Abstract
Purpose :
The molecular mechanisms underlying development of glaucoma are not completely understood. However, profibrotic TGFβ2 signaling has been suggested to be involved in glaucomatous damage to the trabecular meshwork (TM). Our work has shown TGFβ2 and toll-like receptor 4 (TLR4) signaling crosstalk is involved in the development of ocular hypertension and TM damage. We have identified fibronectin extra domain A (FN-EDA) as an important regulator of pathogenic TLR4 and TGFβ2 signaling in the TM. FN-EDA is a known damage associated molecular pattern (DAMP) that can activate TLR4, separate from the classical activator of TLR4, LPS. Here, we examined whether endocytosis of DAMP-activated TLR4 is delayed or prevented compared to LPS activated TLR4, thus preventing negative regulation of TLR4 signaling and contributing to the feed-forward mechanisms of pathogenic TGFβ2-TLR4 signaling crosstalk.
Methods :
Primary human TM cells were grown to confluence and transfected overnight with Ad5.TLR4 to overexpress TLR4. Cells were then treated with LPS (100 ng/ml), cFN-EDA (10 ug/ml), or untreated as a control for 2 hours. After 2 hours, cells were fractionated into cytosolic and membrane components. Equal amounts of each fraction were immunoprecipitated for TLR4 and resulting immunoprecipitants were immunoblotted for TLR4 expression. Cells were similarly treated and probed for TLR4 using on-cell western techniques and total membrane TLR4 immunofluorescence quantified.
Results :
Immunoblotting of fractionated and immunoprecipitated cell lysates from treated primary human TM cells showed control cells had a TLR4 cytosolic to membrane ratio of 0.73, LPS treated had a ratio of 1.15, and cFN-EDA treated had a ratio of 0.82, with a smaller ratio indicative of more TLR4 present in the membrane compared to the cytosol (n=3, p<0.05). Similarly, on cell western techniques indicated increased TLR4 membrane expression in the cFN-EDA treated cells compared to LPS treated cells (n=3, p<0.05).
Conclusions :
This study demonstrates TLR4 is retained in higher amounts at the membrane when activated by the known DAMP, FN-EDA, versus activation by LPS. This retention of TLR4 membrane expression suggests that DAMP-activated TLR4 is resistant to endocytosis, which may contribute to the feed-forward mechanisms of pathogenic TGFβ2-TLR4 signaling crosstalk. This important finding helps to elucidate more therapeutic targets that may combat glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.