Involvement of Insulin/Phosphoinositide 3-Kinase/Akt Signal Pathway in 17β-Estradiol-mediated Neuroprotection

Abstract

In the present study, we tested the hypothesis that 17β-estradiol (βE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H2O2)-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both βE2 and 17α-estradiol (αE2) significantly protected against H2O2-induced retinal neuron degeneration; however, progesterone had no effect. βE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [32γATP] were used as substrate. Phospho-Akt levels were also transiently increased by βE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of βE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of βE2, suggesting that βE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that βE2 and αE2, but not progesterone, identified phosphorylated insulin receptor β-subunit (IRβ) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRβ activation of PI3K. Systemic administration of βE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of βE2 activated retinal IRβ, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection. In the present study, we tested the hypothesis that 17β-estradiol (βE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H2O2)-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both βE2 and 17α-estradiol (αE2) significantly protected against H2O2-induced retinal neuron degeneration; however, progesterone had no effect. βE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [32γATP] were used as substrate. Phospho-Akt levels were also transiently increased by βE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of βE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of βE2, suggesting that βE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that βE2 and αE2, but not progesterone, identified phosphorylated insulin receptor β-subunit (IRβ) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRβ activation of PI3K. Systemic administration of βE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of βE2 activated retinal IRβ, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection. Oxidative stress-induced neuronal cell death has been implicated in different neurological disorders and neurodegenerative diseases (1Moosmann B. Behl C. Expert Opin. Investig. Drugs. 2002; 11: 1407-1435Crossref PubMed Scopus (193) Google Scholar, 2Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2666) Google Scholar). There is substantial evidence that light injures photoreceptors by increasing the formation of reactive oxygen species (3Yamashita H. Horie K. Yamamoto T. Nagano T. Hirano T. Retina. 1992; 12: 59-66Crossref PubMed Scopus (54) Google Scholar) and that antioxidants can rescue light-damaged photoreceptors (4Taguchi H. Ogura Y. Takanashi T. Hashizoe M. Honda Y. Investig. Ophthalmol. Vis. Sci. 1996; 37: 1444-1450PubMed Google Scholar, 5Stoyanovsky D.A. Goldman R. Darrow R.M. Organisciak D.T. Kagan V.E. Curr. Eye Res. 1995; 14: 181-189Crossref PubMed Scopus (88) Google Scholar, 6Blanks J.C. Pickford M.S. Organisciak D.T. Investig. Ophthalmol. Vis. 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Ophthalmol. Vis. Sci. 1996; 37: 1444-1450PubMed Google Scholar, 5Stoyanovsky D.A. Goldman R. Darrow R.M. Organisciak D.T. Kagan V.E. Curr. Eye Res. 1995; 14: 181-189Crossref PubMed Scopus (88) Google Scholar, 6Blanks J.C. Pickford M.S. Organisciak D.T. Investig. Ophthalmol. Vis. Sci. 1992; 33: 2814-2821PubMed Google Scholar), have been used to treat retinal degeneration. However, no effective medical therapy is currently available in humans, although vitamin A supplementation has shown some beneficial effect by slowing down the progression of retinitis pigmentosa (23Berson E.L. Int. Ophthalmol. Clin. 2000; 40: 93-111Crossref PubMed Scopus (47) Google Scholar). The female sex hormone estrogen has a variety of metabolic activities including numerous effects on neurons (24Carrer H.F. Cambiasso M.J. Cell Mol. Neurobiol. 2002; 22: 479-500Crossref PubMed Scopus (78) Google Scholar). Therefore, the notion that estrogen is only important for sex differentiation and maturation has changed, to include its function as a neuromodulator and neuroprotectant. It appears that estrogen specifically maintains verbal memory in women and may prevent the deterioration in short and long term memory which is associated with normal aging (25Sherwin B.B. J. Psychiatry Neurosci. 1999; 24: 315-321PubMed Google Scholar). In addition, estrogen is not restricted to females because the male sex hormone testosterone (and other steroids with a 19-carbon atom structure, so-called C-19 steroids) can be converted chemically to estradiol in various tissues, including the brain, by an aromatase P450 enzyme (26Schumacher M. Akwa Y. Guennoun R. Robert F. Labombarda F. Desarnaud F. Robel P. Nicola A.F. Baulieu E.E. J. Neurocytol. 2002; 29: 307-326Crossref Scopus (174) Google Scholar). 17β-Estradiol (βE2) 1The abbreviations used are: βE2, 17β-estradiol; αE2, 17α-estradiol; ERG, electroretinogram; GST, glutathione S-transferase; IGF-IR, insulin-like growth factor I receptor; IRβ, insulin receptor β-subunit; MTT, 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ONL, outer nuclear layer; PI3K, phosphoinositide 3-kinase; SH2, Src homology 2; TBS, Tris-buffered saline; TUNEL, TdT-mediated digoxigenin-dUTP nick-end labeling. is a steroid hormone synthesized enzymatically mainly in the ovaries from acetate, cholesterol, progesterone, and testosterone, but also by the placenta during pregnancy and, to a lesser extent, in the adrenal cortices, testes, and peripheral tissues (26Schumacher M. Akwa Y. Guennoun R. Robert F. Labombarda F. Desarnaud F. Robel P. Nicola A.F. Baulieu E.E. J. Neurocytol. 2002; 29: 307-326Crossref Scopus (174) Google Scholar). Several lines of evidence suggest that estrogen has neurotrophic and neuroprotective properties (27Wise P.M. Trends Endocrinol. Metab. 2002; 13: 229-230Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 28Garcia-Segura L.M. Azcoitia L. DonCarlos L.L. Prog. Neurobiol. 2001; 63: 29-60Crossref PubMed Scopus (832) Google Scholar). For many years, it has been known that βE2 promotes viability and survival of neurons in primary neuronal cultures. Addition of βE2 to defined culture media increased the viability, survival, and differentiation of primary neuronal cultures from different brain areas including amygdala (29Lorenzo A. Diaz H. Carrer H. Caceres A. J. Neurosci. Res. 1992; 33: 418-435Crossref PubMed Scopus Google Scholar), M.J. Carrer H.F. J. Neurosci. Res. 2001; Scopus Google Scholar), and H. C. Behl C. J.C. J. Neurochem. 1999; PubMed Scopus (122) Google Scholar). 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In the present study, we have used in vitro and in vivo to the role of estrogen as a neuroprotectant in the of were and the retinas of were with the of a in a culture The retinas were in of with in a and for 2 using a on The through a which with and the were through a by a with The in a for the and the cell in of media using a The of with a cell and the with to The were in culture on that had been with The were in with and in The cultures were used in experiments in culture on were for in in Tris-buffered and with of and in that for as described in a W. Chen W. Elias R. McGinnis J.F. J. Neurosci. Res. 2000; PubMed Scopus Google Scholar). binding were by normal for The were with and in with and using to whereas estrogen using by with to The experiments were using normal the as the primary as as primary as for by primary were The on were with and and with an with and and a The were and in a described W. Chen W. Elias R. McGinnis J.F. J. Neurosci. Res. 2000; PubMed Scopus Google Scholar), a of in as of in a of of and of the of to and 2 of of the an of the using a of apoptosis using the digoxigenin-dUTP nick-end with a available in apoptosis as described W. Tombran-Tink J. Chen W. Mrazek D. Elias R. McGinnis J.F. J. Neurosci. Res. 1999; 57: 789-800Crossref PubMed Scopus (178) Google Scholar). for the to the were identified with a and were by a and in a The of by by the of by in the of cell were from for and and were as independent independent experiments were as described W. W. McGinnis J.F. J. Neurosci. Res. 2001; 63: PubMed Scopus Google Scholar). The were in of and using a on for and for the from with the no in the to The with of acetate, and of The were for and the were in of and A to a of and the were for 2 of on a for 2 and were by and on to The were for with and and with in The were with subunit of phosphoinositide 3-kinase (PI3K) receptor β-subunit (IRβ) for 2 primary the were with and by to the PI3K were as described M. B. M. L. Full Text PDF PubMed Scopus Google Scholar, Chen H. Investig. Ophthalmol. Vis. Sci. 1997; Google Scholar). were on in of the phosphoinositide of and The were for and by the of of by of were and on with a of and 2 The were by in in and in an for were to and were and by and Pull-down S-transferase were by of the and a M.E. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The of by of the as by Pull-down experiments were as described Investig. Ophthalmol. Vis. Sci. 2001; 42: Google Scholar), using of that had been retinal neurons were with with for The were in of and for to were by in and to The were to by with were and in a light an of 2 to light for light an of by two light that were the of the During light were in with A in the in the but in the of the on the of βE2 of to light for of by were as described W. Tombran-Tink J. Elias R. Sezate S. Mrazek D. McGinnis J.F. Investig. Ophthalmol. Vis. Sci. 2001; 42: 1646-1652PubMed Google Scholar). were in the were with and were with a were with a in the with A the and a on the The of light and the were with of For the light of by were by an of were and in and were the to of of the nuclear defined the in the and using the as a of The In to the for the retinal of the of to the effects of light different of In of the experiments a from the retinas of of by of βE2, and βE2 in by a enzyme were by of of and by βE2 H2O2-induced tested the effects of βE2 in retinal neuronal culture using to an oxidant We examined the of βE2 on neurons by the cultures with different of βE2 from to A in cell viability only the the role of βE2 in retinal neurons from H2O2-induced we retinal neuron cultures with different of βE2 from to to in cell viability were and of βE2 We used to culture βE2, the of There no of for to as by the of the the effects of βE2 through the estrogen we the experiments described in the of an estrogen receptor that of tamoxifen for did not significantly cell viability, although in cell viability were and Pretreatment with tamoxifen for to the of βE2 did not significantly the neuroprotective effect of βE2 in of cultured primary retinal neurons that a of estrogen were that were not photoreceptor and were the of of and estrogen in estrogen of and on the The estrogen that βE2 Apoptosis by to βE2 can cell death by A were in whereas cultures with for had of apoptosis. However, of retinal neurons with βE2 for to to a in the of A were in the with βE2 to The of in cultures cultures from independent experiments whereas cultures Pretreatment with βE2 significantly the to of apoptosis by βE2 also in a of by of (αE2) and of the βE2 effect by two other αE2, an of βE2 that not the estrogen and to the the of when in the of A were in cultures whereas of cell death were present in cultures with for Pretreatment of retinal neurons with for to a in the of However, with progesterone for to did not to apoptosis by significantly of the of that cultures with progesterone had no cultures However, with the of in cultures from to whereas cultures significantly the of although to a lesser βE2 of the of that it significantly whereas progesterone did not and and of Cell in by systemic administration of βE2 by ERG, and of to light the of the of photoreceptor cell from the normal in A and to in the of the retinas in However, in rescue of photoreceptors with the of of as a function of the retinal a of photoreceptors by systemic of βE2 the βE2 also protected retinal as demonstrated in the in and were in the βE2 in a of different the by βE2 of and as function of rat retinal outer nuclear the and the results as S.D. for and the results as S.D. for The to photoreceptor is through of apoptosis. in the in the of the retinas in to light for an of Systemic of βE2 significantly light-induced photoreceptor cell apoptosis in The of βE2 in βE2 and of of The of βE2 in βE2 of PI3K has been shown to to neuronal H. J. Y. J. Neurosci. Res. 2002; PubMed Scopus Google Scholar). PI3K activity increased by a of cultured retinal neurons with βE2 and a and activity and to the A of PI3K activation in rat systemic of βE2 of phosphoinositide increased to and to by the that the of the regulatory subunit of PI3K did not to βE2 that the increased activity of PI3K not by increased in the Pretreatment with a PI3K inhibitor, for to the of βE2 inhibited the protective effect the LY294002 A and results suggest that the βE2 effect may be through some by activation of the PI3K The effect of the two estrogen and the of a tamoxifen effect as as the of estrogen receptor in the photoreceptor that the estrogen receptor is not involved in the estrogen-mediated photoreceptor it has been shown that IRβ is involved in the of PI3K activity in the M.E. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Investig. Ophthalmol. Vis. Sci. 2001; 42: Google Scholar). We examined IRβ activation may be involved in the estrogen effect. We on the W. R. L. C.E. McGinnis J.F. LaVail M.M. and Scholar) that 1) IRβ is in cultured rat retinal 2) βE2 and activated and progesterone In the present study, using experiments to IRβ phosphorylation we demonstrated that both insulin and βE2 activated The from in vivo experiments that both IRβ and insulin-like growth receptor were present in the Systemic administration of βE2 activated IRβ but not using We also demonstrated by that insulin receptor is present in the and outer of photoreceptor In addition, we that the insulin receptor did not PI3K and of βE2 with a of cultured retinal neurons did not PI3K of IRβ in outer and of photoreceptor in outer of IRβ in outer and of of and on of insulin receptor inhibitor, inhibited estrogen-mediated activation of PI3K. of βE2 with cell for did not It is known that Akt is the of PI3K in and is activated by phosphorylation 2002; PubMed Scopus Google Scholar). Akt can be activated by βE2, retinal neuron cultures were with βE2 for different and and were to with the W. R. L. C.E. McGinnis J.F. LaVail M.M. and shown in of βE2 to the culture for significantly increased phosphorylated Akt and increase for to by Systemic of βE2 the of also transiently increased in vivo the that the of the did not to βE2 that the increased not by increased Akt in the In the present study, we demonstrated that estrogen has a neuroprotective effect in the which is the it has been known that βE2 promotes viability and survival of other primary neuronal culture such as K. Shimohama S. H. T. T. H. A. J. Neurosci. Res. 2001; PubMed Scopus Google Scholar), R. K. W. Metab. Res. 2001; 33: PubMed Scopus Google Scholar), M.J. Carrer H.F. J. Neurosci. Res. 2001; Scopus Google Scholar), is the that βE2 H2O2-induced light-induced photoreceptor and the signaling In some tissues, of βE2 may be by the estrogen receptor and (27Wise P.M. Trends Endocrinol. 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