Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells

Abstract

Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a preformed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM.

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic

“…The FUD peptide and a mutated FUD control peptide, termed mFUD, were successfully expressed using recombinant technology as described previously (9,18). Peptide expression was induced using IPTG at a colony optical density of 0.6, approximately 4 h following colony seeding.…”

“…The FUD peptide and its mFUD control peptide variant (18) were recombinantly expressed in BL21 (DE3) E. coli as a His-tagged pET-ELMER construct using previously described protocol (9) with modifications recently reported by Filla et al pertaining to His-tag removal (18). Briefly, expression of FUD or mFUD was induced by 1 mM isopropyl β-D-1thiogalactopyranoside and cell lysis was facilitated by a lysis buffer (100 mM Sodium Phosphate, 10 mM Tris, 8 M Urea, 5 mM imidazole, pH 8.0).…”

Integrative transcriptomic and proteomic analysis reveals CD9/ITGA4/PI3K‐Akt axis mediates trabecular meshwork cell apoptosis in human glaucoma

“…As a result, a large number of genes and proteins showed an up or down tendency between iHTM and GTM 3 (Figure 2B 15 Furthermore, CTGF was also demonstrated as the matricellular proteins in glaucoma, which plays a role in fibrosis and increased extracellular matrix deposition. 16 CTGF might be relevant for the development of elevated IOP, which is considered as a high-risk factor in glaucoma pathogenesis.…”

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

“…Antibodies against the 40kDa (gelatin-binding) region of fibronectin were described previously [13]. The functional upstream domain (FUD) of streptococcus pyogenes adhesin F1 protein was described previously [14]. The TLR4 (TAK-242) and NF-κB (BAY11-7082) inhibitors were from EMD Millipore (Temecula, CA).…”

“…Cells were grown for 6 days and medium containing FUD was changed every other day. Recombinant FUD was prepared as previously described [14]. On day 8, 3D decellularized matrix was prepared by solubilizing 3T3-L1 cells in 1 mL of extraction buffer (20 mM NH4OH and 0.5% Triton-X in 1X PBS) for 5 minutes.…”