Abstract
BackgroundMyocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro370 to Leu (P370L) mutation of myocilin is associated with severe glaucoma phenotypes and Gln368 stop (Q368X) is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA) activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin.Methodology/Principal FindingsWild type myocilin, when transfected into cultured human TM cells, induced a loss of actin stress fibers as judged by phalloidin staining. Such a loss was averted by treatment of secreted Frizzled-related protein 1 (sFRP1), an inhibitor of Wnt signaling. Consistent with the notion that Wnt pathway mediates the myocilin phenotype, Wnt activation was demonstrated by TOP/FOP-Flash reporter assays. Treatment of human TM cells of a Wnt activator, SB216763, as well as transfection of myocilin P370L and Q368X mutants all resulted in actin stress fiber loss, PKA activation and RhoA inactivation. The PKA elevation was obviated by the sFRP1 treatment, indicating that Wnt signaling was upstream that of PKA.Conclusions/SignificanceThe present study demonstrated that following forced expression of wild type myocilin, Wnt was activated, triggering in turn other myocilin-related alterations. P370L and Q368X mutations induced similar phenotypes, suggesting one possible mechanism how the mutants may lead to TM cell damage and pathology.
Highlights
Glaucoma, one of the leading causes of irreversible blindness worldwide, is characterized by progressive loss of retinal ganglion cells and the accompanying axons, as well as cupping of the optic nerve head [1]
To test whether Wnt signaling is involved in the myocilin phenotype, trabecular meshwork (TM) cells after myocilin transfection were treated with secreted Frizzled-related protein 1 (sFRP1), a Wnt signaling pathway inhibitor [25]
The present study demonstrated that wild type myocilin, when overexpressed at a moderate level, activated the Wnt signaling pathway, which in turn mediated downstream events such as loss of actin stress fibers and elevation of the protein kinase A (PKA) activity
Summary
One of the leading causes of irreversible blindness worldwide, is characterized by progressive loss of retinal ganglion cells and the accompanying axons, as well as cupping of the optic nerve head [1]. The trabecular meshwork (TM), a specialized tissue located next to the cornea, is the major site for regulation of the aqueous humor outflow [3,4]. It is composed of layers of trabecular beams made up of extracellular matrix (ECM) elements. TM cells that cover the beams display an endothelial cell-like morphology and lining property but are of a unique cell type [4] They are avid phagocytes [5], possess contractile and migratory apparatus [6], and have the capacity to produce ECM elements [4,7]. We examined whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants display biological effects similar to those of the wild type myocilin
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