The study investigates the ability of fibronectin's extra domain A (EDA) to activate Toll-like receptor 4 (TLR4), a key receptor involved in innate immunity. EDA, a region of fibronectin produced in response to tissue injury, has been implicated in various physiological and pathological processes, particularly in inflammation. The researchers found that recombinant EDA, but not other fibronectin domains, activates human TLR4 in HEK 293 cells, which normally lack this receptor. This activation requires the co-expression of MD-2, an accessory protein for TLR4. Unlike lipopolysaccharide (LPS), EDA's activity is heat-sensitive and persists in the presence of polymyxin B and the LPS antagonist E5564, suggesting a distinct mechanism from LPS. The study also demonstrates that EDA can induce matrix metalloproteinase 9 (MMP-9) expression in THP-1 cells, a monocyte/macrophage cell line, and that EDA activates TLR4 in a dose-dependent manner. Additionally, EDA-stimulated TLR4 activation is inhibited by polymyxin B and E5564, further confirming its distinct signaling pathway from LPS. These findings suggest that EDA-containing fibronectin fragments may promote inflammatory responses through a mechanism involving TLR4 activation.The study investigates the ability of fibronectin's extra domain A (EDA) to activate Toll-like receptor 4 (TLR4), a key receptor involved in innate immunity. EDA, a region of fibronectin produced in response to tissue injury, has been implicated in various physiological and pathological processes, particularly in inflammation. The researchers found that recombinant EDA, but not other fibronectin domains, activates human TLR4 in HEK 293 cells, which normally lack this receptor. This activation requires the co-expression of MD-2, an accessory protein for TLR4. Unlike lipopolysaccharide (LPS), EDA's activity is heat-sensitive and persists in the presence of polymyxin B and the LPS antagonist E5564, suggesting a distinct mechanism from LPS. The study also demonstrates that EDA can induce matrix metalloproteinase 9 (MMP-9) expression in THP-1 cells, a monocyte/macrophage cell line, and that EDA activates TLR4 in a dose-dependent manner. Additionally, EDA-stimulated TLR4 activation is inhibited by polymyxin B and E5564, further confirming its distinct signaling pathway from LPS. These findings suggest that EDA-containing fibronectin fragments may promote inflammatory responses through a mechanism involving TLR4 activation.