title: Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells.
abstract: OBJECTIVE
  Activation of the IL-1/NF-κB inflammatory stress pathway and induction of SELE expression in the trabecular meshwork (TBM) is a marker for high-tension glaucomas of diverse etiology. Pathway activation stimulates aqueous outflow and protects against oxidative stress, but may be damaging in the long-term. MYOC mutations have been causally linked to high-tension forms of primary open angle glaucoma (POAG). This study investigated a possible link between MYOC mutations and activation of the IL-1/NF-κB pathway and expression of SELE.
  
  
  METHODS
  We constructed MYOC expression vectors with mutations at sites that cause POAG. Mutations (Q368X, Y437H, A427T) were selected to represent proteins with differing POAG-causing potency (Q368X > Y437H > A427T) and intracellular retention behavior (Q368X and Y437H retained, A427T released). The constructs were made in two different kinds of vectors; one a plasmid designed for transient transfection (pCMV6), and one a doxycycline-inducible lentiviral vector (pSLIK) for stable cell transduction. The immortalized human trabecular meshwork line TM-1 was used for all expression studies. Expression of IL1A mRNA was determined by reverse transcription (RT)-PCR, as well as a set of five other genes associated with signaling pathways linked to glaucoma: IL1B and IL6 (NF-κB pathway), TGFB2 and ACTA2 (TGF-β pathway) and FOXO1 (E2F1 apoptotic pathway). An ELISA was used to quantify IL1A protein released into culture media. To quantify intracellular NF-κB activity, we transiently transfected stably transduced cell lines with a luciferase expression vector under control of the IL8 promoter (containing an NF-κB response element).
  
  
  RESULTS
  Transiently expressed wild-type MYOC was released into cell culture media, whereas mutant MYOCs Q368X and Y437H remained within cells. Both mutant MYOCs activated the IL-1/ NF-κB pathway, significantly stimulating expression of IL1A and IL1B. However Y437H, which causes a severe glaucoma phenotype, was less effective than Q368X, which causes a moderate glaucoma phenotype. In addition, the retained mutants stimulated expression of stress response genes ACTA2 and FOXO1. Unexpectedly, wild-type MYOC significantly decreased expression of IL6 and TGFB2, to approximately half of the control levels, and expression of IL1B and ACTA2 was also slightly decreased. Induction of MYOC mutants Q368X and Y437H in stably transduced cell lines significantly stimulated the level of IL1A protein released into culture media. Once again however, the effect of the severe MYOC mutant Y437H was less than the effect of the moderate MYOC mutant Q368X. In contrast, induced expression of the intracellularly retained 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 increased NF-κB activity by 25% over IL1A alone. In contrast, induction of Q368X or A427T plus IL1A treatment did not significantly affect NF-κB activity over IL1A alone. However, wild-type MYOC expression inhibited IL1A-stimulated NF-κB activity. We also observed that endogenous MYOC expression was induced by IL1A in TM-1 cells and primary TBM cell cultures. SELE was co-expressed with MYOC in the primary cell lines.
  
  
  CONCLUSIONS
  These results indicate that POAG-causing MYOC mutants activate the IL-1/NF-κB pathway, with activation levels correlated with intracellular retention of the protein, but not POAG-causing potency. Unexpectedly, it was also discovered that wild-type MYOC inhibits activation of the IL-1/NF-κB pathway, and that activation of the IL-1/NF-κB pathway stimulates expression of MYOC. This is the first evidence that glaucoma-causing MYOC mutants can activate the inflammatory response and that wild-type MYOC has anti-inflammatory activity.
authors.affiliation: USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Itakura
authors.affiliation: Departments of Pathology & Laboratory Medicine and Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Peters
authors.affiliation: USC Institute for Genetic Medicine, USC Eye Institute, and Departments of Cell & Neurobiology and Ophthalmology, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA.
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Fini
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002461:Cell Line, Transformed; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005902:Glaucoma, Open-Angle / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D061251:Primary Cell Culture; D015398:Signal Transduction; D014129:Trabecular Meshwork / Q000378:metabolism / Q000473:pathology; D053781:Transforming Growth Factor beta2 / Q000235:genetics / Q000378:metabolism; D000099179:Myocilin
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D019040:E-Selectin; D005136:Eye Proteins; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D006023:Glycoproteins; D053583:Interleukin-1beta; D015850:Interleukin-6; D016328:NF-kappa B; D053781:Transforming Growth Factor beta2; D000099179:Myocilin; C541116:ACTA2 protein, human; C085126:FOXO1 protein, human; C546936:SELE protein, human; C509041:TGFB2 protein, human