Stability of mRNAs in Human Trabecular Meshwork Cells on Nanoscale Topographically Patterned Substrates Following Oxidative Stress

Abstract

 

Purpose:

 

Oxidative stress is present in glaucoma and is known to increase IL-6 and IL-8 in human trabecular meshwork (HTM) cells after oxidative challenge. We have reported that substratum topographic cues dramatically modulate mRNA levels in HTM cells. The purpose of this study was to determine if topography influences IL-6 and IL-8 mRNA expression and if oxidative stress changes the stability of IL-6 and IL-8 mRNAs in the presence or absence of substratum topographic cues. Finally, we explored the potential role of HuR, a protein that stabilizes mRNA under oxidative stress.

 

Methods:

 

HTM cells were cultured on flat or topographically patterned substrates containing 700nm ridges and grooves. The cells were treated with or without 150µM H₂O₂ for 2 hours in the presence of actinomycin D that prevents additional mRNA synthesis. IL-6 and IL-8 mRNAs were analyzed by Q-PCR. Additionally, immunohistochemistry and Western blots were performed for HuR.

 

Results:

 

Provision of topographic cues failed to alter IL-6 mRNA levels. IL-6 mRNA, that in the absence of H₂O₂ decreased over time. In the presence of H₂O₂, however, IL-6 mRNA was 3 fold higher compared to cells without H₂O₂ on both planar and topographically patterned surfaces. Similarly, topography alone did not influence IL-8 mRNA levels, but IL-8 mRNA levels treated with H₂O₂ was 1.7 fold greater only on 1400nm surfaces compared to the cells without H₂O₂. HuR was detected in HTM cells with Western blot. Immunohistochemistry demonstrated that H₂O₂ treatment induced the translocation of HuR from the nucleus to the cytoplasm.

 

Conclusions:

 

IL-6 mRNA levels were not affected by topographic cues and the stability of IL-6 mRNA was increased after oxidative stress, but. Although topography did not affect IL-8 mRNA expression, it did influence IL-8 mRNA stability after oxidative stress. These effects may be coupled to HuR’s presence in the cytoplasm. Topographic cues may be important in how HTM cells react to glaucoma related oxidative stress.

 

Keywords: trabecular meshwork • oxidation/oxidative or free radical damage