Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons
Introduction
Steroid hormones play a regulatory role in a variety of cellular processes such as reproduction, development, differentiation, apoptosis, and brain function [1]. Many studies have identified that steroid hormones are synthesized not only in peripheral tissues but also in the central nervous system (CNS) as neurosteroids [2].
Progesterone (P4), one of these neurosteroids, has multiple biological functions in the CNS, among which is its ability to afford neuroprotection. For example, P4 protects hippocampal neurons from FeSO4−, amyloid β- as well as glutamate-induced cell death [3], [4], [5], [6]. Further, P4 is effective in reducing secondary neuronal loss and the associated cognitive impairment following cortical contusion injury [7], [8]. Although studies continue to demonstrate the neuroprotective potential of P4, our understanding of the likely numerous mechanisms underlying the neuroprotective effects of P4 remain incomplete.
The goal of the present study was to identify what role the neurosteroid P4 plays in controlling intracellular Ca2+ concentrations and Ca2+ signaling in neurons.
Calcium (Ca2+) contributes to signal transduction and acts as an intracellular second messenger in neurons. Ca2+ signaling is highly regulated and dynamic. Intracellular Ca2+ signaling in neurons plays an important role in regulating numerous cellular processes such as gene expression, cell development, neurotransmitter release, and apoptosis [9], [10]. Even small alterations in Ca2+-dependent homeostatic mechanisms contribute to various diseases that involve functional decline or death of cells [11], [12], [13].
A functional link between P4 and intracellular Ca2+ levels ([Ca2+]i) was seen in non-neuronal systems [14], however, the cellular mediators involved in the P4-mediated changes in [Ca2+]i remain to be determined particularly for neurons. What is known is that intracellular Ca2+ channels (ICCs) such as inositol 1,4,5-trisphosphate receptors (IP3Rs) are major components of the cytosolic Ca2+ regulation machinery [10], [15]. Furthermore, several steroid hormones activate other signaling molecules that might in turn lead to activation of ICCs [15], [16].
IP3Rs are located predominantly in the endoplasmic reticulum (ER). Activation of phospholipase C by extracellular stimuli including hormones and neurotransmitters leads to hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2), generating IP3 and diacylglycerol [13]. IP3 activates IP3R, leading to release of Ca2+ from intracellular stores, and thus, is capable of contributing to the control of intracellular Ca2+ levels. Drugs that alter Ca2+ release from intracellular stores may affect neuronal function [13].
Both estradiol and P4 elicit the phosphorylation of Akt kinase in mouse cerebral cortical cultures [16], a signaling protein that has been implicated in the phosphorylation of IP3Rs in non-neuronal cell types and tissues [17]. Furthermore, Akt activation has been implicated in neuroprotective signaling in neurons [6]. Therefore, the overall goal of this study was to identify the mechanism by which P4 alters IP3R-mediated Ca2+ release in hippocampal neurons.
We tested the hypothesis that Akt-mediated phosphorylation of IP3R leads to increased IP3R-mediated intracellular Ca2+ release. In addition, we tested the second hypothesis that P4 modifies cellular expression levels and/or distribution of certain components of P4 and Ca2+ signaling pathways as well as IP3R activity. We show that P4, through activation of Akt, potentiates IP3R-mediated Ca2+ release. This work is unique in that it identifies a specific Ca2+ signaling protein target that is activated by Akt through the steroid hormone P4. Furthermore, this work reveals a mechanism for how P4 alters Ca2+ signaling in neurons.
Section snippets
Preparation of mouse hippocampal neurons and primary cell culture
Hippocampal tissue from 2-day-old mice was removed, minced in Hank's balanced salt solution (HBSS) and incubated in 0.25% trypsin and 0.1% DNase in phosphate buffered saline (PBS) at 37 °C for 20 min. Tissue from both sexes was used and no differences in parameters assessed by the present study were detected. The tissue was then briefly triturated with a fire-polished glass pipet. After centrifugation, washing and trituration, the cells were filtered through a 70-μm cell strainer and plated on
Subcellular localization and distribution of IP3Rs and proteins involved in P4 signaling in primary cultures of mouse hippocampal neurons
To determine if there were any P4-induced changes in the expression and distribution of IP3R receptors and the neuroprotection-associated signaling protein, Akt, cells were treated cells with 100 nM P4 for 40 min and analyzed using immunocytochemistry. The cells were double-immunolabeled with specific antibodies for each signaling protein along with a neurofilament antibody as a neuronal marker, followed by nuclear counterstaining by DAPI. IP3R1, 2 and 3 (Fig. 1a–c, respectively), PR (Fig. 2),
Discussion
Calcium (Ca2+) is an essential second messenger that mediates multiple cellular responses in neurons to various stimuli including cell proliferation, neurotransmission, and cell death [10], [15]. Tight control of spatial and temporal Ca2+ signaling enables neurons to perform these different tasks appropriately. P4 is involved in multiple processes in neurons including neuroprotection [4], [5], [6], [7], [8]. There is increasing evidence from non-neuronal cell models suggesting that P4 is
Acknowledgements
This study was supported in part by a German National Merit Scholarship Foundation student scholarship (C.M.), by the University of North Texas Health Science Center at Fort Worth Intramural Research Program (P.K. and M.S.), grants EY014227 from NIH/NEI (P.K.), RR022570 from NIH/NCRR (P.K.) and AG010485 (P.K.), AG022550 (P.K.) and AG027956 from NIH/NIA (P.K. and M.S.) as well as by The Garvey Texas Foundation (P.K. and M.S.). We thank Margaret, Richard and Sara Koulen for generous support and
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These authors contributed equally to the manuscript.