The extra domain A (EDA) of fibronectin activates Toll-like receptor 4 (TLR4), which is the receptor stimulated by bacterial lipopolysaccharide (LPS). This study shows that recombinant EDA activates TLR4 in HEK 293 cells, which normally lack this receptor. The activation of TLR4 by EDA requires co-expression of MD-2, an accessory protein. Unlike LPS, EDA activity is heat-sensitive and remains active in the presence of polymyxin B and E5564, which block LPS activation. These findings suggest that EDA-containing fibronectin fragments can induce inflammatory responses through TLR4 activation. The study also demonstrates that EDA activates NF-κB, a key regulator of inflammatory gene expression. Additionally, EDA activates TLR4 in C3H/HeJ mice, which have a Tlr4 mutation, indicating that EDA acts through TLR4. The study further shows that EDA activation of TLR4 is not due to LPS contamination in the recombinant protein. The results suggest that EDA-containing fibronectin fragments may serve as signaling molecules that activate TLR4 through a distinct pathway. The study also highlights the importance of MD-2 in EDA activation of TLR4 and the role of CD14 in enhancing EDA activation. The findings provide a mechanism by which EDA-containing fibronectin fragments promote inflammatory responses. The study also shows that EDA activates MMP-9 production in THP-1 cells, which is a key enzyme in tissue remodeling. The results indicate that EDA can activate TLR4 independently of LPS and that the effects of EDA can be distinguished from those of LPS. The study also shows that EDA activates TLR4 in the absence of CD14, suggesting that EDA may use a different pathway for TLR4 activation. The study concludes that EDA-containing fibronectin fragments can activate TLR4 and induce inflammatory responses, providing a new mechanism for fibronectin's role in inflammation.The extra domain A (EDA) of fibronectin activates Toll-like receptor 4 (TLR4), which is the receptor stimulated by bacterial lipopolysaccharide (LPS). This study shows that recombinant EDA activates TLR4 in HEK 293 cells, which normally lack this receptor. The activation of TLR4 by EDA requires co-expression of MD-2, an accessory protein. Unlike LPS, EDA activity is heat-sensitive and remains active in the presence of polymyxin B and E5564, which block LPS activation. These findings suggest that EDA-containing fibronectin fragments can induce inflammatory responses through TLR4 activation. The study also demonstrates that EDA activates NF-κB, a key regulator of inflammatory gene expression. Additionally, EDA activates TLR4 in C3H/HeJ mice, which have a Tlr4 mutation, indicating that EDA acts through TLR4. The study further shows that EDA activation of TLR4 is not due to LPS contamination in the recombinant protein. The results suggest that EDA-containing fibronectin fragments may serve as signaling molecules that activate TLR4 through a distinct pathway. The study also highlights the importance of MD-2 in EDA activation of TLR4 and the role of CD14 in enhancing EDA activation. The findings provide a mechanism by which EDA-containing fibronectin fragments promote inflammatory responses. The study also shows that EDA activates MMP-9 production in THP-1 cells, which is a key enzyme in tissue remodeling. The results indicate that EDA can activate TLR4 independently of LPS and that the effects of EDA can be distinguished from those of LPS. The study also shows that EDA activates TLR4 in the absence of CD14, suggesting that EDA may use a different pathway for TLR4 activation. The study concludes that EDA-containing fibronectin fragments can activate TLR4 and induce inflammatory responses, providing a new mechanism for fibronectin's role in inflammation.