptide representing an unfolded intermediate structure of the first Type III domain has been shown to initiate the toll-like receptor-4 (TLR4)-NFκB-dependent release of cytokines from human dermal fibroblasts (You, et al., J. Biol. Chem., 2010). Here we demonstrate that FnIII-1c and the alternatively spliced FnEDA domain induce a TLR4 dependent activation of p38 MAP kinase and its downstream effector, MAPKAP Kinase-2 (MK-2), to regulate cytokine expression in fibroblasts. RT-qPCR analysis indicated that the p38-MK-2 pathway regulates IL-8 mRNA stability. Interestingly, addition of FnIII-1c and FnEDA synergistically enhanced TLR4-dependent IL-8 release. These data indicate that Fn contains two Type III domains which can activate TLR signaling to induce an inflammatory response in fibroblasts. Furthermore, our data identifies the NF-κB and p38/MK2 signaling pathways as transducers of signals initiated in response to structural changes in fibronectin.

Conformational remodeling of the fibronectin matrix selectively regulates VEGF signaling.

The fibronectin matrix plays a crucial role in the regulation of angiogenesis during development, tissue repair and pathogenesis. Previous work has identified a fibronectin-derived homophilic binding peptide, anastellin, as an effective inhibitor of angiogenesis; however, its mechanism of action is not well understood. In the present study, we demonstrate that anastellin selectively inhibits microvessel cell signaling in response to the VEGF165 isoform, but not VEGF121, by preventing the assembly of the complex containing the VEGF receptor and neuropilin-1. Anastellin treatment resulted in the inactivation of α5ß1 integrins but was not accompanied by a change in either adhesion complexes or adhesion-based signaling. Integrin inactivation was associated with a masking of the fibronectin synergy site within the extracellular matrix (ECM), indicating that α5ß1 inactivation resulted from a decrease in available ligand. These data demonstrate that anastellin influences the microvessel cell response to growth factors by controlling the repertoire of ligated integrins and point to anastellin as an effective regulator of fibronectin matrix organization. These studies further suggest that homophilic fibronectin binding peptides might have novel applications in the field of tissue regeneration as tools to regulate neovascularization.

Topographical changes in extracellular matrix: Activation of TLR4 signaling and solid tumor progression.

The relationship between cancer progression and chronic inflammation is well documented but poorly understood. The innate immune system has long been recognized as the first line of defense against invading pathogens. More recently, endogenous molecules released from tissue matrix (Damage Associated Molecular Patterns [DAMPs]) following tissue injury or periods of active matrix remodeling have also been identified as regulators of innate immunity. DAMPs have been identified as ligands for Toll-like receptors (TLRs), a family of cell-surface proteins which regulate the immune response. TLRs have been identified on resident tissue cells as well as most tumor cells. Therefore, dysregulation of the innate immune response secondary to biochemical and mechanical driven changes in the extracellular matrix of the tumor microenvironment may be a critical component of the chronic inflammation associated with tumor progression. Here we review the role of extracellular matrix (ECM)-derived DAMPS in the activation of TLR4 signaling in the context of tumor progression. We also explore the various types of topographical changes that can lead to ECM-derived DAMPs and their contribution to TLR4 activation.

Epidermal growth factor (EGF) regulates α5ß1 integrin activation state in human cancer cell lines through the p90RSK-dependent phosphorylation of filamin A.

BACKGROUND: Regulation of integrin activation has important implications for tumor cell invasion and metastasis. RESULTS: EGF activates ERK/p90RSK and Rho/Rho kinase signaling in A431 and DiFi colon cancer cells, leading to phosphorylation of filamin A (FLNa) and inactivation of the α5ß1 integrin receptor. CONCLUSION: EGF promotes α5ß1 inactivation through the p90RSK-dependent phosphorylation of FLNa. SIGNIFICANCE: We have identified a novel EGF-dependent mechanism controlling the α5ß1 integrin activation state. Cell adhesion, motility, and invasion are regulated by the ligand-binding activity of integrin receptors, transmembrane proteins that bind to the extracellular matrix. Integrins whose conformation allows for ligand binding and appropriate functional activity are said to be in an active state. Integrin activation and subsequent ligand binding are dynamically regulated by the association of cytoplasmic proteins with integrin intracellular domains. In this study, we evaluated the role of EGF in the regulation of the activation state of the α5ß1 integrin receptor for fibronectin. The addition of EGF to either A431 squamous carcinoma cells or DiFi colon cancer cells resulted in loss of α5ß1-dependent adhesion to fibronectin but no loss of integrin from the cell surface. EGF activated the EGF receptor/ERK/p90RSK and Rho/Rho kinase signaling pathways. Blocking either pathway inhibited EGF-mediated loss of adhesion, suggesting that they work in parallel to regulate integrin function. EGF treatment also resulted in phosphorylation of filamin A (FLNa), which binds and inactivates ß1 integrins. EGF-mediated FLNa phosphorylation was completely blocked by an inhibitor of p90RSK and partially attenuated by an inhibitor of Rho kinase, suggesting that both pathways converge on FLNa to regulate integrin function. A431 clonal cell lines expressing non-phosphorylated dominant-negative FLNa were resistant to the inhibitory effects of EGF on integrin function, whereas clonal cell lines overexpressing wild-type FLNa were more sensitive to the inhibitory effect of EGF. These data suggest that EGF-dependent inactivation of α5ß1 integrin is regulated through FLNa phosphorylation and cellular contractility.

The First Type III Domain of Fibronectin is Associated with the Expression of Cytokines within the Lung Tumor Microenvironment.

Recent studies have pointed to changes in tissue mechanics as a contributory element to the development of malignancies. Increased tissue rigidity is associated with the unfolding of the Type III domains of fibronectin within the extracellular matrix. The consequences of this unfolding on cellular functions within the lung are not well understood. In the present study, we evaluated the effect of a peptide representing a partially unfolded intermediate of the first Type III repeat of fibronectin (FnIII-1c) on inflammatory gene expression in adult human lung fibroblast cells. FnIII-1c induced expression of cytokines, CXCL1-3, IL-8 and TNF-α, by lung fibroblast cells. The increase in IL-8 expression was dependent on Toll-like receptor 2 and NFκB. Immunohistochemistry of tissue arrays representing squamous cell carcinoma of the lung revealed extensive stromal staining for IL-8 and fibronectin fibrils which were co-aligned with myofibroblasts. These data suggest a model in which unfolding of FnIII domains secondary to myofibroblast-generated tension may induce the release of cytokines by stromal fibroblasts present within the lung tumor.

The first type III repeat in fibronectin activates an inflammatory pathway in dermal fibroblasts.

Remodeling of the fibronectin matrix occurs during a variety of pathological and regenerative processes. Cellular generated tensional forces can alter the secondary and tertiary structure of the fibronectin matrix and regulate the exposure of cryptic activities that directly impact cell behavior. In the present study, we evaluated the effect of the partially unfolded Type III fibronectin module, FnIII-1c, on gene expression in dermal fibroblasts. Microarray and PCR analysis indicated that the addition of FnIII-1c to human dermal fibroblasts induced the expression of several inflammatory genes including the cytokines, IL-8 and TNF-α. ELISA analysis indicated that the increased gene expression was accompanied by the secretion of IL-8 and TNF-α protein. FnIII-1c-induced gene expression was preceded by increased phosphorylation of IκB kinase (IKK) and IκBα as well as the nuclear translocation of NFκB. PCR and ELISA analysis showed that inhibition of the NFκB signaling pathway completely blocked the induction of IL-8 and TNF-α. Blocking antibodies to Toll-like receptor 4 inhibited both the activation of the NFκB signaling pathway as well as cytokine expression in response to FnIII-1c. These data suggest that fibronectin matrix remodeling can induce the expression of cytokines by stromal cells present in the tissue microenvironment.

Regulation of p38 MAP kinase by anastellin is independent of anastellin's effect on matrix fibronectin.

Anastellin is an angiogenesis inhibitor derived from the first type III repeat of fibronectin (FN). Anastellin binds to fibronectin and promotes the polymerization of soluble fibronectin into a highly polymerized form termed superfibronectin. In addition, anastellin also causes remodeling of pre-existing fibronectin matrix and modulates cell signaling pathways in both endothelial cells and fibroblasts. In the present study, we address the relationship of anastellin's effects on fibronectin matrix to its effects on p38 MAP kinase (MAPK) activation. Using a mutant form of anastellin which binds to fibronectin matrix, but does not stimulate formation of superfibronectin, we demonstrate that the activation of p38 MAPK by anastellin is not dependent on the formation of superfibronectin. The mutant form of anastellin does stimulate matrix remodeling, but experiments using FN(-/-) cells show that the effect of anastellin on p38-MAPK activation is completely independent of fibronectin. Anastellin was able to activate p38 MAPK on cells in suspension as well as on cells null for beta1 integrins, suggesting that anastellin activity did not require ligation of integrins. These data suggest that the activation of p38 MAPK by anastellin is independent of anastellin's effects on fibronectin matrix organization.