Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork.
OBJECTIVE
Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells.


METHODS
Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining.


RESULTS
Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 μl/min/mm Hg in Tg-MYOCY437H vs. 0.0332 μl/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells.


CONCLUSIONS
These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.
North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Texas, United States.
Ramesh B
RB
Kasetti
North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Texas, United States.
Tien N
TN
Phan
North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Texas, United States.
J Cameron
JC
Millar
North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, Texas, United States.
Gulab S
GS
Zode
D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005902:Glaucoma, Open-Angle / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007429:Intraocular Pressure; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000378:metabolism* / Q000473:pathology; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D004247:DNA; D000091342:Endoplasmic Reticulum Chaperone BiP; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin; C000717247:HSPA5 protein, human; C000717248:Hspa5 protein, mouse