Altered stability of mRNAs associated with glaucoma progression in human trabecular meshwork cells following oxidative stress.

PURPOSE

The goals of this study were to determine if oxidative stress on human trabecular meshwork (HTM) cells influences the stability of key mRNAs containing AU rich elements (AREs) known to be associated with glaucoma progression, and if the presence of topographic cue alters the stability of these mRNAs.

METHODS

HTM cells were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 1 hour in the presence of 5 μg/mL actinomycin D and compared with untreated cells. The selected mRNAs (IL-6, IL-8, myocilin, SPARC [secreted protein, acidic and rich in cysteine], matrix metalloproteinase [MMP]-3, and MMP-9) from the cells were analyzed by using relative quantitative PCR. Immunohistochemistry for Hu antigen R (HuR) was performed in addition to Western blots of HuR. HTM cells were also grown on topographically patterned surfaces, and IL-6 mRNA was analyzed by quantitative PCR.

RESULTS

H(2)O(2) increased IL-6 mRNA stability 0.145 (0.095-0.27) to 0.345 (0.2-0.48) (normalized ratio, median [interquartile range]) (n = 5), while IL-8 mRNA was increased from 0.565 (0.408-0.6) to 0.775 (0.486-0.873) (n = 5). These differences were statistically significant (P = 0.0313, for both IL-6 and IL-8; Wilcoxon signed-rank test). The mRNAs of myocilin, SPARC, and MMP-3, which do not have AREs, were more stable after actinomycin D treatment and were not altered with oxidation. Western blot and immunohistochemistry demonstrated that H(2)O(2) treatment induces the translocation of HuR from the nucleus to the cytoplasm. Nanopatterned surfaces did not alter IL-6 mRNA stability.

CONCLUSIONS

Oxidative stress stabilizes IL-6 and IL-8 mRNAs significantly. The decay of certain mRNAs associated with glaucoma may be altered in the trabecular meshwork of glaucoma patients.