Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons

doi:10.1016/j.ceca.2008.10.006

Cell Calcium

Volume 45, Issue 3, March 2009, Pages 233-242

https://doi.org/10.1016/j.ceca.2008.10.006 Get rights and content

Abstract

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca²⁺) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP₃Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca²⁺ signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP₃R-mediated intracellular Ca²⁺ responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca²⁺ signaling and its related functions.

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

References (32)

E.E. Baulieu
Steroid hormones in the brain: several mechanisms
E.T. Asbury et al.
Progesterone facilitates the acquisition of avoidance learning and protects against subcortical neuronal death following prefrontal cortex ablation in the rat
Behav. Brain Res.
(1998)
R.L. Roof et al.
Progesterone facilitates cognitive recovery and reduces secondary neuronal loss caused by cortical contusion injury in male rats
Exp. Neurol.
(1994)
L. Missiaen et al.
Abnormal intracellular ca(2+)homeostasis and disease
Cell Calcium
(2000)
M.T. Khan et al.
Akt kinase phosphorylation of inositol 1,4,5-trisphosphate receptors
J. Biol. Chem.
(2006)
J.C. Kirkman-Brown et al.
Biphasic elevation of [Ca(2+)](i) in individual human spermatozoa exposed to progesterone
Dev. Biol.
(2000)
R.S. Duncan et al.
Differential inositol 1,4,5-trisphosphate receptor signaling in a neuronal cell line
Int. J. Biochem. Cell. Biol.
(2007)
R.J. Wojcikiewicz et al.
Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. Type I, II, and III receptors are differentially susceptible to phosphorylation and are phosphorylated in intact cells
J. Biol. Chem.
(1998)
G.R. Bai et al.
Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase
Biochem. Biophys. Res. Commun.
(2006)
C.P. Bagowski et al.
The classical progesterone receptor associates with p42 MAPK and is involved in phosphatidylinositol 3-kinase signaling in Xenopus oocytes
J. Biol. Chem.
(2001)

P. Thomas et al.

Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: a review with some new findings

Steroids

(2002)

M.E. Stokely et al.

Microfluorimetry defines early axonal damage in a rat model of optic neuritis: a novel method targeting early CNS autoimmunity

J. Neurosci. Meth.

(2007)

M.J. Tsai et al.

Molecular mechanisms of action of steroid/thyroid receptor superfamily members

Annu. Rev. Biochem.

(1994)

Y. Goodman et al.

Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid beta-peptide toxicity in hippocampal neurons

J. Neurochem.

(1996)

J. Nilsen et al.

Impact of progestins on estrogen-induced neuroprotection: synergy by progesterone and 19-norprogesterone and antagonism by medroxyprogesterone acetate

Endocrinology

(2002)

J. Nilsen et al.

Divergent impact of progesterone and medroxyprogesterone acetate (Provera) on nuclear mitogen-activated protein kinase signaling

Proc. Natl. Acad. Sci. U.S.A.

(2003)

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Progesterone is a gonadal steroid hormone that is synthesized primarily by the ovary in the female, and also the testes and adrenal cortex in the male [12]. Many studies have identified that steroid hormones are synthesized not only in peripheral tissues but also in the central nervous system (CNS) [13], and because of this reason; these hormones have also been called “neurosteroids”. Progesterone has multiple biological functions in the CNS including the modulation of neuroprotection, neuroplasticity, mood, neurogenesis and neuroinflammation [14,15].
Progesterone has been shown to have neuroprotective effects in experimental acute brain injury models, but little is known about the effects of steroid sex hormones in models of retinitis pigmentosa (RP). The aim of this study was to asses whether progesterone had a protective effect in one animal model of RP (the rd1 mice), and whether its action was due at least in part, to its ability to reduce free radical damage or to increase antioxidant defences.
Rd1 and wild type (wt) mice received an oral administration of 100 mg/kg body/weight of progesterone on alternate days starting at postnatal day 7 (PN7) and were sacrificed at different postnatal days.
Our results show that progesterone decreases cell death, as the number of TUNEL-positive cells were decreased in the ONL of the retina from treated rd1 mice. At PN15, treatment with progesterone increased values of ERG b-wave amplitude (p < 0,5) when compared with untreated mice. Progesterone also decreased the observed gliosis in RP, though this effect was transient.
Treatment with progesterone significantly reduced retinal glutamate concentrations at PN15 and PN17. To clarify the mechanism by which progesterone is able to decrease retinal glutamate concentration, we examined expression levels of glutamine synthase (GS). Our results showed a significant increase in GS in rd1 treated retinas at PN13.
Treatment with progesterone, significantly increase not only GSH but also oxidized glutathione retinal concentrations, probably because progesterone is able to partially increase glutamate cysteine ligase c subunit (GCLC) at PN15 and PN17 (p < 0,05).
In summary, our results demonstrate that oral administration of progesterone appears to act on multiple levels to delay photoreceptor death in this model of RP.
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Taken together, IP3R2 is the major subtype of IP3Rs in ONHAs; Ca2 + release from the nuclear envelope is likely amplified through RyR2. This differential pattern of Ca2 + signaling in ONHAs therefore potentially regulates gene expression (Hwang et al., 2009; Koulen et al., 2008) (Fig. 8) to adapt to changes in the astrocytic milieu and synaptic signaling, in accordance with the generally accepted view of astrocytes contributing critically to synaptic signaling and homeostasis (Dallerac et al., 2013). Our current knowledge regarding signaling pathways in ONHAs upstream and downstream of intracellular Ca2 + release is very limited.
Recent evidence suggests that astrocytes do not serve a mere buffering function, but exhibit complex signaling pathways, disturbance of which contributes significantly to the pathophysiology of CNS diseases. Little is known regarding the intracellular signaling pathways in the specialized optic nerve head astrocytes (ONHAs), the major glia cell type in non-myelinated optic nerve head. Here we show the differential subcellular expression of intracellular Ca^2 + channels in ONHAs. Expression of type 1 and type 3 inositol-1-4-5,-trisphosphate receptors (IP₃Rs) in the endoplasmic reticulum and type 2 IP₃Rs in the nuclear envelope causes differential Ca^2 + release from intracellular stores in nuclear vs. cytosolic compartments. Our study identifies differential distribution and activity of Ca^2 + channels as molecular substrate and mechanism by which astrocytes independently regulate Ca^2 + transients in both cytoplasm and nucleoplasm, thereby controlling genomic and non-genomic cellular signaling, respectively. This provides excellent targets for therapeutics restoring pathological disturbances of intracellular Ca^2 + signaling present in glaucoma and other neurodegenerative disorders with astrocyte involvement.
Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling
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Thus progesterone may exert anticonvulsive or inhibitory actions independent of its metabolism to DHP/THP. In addition, progesterone has been reported to regulate brain activity via GABAA receptor-independent mechanisms (Zhu et al., 2008; Hwang et al., 2009; Zheng, 2009; Johannessen et al., 2011; Kelley and Mermelstein, 2011; Luoma et al., 2011; Singh and Su, 2013; King, 2013). Our in vitro observations are in keeping with these previous findings.
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Adult male mice were kindled via a daily stimulation protocol. Electroencephalographic (EEG) discharges were recorded from the hippocampus or cortex to assess “focal” or “generalized” seizure activity. Kindled mice were treated with intra-peritoneal injections of progesterone (10, 35, 100 and 160 mg/kg) with or without finasteride pretreatment (50 or 100 mg/kg), THP (1, 3.5, 10 and 30 mg/kg), midazolam (2 mg/kg) and carbamazepine (50 mg/kg). Entorhinal cortical slices were prepared from naïve young mice, and repetitive epileptiform potentials were induced by 4-aminopyridine (100 μM), picrotoxin (100 μM) and finasteride (1 μM).
Pretreatment with finasteride did not abolish the anticonvulsant effects of progesterone. In finasteride-pretreated mice, progesterone at 100 and 160 mg/kg decreased cortical but not hippocampal afterdischarges (ADs). Carbamazepine mimicked the effects of progesterone with finasteride pretreatments in decreasing cortical discharges and motor seizures, whereas midazolam produced effects similar to progesterone alone or THP in decreasing hippocampal ADs and motor seizures. In brain slices, progesterone at 1 μM inhibited entorhinal epileptiform potentials in the presence of picrotoxin and finasteride.
We suggest that progesterone may have THP/GABA_A-dependent and independent anticonvulsive actions in the hippocampal-kindled mouse model.
Distribution of membrane progesterone receptor alpha in the male mouse and rat brain and its regulation after traumatic brain injury
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Progesterone has been shown to exert pleiotropic actions in the brain of both male and females. In particular, after traumatic brain injury (TBI), progesterone has important neuroprotective effects. In addition to intracellular progesterone receptors, membrane receptors of the hormone such as membrane progesterone receptor (mPR) may also be involved in neuroprotection. Three mPR subtypes (mPRα, mPRβ, and mPRγ) have been described and mPRα is best characterized pharmacologically. In the present study we investigated the distribution, cellular localization and the regulation of mPRα in male mouse and rat brain. We showed by reverse transcription-PCR that mPRα is expressed at similar levels in the male and female mouse brain suggesting that its expression may not be influenced by steroid levels. Treatment of males by estradiol or progesterone did not modify the level of expression of mPRα as shown by Western blot analysis. In situ hybridization and immunohistochemistry analysis showed a wide expression of mPRα in particular in the olfactory bulb, striatum, cortex, thalamus, hypothalamus, septum, hippocampus and cerebellum. Double immunofluorescence and confocal microscopy analysis showed that mPRα is expressed by neurons but not by oligodendrocytes and astrocytes. In the rat brain, the distribution of mPRα was similar to that observed in mouse brain; and after TBI, mPRα expression was induced in oligodendrocytes, astrocytes and reactive microglia. The wide neuroanatomical distribution of mPRα suggests that this receptor may play a role beyond neuroendocrine and reproductive functions. However, in the absence of injury its role might be restricted to neurons. The induction of mPRα after TBI in microglia, astrocytes and oligodendrocytes, points to a potential role in mediating the modulatory effects of progesterone in inflammation, ion and water homeostasis and myelin repair in the injured brain.
Distribution of mRNAs encoding classical progestin receptor, progesterone membrane components 1 and 2, serpine mRNA binding protein 1, and progestin and ADIPOQ receptor family members 7 and 8 in rat forebrain
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Citation Excerpt :
Protein kinase G can activate additional kinases such as phosphoinositide 3-kinase, resulting in phosphorylation of Akt (Kandel and Hay, 1999; Brazil and Hemmings, 2001). The rapid phosphorylation of Akt in response to P4 has been observed in cells from cortex and hippocampus (Singh, 2001; Hwang et al., 2009), and our study shows that both brain regions contain Pgrmc1 and Serbp1. Taken together, these observations suggest that phosphorylation events initiated by P4 may involve the Pgrmc1/Serbp1 complex.
Several lines of evidence suggest the existence of multiple progestin receptors that may account for rapid and delayed effects of progesterone in the CNS. The delayed effects have been long attributed to activation of the classical progestin receptor (Pgr). Recent studies have discovered novel progestin signaling molecules that may be responsible for rapid effects. These include progesterone receptor membrane component 1 (Pgrmc1), Pgrmc2, progestin and adipoQ receptor 7 (Paqr7) and Paqr8. The functions of these molecules have been investigated extensively in non-neural, but not in neural tissues, partly because it is unclear which are expressed in the brain and where they are expressed. To address these issues, we compared the distributions of mRNAs encoding Pgr, Pgrmc1, Pgrmc2, Paqr7 and Paqr8 using in situ hybridization with radiolabeled oligodeoxynucleotidyl probes in forebrain tissues of estradiol-treated female rats. We also examined the distribution of serpine mRNA binding protein 1 (Serbp1), a putative binding partner of Pgrmc1. Analyses of adjacent brain sections showed that the highest expression of mRNAs encoding Pgr, Pgrmc1, Pgrmc2 and Serbp1 was detected in several hypothalamic nuclei important for female reproduction. In contrast, expression patterns of Paqr7 and Paqr8 were low and homogeneous in the hypothalamus, and more abundant in thalamic nuclei. The neuroanatomical distributions of these putative progestin signaling molecules suggest that Pgrmc1 and Pgrmc2 may play roles in neuroendocrine functions while Paqr7 and Paqr8 are more likely to regulate sensory and cognitive functions.
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Progesterone and its derivatives bind to cell membranes of various rat brain tissues, suggesting non-classical mechanisms of action [53–55]. Non-genomic and rapid effects of progesterone within the central nervous system have been observed, including: effects on ion transport [56,57], neurotransmitter release [58], protein kinase activity [59,60] and nuclear translocation [57]. Early speculation regarding the identity of the receptor mediating the non-genomic effects of progesterone in the brain led to the discovery of a steroid binding site on the GABAA/benzodiazepine receptor chloride channel complex in rat brains [61].
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¹: These authors contributed equally to the manuscript.

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Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons

Abstract

Introduction

Section snippets

Preparation of mouse hippocampal neurons and primary cell culture

Subcellular localization and distribution of IP3Rs and proteins involved in P4 signaling in primary cultures of mouse hippocampal neurons

Discussion

Acknowledgements

Behav. Brain Res.

Exp. Neurol.

Cell Calcium

J. Biol. Chem.

Dev. Biol.

Int. J. Biochem. Cell. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Steroids

J. Neurosci. Meth.

Molecular mechanisms of action of steroid/thyroid receptor superfamily members

Annu. Rev. Biochem.

Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid beta-peptide toxicity in hippocampal neurons

J. Neurochem.

Impact of progestins on estrogen-induced neuroprotection: synergy by progesterone and 19-norprogesterone and antagonism by medroxyprogesterone acetate

Endocrinology

Divergent impact of progesterone and medroxyprogesterone acetate (Provera) on nuclear mitogen-activated protein kinase signaling

Proc. Natl. Acad. Sci. U.S.A.

Subcellular localization and distribution of IP₃Rs and proteins involved in P4 signaling in primary cultures of mouse hippocampal neurons