GTx-822, an ERβ-selective agonist, protects retinal pigment epithelium (ARPE-19) from oxidative stress by activating MAPK and PI3-K pathways

Anand Giddabasappa, Matthew N. Bauler, Christina M. Barrett, Christopher C. Coss, Zhongzhi Wu, Duane Miller, James T. Dalton, Jeetendra R. Eswaraka

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

PURPOSE. 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 2O 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 2O 2) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H 2DCFDA 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.

Original languageEnglish (US)
Pages (from-to)5934-5942
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number11
DOIs
StatePublished - Nov 1 2010

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Retinal Pigment Epithelium
Estrogens
Oxidative Stress
Apoptosis
Estrogen Receptors
Hydrogen Peroxide
Transcriptional Activation
Gene Expression
GTx-822
Protein Array Analysis
Cytoprotection
Eye Diseases
Mitochondrial Membrane Potential
Protein Kinases
Reactive Oxygen Species
Proteins
Cell Death
Up-Regulation
Antioxidants
Fluorescence

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

GTx-822, an ERβ-selective agonist, protects retinal pigment epithelium (ARPE-19) from oxidative stress by activating MAPK and PI3-K pathways. / Giddabasappa, Anand; Bauler, Matthew N.; Barrett, Christina M.; Coss, Christopher C.; Wu, Zhongzhi; Miller, Duane; Dalton, James T.; Eswaraka, Jeetendra R.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 11, 01.11.2010, p. 5934-5942.

Research output: Contribution to journalArticle

Giddabasappa, Anand ; Bauler, Matthew N. ; Barrett, Christina M. ; Coss, Christopher C. ; Wu, Zhongzhi ; Miller, Duane ; Dalton, James T. ; Eswaraka, Jeetendra R. / GTx-822, an ERβ-selective agonist, protects retinal pigment epithelium (ARPE-19) from oxidative stress by activating MAPK and PI3-K pathways. In: Investigative Ophthalmology and Visual Science. 2010 ; Vol. 51, No. 11. pp. 5934-5942.
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abstract = "PURPOSE. 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 2O 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 2O 2) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H 2DCFDA 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.",
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T1 - GTx-822, an ERβ-selective agonist, protects retinal pigment epithelium (ARPE-19) from oxidative stress by activating MAPK and PI3-K pathways

AU - Giddabasappa, Anand

AU - Bauler, Matthew N.

AU - Barrett, Christina M.

AU - Coss, Christopher C.

AU - Wu, Zhongzhi

AU - Miller, Duane

AU - Dalton, James T.

AU - Eswaraka, Jeetendra R.

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N2 - PURPOSE. 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 2O 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 2O 2) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H 2DCFDA 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.

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