Regulation of the Innate Immune Response by Fibronectin: Synergism between the III-1 and EDA Domains

Abstract

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.

Toll-Like Receptor-4 Signaling Drives Persistent Fibroblast Activation and Prevents Fibrosis Resolution in Scleroderma

“…It was shown that exposure of fibroblasts to FNIII1c, a stable unfolded intermediate of FNIII1, can elicit TLR4-dependent inflammatory signaling and induce cytokine synthesis. 41,42 This region of the fibronectin molecule is exposed through tensional forces within the rigid fibrotic microenvironment. These observations have clear implications for fibrosis, since fibrotic tissue is characterized by increased stiffness and mechanical forces.…”

A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans

“…Briefly, a large plate of FUDRsterilized, synchronized day 1 adult worms from each strain were freeze-cracked in RIPA buffer, sonicated, and spun down at 4°to collect supernatant. This supernatant was then used for Western analysis using previously established methods (Kelsh et al 2014). R-GECO1 levels were probed using a DsRed Polyclonal Antibody (Clontech catalog no.…”

Experimental and computational examination of anastellin (FnIII1c)–polymer interactions

“…7 Another area of Fn that has been shown to elicit potentially useful interactions is the Cterminal fragment of the first FnIII domain (FnIII1c), known as anastellin, which has 76 amino acid residues. [8][9][10][11][12] The FnIII1 domain has been identified as a region associated with matrix formation by Fn-Fn interactions. 8 When mixed with whole Fn molecules, anastellin can create a different form of Fn known as superfibronectin which is adept at supporting cell adhesion and spreading.…”