Glucocorticoids exacerbate hypoxia-induced expression of the pro-apoptotic gene Bnip3 in the developing cortex

Abstract

Neonatal administration of the synthetic glucocorticoid, dexamethasone (DEX) retards brain growth, alters adult behaviors and induces cell death in the rat brain, thereby implicating glucocorticoids as developmentally neuroendangering compounds. Glucocorticoids also increase expression of proapoptotic Bcl-2 family members and exacerbate expression of hypoxic responsive genes. Bnip3 is a pro-apoptotic Bcl-2 family member that is upregulated in response to hypoxia. In these studies, we investigated the interactions of glucocorticoid receptor and hypoxia in the regulation of Bnip3 mRNA in cortical neurons. Using quantitative real time RT-PCR, we found that DEX treatment of postnatal day 4-6 rat pups caused a significant increase in Bnip3 mRNA expression compared to vehicle controls. A significant increase in Bnip3 mRNA was also measured in primary cortical neurons 72 hours after treatment with RU28362, a glucocorticoid receptor selective agonist. In primary cortical neurons, hypoxia increased Bnip3 mRNA expression and this was exacerbated with RU28362 treatment. To elucidate the mechanism of glucocorticoid and hypoxia mediated regulation of Bnip3 transcription, a Bnip3 promoter -luciferase reporter construct was utilized in primary cortical neurons. Upregulation of the Bnip3 promoter was mediated by a single glucocorticoid response element and a hypoxic response element. Bnip3 overexpression in primary cortical neurons significantly increased cell death, which is dependent on the Bnip3 transmembrane domain. However, despite the increased expression of Bnip3 following glucocorticoid and hypoxia treatment, corresponding decreases in cell survival were minimal. These studies identify a novel pathway in the developing cortex through which glucocorticoids may enhance a metabolic insult, such as hypoxia. KeywordsBcl-2; glucocorticoid receptor; dexamethasone; primary cortical neurons; postnatal; apoptosis Corticosterone, the endogenous glucocorticoid secreted by the rat adrenal gland, binds with high affinity to two different receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) (Reagan and McEwen, 1997). MR activation is associated with a neuroprotective phenotype (Almeida et al., 2000), whereas GR activation is implicated in the induction of an endangered neural phenotype (Sapolsky et al., 1986, Reagan andMcEwen, 1997). For example, in the adult, GR activation by the synthetic glucocorticoid Name and address of corresponding author: Robert J. Handa, Colorado State University, Department of Biomedical Sciences, W103 Anatomy, 1617 Campus Delivery, Fort Collins, CO 80523-1617, Phone: (970) Facsimile: (970) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process er...

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