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Tatsuo Itakura, Donna M Peters, M Elizabeth Fini; MYOC Involves the NF-κB Pathway by Regulation of an IL1A Feedback Loop. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2009.
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© ARVO (1962-2015); The Authors (2016-present)
Activation of an ELAM-1/IL-1/NF-κB inflammatory stress pathway in the trabecular meshwork (TBM) is a marker for high-tension glaucomas of diverse etiology. Activation of the pathway stimulates aqueous outflow and protects against oxidative stress, but may be damaging in the long term (Nat Med, 7:304, 2001). MYOC mutations are causatively linked to high-tension forms of primary open angle glaucoma (POAG). Previously, we showed that glaucoma-causing MYOC mutations induce IL1A mRNA (ARVO 2011). Here we compared the effects of mutant MYOC proteins to wild-type MYOC.
We cloned wild type and mutant MYOCs into a lentivirus vector (pSLIK) under control of the tet-on system, infected immortalized human TBM cells (TM-1), and selected stable cell lines. MYOC mutants were selected to represent proteins with differing intracellular retention behavior and POAG-causing potency (Q368X and Y437H = high retention/potency; A427T = low retention/potency). To quantify IL1A protein released into culture media, we used an ELISA kit (Life Technologies). To quantify intracellular NF-κB activity, we transiently transfected lenti-cell lines with a luciferase expression vector under control of the IL8 promoter (containing an NF-κB response element).
Doxycycline (dox) induction (1 ug/ml) of MYOC mutants Q368X and Y437H increased IL1A protein released into culture media by ~25-fold (p<0.0007) and 10-fold (p<0.002), respectively. On the other hand, expression of mutant MYOC (A427T) or wild type MYOC did not change the amount of IL1A protein in culture media. Induction of Y437H MYOC plus IL1A treatment (10 ug/ml) increased NF-κB activity by 25% over IL1A alone (p<0.05). Induction of Q368X or A427T plus IL1A treatment did not significantly affect NF-κB activity over IL1A alone. However, the wild type MYOC expression inhibited IL1A-stimulated NF-κB activity by 31% (p<0.04). IL1A treatment induced MYOC expression in TM-1 cells and primary TBM cell cultures, as previously shown by another lab (IOVS 52:7548, 2011).
These results provide additional evidence that glaucoma-causing MYOC mutants activate the IL-1/NF-κB pathway, with activation levels correlated with cell retention and potency in causing POAG. Unexpectedly, it was also discovered that wild type MYOC inhibits activation of NF-κB via a negative IL1A feedback loop. This is the first evidence that MYOC has anti-inflammatory activity.
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