Date of Award

8-1-2003

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Graduate School of Biomedical Sciences

First Advisor

Thomas Yorio

Abstract

Zhang, Xinyu, Mechanisms of glucocorticoid-induced ocular hypertension. Doctor of Philosophy (Pharmacology & Neuroscience). August 2003; 163p; 4 tables; 24 figures; 102 titles. Glucocorticoids, frequently used anti-inflammatory and immunosuppressive agents, are associated with ocular hypertension and glaucoma. Endothelin-1 (ET-1) is also implicated in glaucoma pathology and optic neuropathy as its concentration is elevated in glaucoma patients and in animal models of glaucoma and chronic administration of ET-1 produces damage to the optic nerve head in rats. Glucocorticoids have been reported to regulate the expression of ET-1 gene and ET receptors in the cardiovascular system. However in the eye, the interactions between glucocorticoids and ET-1 have been implicated in the regulation of intraocular pressure and contribute to glaucoma pathology. Therefore, the purpose of the investigations described herein was to determine the novel mechanisms that may be involved in the regulation of intraocular pressure by glucocorticoids with interactions with ET-1 and ET receptors in NPE cells, a source of ET-1, and in TM cells where both glucocorticoids and ET-1 effect aqueous humor outflow. The hypothesis was that ET-1 exacerbates the actions of glucocorticoids on TM cells and contributes to increased outflow resistance. Furthermore, individual sensitivities to glucocorticoids differ considerably. About one in every three people in the general population is considered potential steroid responders while almost all primary open angle glaucoma (POAG) patients are steroid responders and develop ocular hypertension after ocular administration of glucocorticoids. The molecular mechanisms underlying the higher glucocorticoid responsiveness among POAG patients remain unknown. The glucocorticoid receptor beta isoform (hGRβ) has become a candidate for glucocorticoid resistance in some diseases, especially in asthma, based on the reports of its negative activity. The purpose of this segment of the investigations was to test the hypothesis that glucocorticoid responsiveness was regulated by the expression of hGRβ in TM cells. We demonstrated that dexamethaosone (Dex), a synthetic glucocorticoid, increased ET-1 synthesis and release from human non-pigmented ciliary epithelial (HNPE) cells. Dex also suppressed ETB receptor protein expression and attenuated ET-1 mediated increase in nitric oxide (NO) while Dex had no effect on ETA receptor expression and ETA receptor mediated intracellular Ca2+ mobilization in TM cells. The increase in the release of ET-1 from HNPE cells with a concomitant decrease of ETB receptor protein expression and ETB receptor mediated NO release by Dex in TM could result in an increase in the contraction and decrease in relaxation of trabecular meshwork thus reducing the intratrabecular space. Such actions by ET-1 may exacerbate Dex effects on the outflow pathway leading to increased outflow resistance and consequently elevated intraocular pressure that typically is associated with glucocorticoids. We have also found a significant difference in hGRβ levels among normal versus glaucomatous TM cell lines, with the POAG TM cell lines having lower hGRβ receptor expression. This is coincidence with the fact that in the normal population, there is a low rate of glucocorticoid responders as compared to almost all POAG patients considered as glucocorticoid responders. Overexpression of hGRβ in TM cells, produced by transfecting a hGRβ expression construct, inhibited Dex-induced expression of myocilin, a glaucomatous gene, supporting the contention that hGRβ acts as a negative regulator of glucocorticoid activity. In addition, we studied the machinery of cytoplasm to nuclear transport of hGRβ. We identified that a chaperon protein, hsp90, is a requirement for the nuclear translocation of hGRβ. In conclusion, we have described a novel-signaling pathway for glucocorticoids through the regulation of ET-1 and ET receptors in the anterior segment which have consequences on aqueous humor outflow. We have also demonstrated a possible molecular mechanism by which glucocorticoid responsiveness in POAG patients is achieved as a result of the low level of nuclear hGRβ receptor isoform expression. Furthermore, we have, for the first time, identified hap90 as a chaperon protein for the translocation of hGRβ from the cytoplasm to the nucleus.

Comments

Zhang, Xinyu, Mechanisms of glucocorticoid-induced ocular hypertension. Doctor of Philosophy (Pharmacology & Neuroscience). August 2003; 163p; 4 tables; 24 figures; 102 titles

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