Accumulation of Asn450Tyr mutant myocilin in ER promotes apoptosis of human trabecular meshwork cells

Mol Vis. 2020 Jul 29:26:563-573. eCollection 2020.

Abstract

Purpose: In a previous study, we identified the Asn450Tyr mutant myocilin gene (Myoc-N450Y) in the pedigree of families with juvenile open angle glaucoma (JOAG), but whether N450Y is a pathogenic mutation remained to be determined. The present study aimed at exploring the role of Myoc-N450Y in primary human trabecular meshwork (HTM) cells.

Methods: Primary HTM cells were infected with lentivirus with wild-type myocilin (Myoc-WT) or Myoc-N450Y. Primary HTM cells overexpressing Myoc-WT or Myoc-N450Y was treated with sodium 4-phenylbutyrate (4-PBA) or not. The secretion and intracellular distribution of Myoc were analyzed with western blotting and immunofluorescence. Expression of endoplasmic reticulum (ER) stress-related proteins was detected with quantitative real-time PCR (qRT-PCR) and western blotting. Cell viability, apoptosis, and expression of the related proteins were examined with Cell Counting Kit-8 (CCK-8), flow cytometry analysis, and western blotting, respectively.

Results: We found that non-secretion of Myoc-N450Y induced ER stress by colocalization with the ER marker calreticulin (CALR), and upregulating the expression of ER stress markers in primary HTM cells. Moreover, overexpression of Myoc-N450Y inhibited the viability and induced apoptosis of primary HTM cells, and inhibition of PI3K/AKT signaling was induced by ER stress. Reduction in ER stress with 4-PBA decreased the level of ER stress markers, promoted secretion, and prevented accumulation of myocilin in the Myoc-N450Y group. Apoptosis was rescued, and inhibition of PI3K/AKT signaling was reversed, after PBA treatment in primary HTM cells with Myoc-N450Y overexpression.

Conclusions: The study results suggest that Myoc-N450Y promotes apoptosis of primary HTM cells via the ER stress-induced apoptosis pathway, in which the PI3K/AKT signaling pathway plays a crucial role.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / genetics
  • Aqueous Humor / metabolism
  • Calreticulin / genetics
  • Calreticulin / metabolism
  • Cell Survival
  • Cytoskeletal Proteins / deficiency
  • Cytoskeletal Proteins / genetics*
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism
  • Endoplasmic Reticulum Stress / drug effects
  • Endoplasmic Reticulum Stress / genetics
  • Eye Proteins / genetics*
  • Gene Expression Regulation
  • Glaucoma, Open-Angle / genetics*
  • Glaucoma, Open-Angle / metabolism
  • Glaucoma, Open-Angle / pathology
  • Glycoproteins / deficiency
  • Glycoproteins / genetics*
  • Humans
  • Intraocular Pressure
  • Mutation*
  • Phenylbutyrates / pharmacology
  • Phosphatidylinositol 3-Kinases / genetics*
  • Phosphatidylinositol 3-Kinases / metabolism
  • Primary Cell Culture
  • Proto-Oncogene Proteins c-akt / genetics*
  • Proto-Oncogene Proteins c-akt / metabolism
  • Signal Transduction
  • Trabecular Meshwork / drug effects
  • Trabecular Meshwork / metabolism*
  • Trabecular Meshwork / pathology

Substances

  • CALR protein, human
  • Calreticulin
  • Cytoskeletal Proteins
  • Eye Proteins
  • Glycoproteins
  • Phenylbutyrates
  • trabecular meshwork-induced glucocorticoid response protein
  • 4-phenylbutyric acid
  • Proto-Oncogene Proteins c-akt