Impact of Progestins on Estrogen-Induced Neuroprotection: Synergy by Progesterone and 19-Norprogesterone and Antagonism by Medroxyprogesterone Acetate

0013-7227/02/$03.00/0 Printed in U.S.A. Endocrinology 143(1):205–212 Copyright © 2002 by The Endocrine Society Impact of Progestins on Estrogen-Induced Neuroprotection: Synergy by Progesterone and 19-Norprogesterone and Antagonism by Medroxyprogesterone Acetate JON NILSEN AND ROBERTA DIAZ BRINTON Department of Molecular Pharmacology and Toxicology and the Program in Neuroscience, University of Southern California, Pharmaceutical Sciences Center, Los Angeles, California 90033 Estrogen replacement therapy is associated with improvement of cognitive deficits and reduced incidence of Alzheimer’s disease. To compare the impact of therapeutically relevant progestins on estrogen-induced neuroprotection, we treated primary hippocampal neuron cultures with 17␤-E2 and progestin, alone and in combination, 48 h before glutamate insult. Estrogen, progesterone, and 19-norprogesterone, alone or in combination, protected against glutamate toxicity. In contrast, medroxyprogesterone acetate (MPA) failed to protect against glutamate toxicity. Not only was MPA an ineffective neuroprotectant but it attenuated the estrogeninduced neuroprotection when coadministered. We addressed the role of MAPK activation in neuroprotection by ovarian ste- roids. Estrogen and all three progestins tested, alone or in combination, activated MAPK, indicating another mechanism of protection. Bcl-2 expression has been shown to prevent cell death and is up-regulated by 17␤-E2. Progesterone and 19-norprogesterone, alone or in combination with estrogen, increased Bcl-2 expression. In contrast, MPA blocked estrogen-induced Bcl-2 expression when coadministered. These results may have important implications for the effective use of hormone replacement therapy in the maintenance of neuronal function during menopause and aging and for protection against neurodegenerative diseases such as Alzheimer’s disease. (Endocrinology 143: 205–212, 2002) A LTHOUGH THE RECEPTORS for estrogens and progestins belong to different nuclear receptor subfamilies and recognize distinct hormone response elements, there is considerable cross-talk between the estrogen and progestin signaling pathways. Progestins have been shown to block the estrogen-stimulated expression of both c-fos and progesterone receptor mRNAs in uterine cells (1, 2). Furthermore, progestins and antiprogestins have been shown to inhibit estrogen-stimulated uterine proliferation (2, 3). Thus, progestins have been added to hormone replacement therapy (HRT), in part, to reduce the risk of uterine cancers associated with unopposed estrogen (4). Progestins have been added to some formulations of HRT to prevent hyperplasia of the endometrium and tumorogenesis (5). In addition to prevention of endometrial hyperplasia, inclusion of progestins in HRT was thought to reduce estrogenic effects in breast that may be associated with increased risk of breast cancer (6). Contrary to this long-held belief, a recent study found that an estrogen-progestin regimen increased cancer risk beyond that associated with estrogen alone (7). This emphasizes the necessity of studying the modulation of estrogenic effects by progestins in various systems. It is becoming increasingly apparent that HRT is beneficial in the central nervous system, in part, because of the neurological effects of estrogens, including protection against neurologic insults, reduced risk of Alzheimer’s disease (AD), and improved cognitive function (8, 9). In vitro models for these effects have shown that estrogen enhances neuronal survival resulting from oxidative stress, excitotoxic insults, and ␤-amyloid (10, 11). Although all the mechanisms underlying these effects remain to be identified, recent studies have shown that the tyrosine kinase/MAPK signal transduction cascade may be involved in estrogen-mediated neuroprotection. Estrogen rapidly activated tyrosine kinase and MAPK activity (12–14), and the neuroprotective effect of estrogen against glutamate toxicity was blocked by inhibitors of tyrosine kinase and MAPK (15). The MAPK pathway is thought to play an important role in the actions of neurotrophins, and its activation could lead to increased expression of antiapoptotic genes. One such antiapoptotic gene, bcl-2, is involved in survival of nerve growth factor (NGF)dependent sensory neurons (16) and inhibits death in response to glutamate toxicity (17). Additionally, Bcl-2 expression is increased in response to estrogen treatment of neuronal cells (18 –20). Very little research on hormonal modulation of neuronal survival has studied the effects of progestins only or progestins in conjunction with estrogen. It is currently unknown what effect different progestins have on estrogen-mediated modulation of MAPK activation and Bcl-2 expression in neuronal cells. It is not clear whether progestins are neuroprotective or not, nor whether they impact the neuroprotective effects of estrogen. Previous studies reported no neuroprotective effect of progestins (21), whereas others have seen positive effects of progestin on neuronal survival (22, 23). Direct comparison between these studies is difficult, because Abbreviations: AD, Alzheimer’s disease; ERT, estrogen replacement therapy; HRT, hormone replacement therapy; LDH, lactate dehydrogenase; MPA, medroxyprogesterone acetate; NGF, nerve growth factor; PBS-Tween, PBS containing 0.05% Tween-20. 205 206 Endocrinology, January 2002, 143(1):205–212 each differed in the neuroprotective model studied and in the progestin used. The purpose of this study was to determine the effects of three widely clinically used progestins [progesterone, 19norprogesterone, and medroxyprogesterone acetate (MPA)] on a well-established model of estrogen-induced neuroprotection. Results of the present study demonstrate that progesterone and 19-norprogesterone were neuroprotective, alone or when administered in conjunction with 17␤-E2, whereas MPA was not. Not only was MPA an ineffective neuroprotectant, it blocked estrogen-induced neuroprotection when coadministered. Although all three progestins tested activated MAPK, only the neuroprotective progesterone and 19-norprogesterone increased the expression of Bcl-2. These data point out the necessity of examining the composition of hormones used in HRT formulations, especially with regard to which progestin is included. This work belies a common underlying theme in endocrinology, that although structurally different ligands behave the same in some systems, they do not necessarily do so in all systems. Materials and Methods Animals All studies were approved by the University of Southern California Institutional Review Board for animal care. Timed-pregnant Sprague Dawley rats were purchased from Harlan Sprague Dawley, Inc., Indianapolis, IN. They were housed under controlled conditions of temperature (22 C), humidity, and light (14 h light, 10 h dark); and water and food were available ad libitum. Neuronal culture Primary cultures of dissociated h (...truncated)