GTx-822, an ER{beta}-selective agonist, protects retinal pigment epithelium (ARPE-19) from oxidative stress by activating MAPK and PI3-K pathways.
OBJECTIVE
The goal of this study was to determine whether an estrogen receptor-β (ERβ)-selective agonist (GTx-822; GTx, Inc., Memphis, TN) could prevent hydrogen peroxide (H(2)O(2))-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection.


METHODS
The selectivity of GTx-822 for ERβ was determined by receptor-binding assay (RBA) and transactivation assay. Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide (t-BH) or hydrogen peroxide (H(2)O(2)) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell death ELISA. Mitochondrial membrane potential was measured with the JC-1 assay. Gene expression and protein expression and activation were quantitated with qRT-PCR and Western blot analysis. Phospho-protein arrays elucidated the activation of protein kinases.


RESULTS
The RBA and transactivation assay revealed that GTx-822 is an ERβ-selective agonist (K(i) = 0.53 nM). GTx-822 prevented oxidative stress in ARPE-19 cells. It preserved mitochondrial function and prevented cellular apoptosis. Pretreatment with GTx-822 increased ERβ gene and protein expression during oxidative stress. Upregulation of the phase II antioxidant genes GPx-2 and HO-1 was also seen in an ERβ-dependent mechanism. GTx-822 pretreatment induced phosphorylation of ERK1/2, PI3-K, and Bad.


CONCLUSIONS
This is the first report to show that GTx-822, an ERβ agonist, can protect ARPE-19 cells from the cellular apoptosis induced by oxidative stress. GTx-822 mediated cytoprotection was mediated through induction of both genomic and nongenomic pathways. The results of this study open new avenues for the use of a selective ERβ agonist in treatment of ocular diseases like AMD where oxidative stress plays a major role in disease pathogenesis.
Giddabasappa
Anand
A
Preclinical Research and Development, GTx, Inc, Memphis, Tennessee 38163, USA.
Bauler
Matthew N
MN
Barrett
Christina M
CM
Coss
Christopher C
CC
Wu
Zhongzhi
Z
Miller
Duane D
DD
Dalton
James T
JT
Eswaraka
Jeetendra R
JR
D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D019610:Cytoprotection; D004797:Enzyme-Linked Immunosorbent Assay; D047629:Estrogen Receptor beta / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011804:Quinolines / Q000494:pharmacology*; D011869:Radioligand Assay; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D020122:tert-Butylhydroperoxide / Q000633:toxicity
D047629:Estrogen Receptor beta; C553877:GTx-822; D011804:Quinolines; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases