Abstract
Purpose :
Although the ECM in TM cells is known to be important in IOP regulation, the molecular mechanisms involved in generating a glaucomatous environment in the TM are not completely understood. Recently we identified a molecular pathway, TGFβ2-TLR4 signaling crosstalk, as an important regulator of glaucomatous damage in the TM which contributes to fibrosis. Here we continued our studies on a novel molecular target, A20 (also known as TNFAIP3), within this pathway which may help to block pathological TGFβ2-TLR4 signaling, ultimately leading to new treatments to prevent the development and progression of glaucoma.
Methods :
Primary human trabecular meshwork (TM) cells were grown to confluence, switched to serum free media, and then treated for 48hrs with TGFβ2 (5ng/mL), TLR4 inhibitor (TAK-242, 15μM), A20 inducer (Vitamin E, 100μM), TGFβ2 and TLR4 inhibitor, TGFβ2 and Vitamin E, or PBS as a control. Lysates were either collected and immunoblotted for A20 and fibronectin expression, or for A20 analysis by RT-PCR.
Results :
RT-PCR and immunoblotting showed that A20 is expressed in primary human TM cells. A20 message increased (p=0.0052) when the TLR4 pathway was inhibited. Immunoblotting of cell lysates from primary TM cell strains showed that TGFβ2, a known inducer of fibrosis, increases fibronectin expression (p=0.0035), while at the same time decreasing the expression of A20 (p=0.0019). Both direct inhibition of TLR4 (with TAK-242) and direct induction of A20 (with Vitamin E) showed a trending increase in A20 expression on immunoblots. Concurrent treatment with both TGFB-2 and Vitamin E also showed an increase in A20 levels.
Conclusions :
This ongoing study reports the expression of A20 in primary cells as it relates to the TGFβ2-TLR4 crosstalk pathway. A20 was easily detectable by PCR analysis and increased with TLR4 inhibition. Low basal expression of A20 was detected at the protein level, which has also been reported in other cell types. However, treatment with TGFβ2, TLR4 inhibitor, or Vitamin E did modulate A20 protein expression. Therefore, TGFβ2-TLR4 crosstalk may be an important mechanistic pathway in the development of fibrotic TM damage and A20 may be able to alleviate some fibrotic responses by attenuating the TLR4 pathways ability to induce fibrosis.
This is a 2021 ARVO Annual Meeting abstract.