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Up-regulation of ASIC3 expression by β-estradiol
Accepted Manuscript Title: Up-regulation of ASIC3 expression by -estradiol Authors: Ping Ren, Wen-Bin Wang, Hai-Hua Pan, Chun-Yu Qiu, Wang-Ping Hu PII: DOI: Reference:
S0304-3940(18)30542-1 https://doi.org/10.1016/j.neulet.2018.08.012 NSL 33747
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
15-6-2018 8-8-2018 11-8-2018
Please cite this article as: Ren P, Wang W-Bin, Pan H-Hua, Qiu C-Yu, Hu WPing, Up-regulation of ASIC3 expression by -estradiol, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.08.012 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 form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Up-regulation of ASIC3 expression by β-estradiol Ping Rena1, Wen-Bin Wang b1, Hai-Hua Pana, Chun-Yu Qiua, Wang-Ping Huc* a
Department of Pharmacology, School of Pharmacy, Hubei University of Science and Technology, 88
Xianning Road, Xianning 437100, Hubei, P R China Xianning Central Hospital, The First Affiliated Hospital of Hubei University of Science, 228 jingui Road,
Xianning 437100, Hubei, P R China c
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Research Center of Basic Medical Sciences, School of Basic Medical Sciences,Hubei University of
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Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, P R China 1 The first two authors contributed equally to the work.
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* Corresponding author
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Correspondence should be addressed to:
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School of Basic Medical Sciences
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Research Center of Basic Medical Sciences
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Wang-Ping Hu
88 Xianning Road
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Hubei University of Science and Technology
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Xianning 437100, Hubei, PR China Phone: +86-715-8268210
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Fax: +86-715-82256221
Email: [email protected]
Highlights:
17β-estradiol (E2) up-regulates acid-sensing ion channel 3 (ASIC3) protein expression via estrogen receptor α.
There are sex differences in ASIC3 expression in rat dorsal root ganglia (DRG). And ASIC3 protein expression in female rat DRG is higher than those in male rat DRG.
ASIC3 protein expression in DRG decreases significantly after ovariectomy, but not after orchiectomy.
ABSTRACT
Sex differences occur in nociceptive pain, and estrogens are involved in the sex differences. Our previous study shows sex differences exist in acidosis-induced nociception in rats, with females being more sensitive than males to acetic acid. However, the mechanisms underlying the sex differences remain unclear. We report here17β-estradiol (E2) up-regulates expression of acid-sensing ion channel 3 (ASIC3), which can mediate the acidosis-induced events. The recombinant plasmid of pCDNA3.1-ASIC3-GFP and pCDNA3.1-estrogen receptor α (ERα) were cotransfected to 293T cells by lipid transfection method. And western blot assays showed expression of ASIC3. We found that E2 markedly increases ASIC3 protein
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expression in a dose- and time- dependent manner in 293T cells expressing ASIC3 and ERα. The upregulating effect of E2 on ASIC3 protein expression is almost completely blocked by the addition of MPP, a specific ERα antagonist. We also observed that sex differences occur in ASIC3 expression in rat dorsal root
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ganglia (DRG) and in acetic acid-induced nociceptive responses. ASIC3 protein expression in female rat DRG is higher than those in male rat DRG. And female rats are more sensitive to acetic acid-induced nociception than males. ASIC3 protein expression in DRG decreases significantly after ovariectomy, but not after
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orchiectomy. These results suggest that E2 up-regulates ASIC3 expression through ERα, which may
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contribute to sex differences in acetic acid-induced nociception.
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KeyWords: 17β-estradiol; acid-sensing ion channel 3; expression; dorsal root ganglia; sex difference 1. Introduction
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Sex differences occur in pain perception, and women are in general more sensitive to pain than men [1-6]. Although sex differences in pain have been linked to many factors, sex hormones are one of many
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important factors influencing pain sensitivity. Among all sex hormones, many studies implicate estrogen may be a key modulator of pain in adults [7, 8].
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Estrogens are the primary female sex hormones which are chiefly produced by the secretions of the ovaries and placentas in female animals. There are three major endogenous estrogens in females: estrone, estradiol (E2), and estriol, of which the activity of E2 is the strongest. It has been shown that E2 has a wide
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and important physiological function, for example, the development and regulation of the female reproductive system and secondary sex characteristics. Numerous studies have also demonstrated the potential effects of E2 on pain processing, although with considerable variability depending on the species, tissue, and the type of test employed [9]. Estrogen receptors, mainly consisting of two subtypes (ERα and ERβ), have been demonstrated to be distributed in peripheral nervous system including dorsal root ganglia (DRG) neurons which are involved in pain perception. ERα is selectively localized on small-diameter sensory neurons, which have been regarded as nociceptors [10]. Therefore, estrogens can potentially alter the
nociceptive process at the primary level. Acid-sensing ion channels (ASICs) are voltage-independent and proton-activated cation channels that have high permeability to Na＋ ions [11, 12]. To date, at least six ASIC subunits encoded by four genes have been identified in mammals [13]. Among the ASIC subunits, ASIC3 displays higher sensitivity to extracellular protons than other ASICs [13, 14]. Most of the ASIC subunits are expressed in both DRG cell bodies and sensory terminals [15, 16]. And ASIC3 is specifically localized in nociceptive fibers [17, 18] .
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Increasing evidences have shown that ASIC3 plays an important role in various pain conditions such as inflammatory pain, postoperative pain, and migraine [19-21]. Our previous studies have indicated that the acidosis-induced nociceptive responses are mediated by ASICs, especially by ASIC3, and there exist sex
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differences in the responses [14]. However, the exact mechanism of ASIC3 underling the sex differences has not been clarified.
In this study, we have investigated that E2 up-regulates ASIC3 expression in 293T cells co-transfected
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with ASIC3 and ERα, and there are sex differences in the ASIC3 expression from rat DRGs and in acidosis-
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evoked pain.
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2. Materials and Methods 2.1. Plasmid construction
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Acid-sensing ion channels 3 gene is cloned into pCDNA3.1-GFP vector, allowing GFP (green fluorescence protein) fusion at the C terminus of ASIC3 by the gene recombinant technology. Its chimeric
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protein has double bio-activities of ASIC3 and GFP. Meanwhile, a recombinant eukaryotic expression vector contained ERα (pCDNA3.1-ERα) is constructed. All plasmids are kindly provided by Dr Li at Middle
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and Southern University of China. 2.2. Cell culture and transfection
Human embryonic kidney (HEK) 293T cells are presented by Dr Li at Middle and Southern University of
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China and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 10% fetal bovine serum at 37℃with 5% CO2. The cells are plated in a 6-well plate at 50-60% confluency a day before transfection. The next day pCDNA3.1-ASIC3-GFP and pCDNA3.1- ERα plasmid are transiently transfected into 293T cells with Lipofectamine® 2000 transfection reagent (Lip2000, Invitrogen), and the DNA-to-Lip 2000 ratio is one to three. When the plasmid pCDNA3.1-ASIC3-GFP is co-transfected with pCDNA3.1- ERα plasmid, the ratio is kept at 1:1. After 12 hours of the transfection, E2 (10-8、10-7、10-6、10-5 M) is added to the cells and cultured continuously for 2, 8, 12 hours and then removed, with all cells waiting until the
same time point before ASIC3 levels were measured, while control group receives vehicle treatment. Meanwhile, E2 (10-5 M) and specific ERα antagonists methyl-piperidino-pyrazole (MPP, 3×10-5 M) are coadded to 293T cells co-transfected with ASIC3 and ERα for 12 hours. Finally, all the cells are collected to assay the level of ASIC3 expression by immunoblotting using anti-GFP antibody (1:1000, Cell Signaling Technology), because GFP can act as a reporter gene. 2.3. Animals and operation
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The experimental protocol is approved by the Animal Research Ethics Committee of Hubei University of Science and Technology. Adult Sprague-Dawley rats (8-10 wk old) are used in all experiments. All animals are kept on a 12-hour light/dark cycle with food and water available ad libitum. After anesthesia, female
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rats in the ovariectomized (OVX) group are treated with bilateral ovariectomy, and those in the shamoperated group are only given laparotomy during the proestrus stage. Meanwhile, bilateral testes of male rats are extirpated (castrated group) or are subjected to a sham surgical procedure. Later 4 weeks after the
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operation, all rats are sacrificed after anesthetized and the rat DRGs are taken out for the next experiment
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study [14].
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2.4. Isolation of DRGs and Western blotting
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The rat DRGs are taken out and transferred immediately into DMEM at pH 7.4. After the removal of the surrounding connective tissues, the rat DRGs are lysed using the RIPA lysis buffer (Beyotime, China) and
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then total protein is isolated. The content of protein is quantified by BCA Protein assay (Thermo Fisher) and then 20 µg protein is boiled with loading buffer. The protein samples are separated by SDS-PAGE using 10%
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gels. The separated proteins are transferred to the PVDF membranes (Merck Millipore). Polyclonal antibody against ASIC3 (1:2000, GeneTex) is used to probe the ASIC3 proteins. The loading control is
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polyclonal antibody against β-actin (1:1000, Cell Signaling Technology). After primary antibodies incubation for overnight, the PVDF membranes (Merck Millipore) are washed with TBS Tween-20 (Tris-buffered saline with Tween-20 detergent) buffers, and then incubated by horseradish peroxidase conjugated antirabbit
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IgG (1: 10,000, Santa Cruz) for 2 h. At last the proteins are visualized using an ECL detection system (Thermo Fisher). 2.5. Nociceptive behavior induced by acetic acid Rats are placed in a 30 × 30 × 30 cm3 Plexiglas chamber and allowed to habituate for at least 30 min before nociceptive behavior experiments. Separate groups of rats are pretreated with 20 μL capsazepine (100 μM, Sigma-Aldrich) in ipsilateral hind paw before injection of acetic acid. After 5 min, rats were restrained lightly and injected subcutaneously with acetic acid solution (0.6%, 20 μL, Sigma-Aldrich) into
the hind paw using a 30-gauge needle connected to a 100 μL Hamilton syringe. Nociceptive behavior (that is, number of flinches) is counted over a 5 min period starting immediately after the injection [22, 23]. 2.6. Drugs and chemicals Trypsin digestion solution contains 5 mL of DMEM in which 0.5 mg/mL trypsin (type Ⅱ-S; Sigma-Aldrich), 1.0 mg/mL collagenase (type Ⅰ-A; Sigma-Aldrich), 0.1 mg/mL deoxyribonuclease (type Ⅰ-A; Sigma-Aldrich) and 1.25 mg/mL soybean trypsin inhibitor (type II-S; Sigma-Aldrich). Capsaicin (Sigma-Aldrich) is dissolved
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in DMSO to obtain 10 mM stock solution. Further dilutions are made with normal saline (NS) to reach final concentrations of 100 μM working solution. MPP (Sigma-Aldrich) and E2(Sigma-Aldrich) are dissolved in ethanol solution to obtain 10 mM stocks of the drugs. Stocks of MPP and E2 are further diluted with cell
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culture medium to reach final concentrations of 3×10-5 M and 10-8 to 10-5 M respectively. Hydrochloric acid and DMEM are purchased from Sigma-Aldrich. Fetal bovine serum is purchased from Gibco (Grand Island, N.Y. USA). Lip2000 is purchased from Thermon fisher.
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2.7. Statistical analysis
Student's t-test or unpaired t-test is used for the analysis of differences in two groups (MPP and 10-5
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whereas statistical analysis of concentration- and time-dependent regulatory changes data was performed using a one-way analysis of variance followed by Bonferroni’s post hoc tests. Data are presented as mean ±
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S.E.M. P-values < 0.05 are considered statistically significant.
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3. Results
3.1. E2 increases the ASIC3 expression in a concentration-dependent manner.
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We firstly treat 293T cells co-expressing ASIC3-GFP and ERα with various concentrations of E2 (10-8、 10-7、10-6、10-5 M ) for 12 h. Figure 1A shows that the effect of different concentrations of E2 on ASIC3 protein expression. To analyze ASIC3 protein expression quantitatively, its expression is normalized to the
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inner reference (β-actin) in its respective group. Figure 1B shows that the level of ASIC3 protein expression gradually increases as concentration of treated E2 increased from 10-8 to 10-5 M. ASIC3 protein expression increases 30.5%±0.03% and 93.4%±0.13% after treatment of 10-8 M and 10-5 M E2, respectively, compared with the control group (n=5, P < 0.05). These results indicate that E2 increases the ASIC3 expression in a concentration-dependent manner. To further verify whether the E2 up-regulation of ASIC3 protein expression is mediated by ERα, we co-apply E2 with and MPP, a specific ERα antagonist, in 293T cells co-expressing ASIC3-GFP and ERα. After co-treatment of E2 (10-5 M) with and MPP (3×10-5M), the
ASIC3 protein expression is close to that of control group, unlike an increase of 93.4%±0.13% in treatment with E2 (10-5 M) alone (n=5, P < 0.01). Thus, MPP obviously blocks the up-regulating effect of E2 on ASIC3 protein expression. The results suggest that E2 concentration-dependently increases the expression of ASIC3 via ERα. The ASIC3-GFP fusion plasmid that we use here comes fully made from Dr Li at Middle and Southern University of China. 3.2. E2 enhances the ASIC3 expression in a time-dependent manner
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We then investigate whether up-regulation of ASIC3 protein expression is dependent upon E2 exposure time. As shown in figure 2, E2 (10-5 M) up-regulates the expression of ASIC3 in 293T cells co-expressing ASIC3 and ERα in time-dependent manner (0-12 h). ASIC3 expression increases 1.7-fold and 2.1-fold at 8
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and 12 h after E2 exposure, while ASIC3 expression has not significant change at 2 h after E2 exposure (figure 2A). The date of semi-quantitive western blotting shows that the ratio of GFP/β-actin increases with the increase of E2 exposure time (n=5, P < 0.01, figure 2B). These results indicate that E2 enhances the
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ASIC3 expression in a time-dependent manner. In addition, E2 does not induce any significant change of
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ASIC3 expression in 293T cells untransfected ERα (data not shown).
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3.3. Sex differences occur in the ASIC3 expression in rat DRGs.
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To verify whether there are sex differences in ASIC3 protein expression in vivo, we detect the level of ASIC3 protein in DRGs of OVX, castrated and sham-operated rats. As shown in figure 3, the expression of
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ASIC3 protein in sham male rats is significantly different compared to sham female rats (n=8, P < 0.01). Moreover, the expression of ASIC3 protein significantly decreases in female rats after removing ovary (n=8,
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P < 0.01）. In contrast, the change of ASIC3 expression in the castrated males group is not obvious compared with sham males group (n=8, P > 0.05). These results indicate that sex differences occur in the
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ASIC3 expression in rat DRGs, which may involve in estrogens. 3.4. Sex differences occur in the nociceptive responses evoked by acetic acid in rats.
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As previously reported, intense flinch/shaking responses are produced by injection of acetic acid on the dorsal surface of the rat hind paw [14]. To investigate whether there are gender differences in the nociceptive responses evoked by acetic acid in rats, we observe the nociceptive responses induced by intradermal injection of pH 6.0 acetic acid in OVX and sham females. As shown in figure 4, OVX females display less nociceptive responses compared with sham females (n =10, P < 0.01). In contrast, there is no significant difference between castrated and sham males (n =10, P > 0.05). Interestingly, compared with sham males, sham females exhibit greater nociceptive responses to intraplantar injection of acetic acid (n
=10, P < 0.01). These results suggest that female rats display an enhanced sensitivity to the acetic acidinduced nociceptive response compared with male and OVX female rats. 4. Discussion In the present study, we provide data for the first time to show that E2 concentration- and timedependently up-regulates ASIC3 protein expression via ERα. We also observe that sex differences occur in ASIC3 expression in rat DRGs and in acetic acid-induced nociceptive responses.
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Estrogens exert their biological effects through genomic and nongenomic mechanisms [24]. Estrogens can freely pass through the cell membrane and bind to ERs located in the cytoplasm and nucleus, and form an estrogen-ER complex. This complex binds to estrogen-response element sequences in the promoter
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region of target genes and modulates the levels of associated mRNAs and proteins [25, 26]. This genomic mechanism plays a regulatory role from hours to days after the initiation of hormonal manipulation [27]. In the present study, we find that E2 concentration-dependently increases the expression of ASIC3 protein.
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The up-regulation of ASIC3 expression is dependent upon E2 exposure time. If the exposure time of E2 is
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too short, less than 2 hours, E2 has no significant effect on ASIC3 expression. Only when the exposure time
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of E2 is longer than 8 hours, E2 significantly increases ASIC3 expression. Thus, we consider that E2 up-
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regulates ASIC3 expression through a genomic mechanism.
We find that MPP, a specific ERα antagonist, almost completely abrogates the up-regulation of ASIC3
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expression by E2 in 293T cells co-expressing ASIC3 and ERα. Moreover, E2 does not induce any significant change of ASIC3 expression in 293T cells untransfected ERα. These results indicate E2 up-regulation of
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ASIC3 expression is mediated by ERα. It has been shown that ERα is also expressed in the rat DRG neurons, and its activation alters the expression of certain mRNA and protein in primary sensory neurons [28] .
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Among ASICs, ASIC3 is highly expressed in both DRG cell bodies and sensory terminals[29] . The present study shows that sex differences occur in the ASIC3 expression in rat DRGs. The level of ASIC3 protein expression in female rat DRGs is higher than those in male rat DRGs. Our result is agreed with result of
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Kobayashi et al. who reported that ASIC3 mRNA expression in female mouse DRGs is more abundant than those in male DRGs [30]. We find that the expression of ASIC3 protein from DRGs decreases after ovariectomy in female rats, but not orchiectomy in male rats. The results suggest that ASIC3 expression in rat DRGs involves in circulating female sex steroids. Combined with the results from cell line expression in the present experiment, E2 may play a key role in sex differences in the ASIC3 protein expression from rat DRGs. It is reported recently that E2 up-regulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed temporomandibular joint likely through the classical genomic
mechanism of ER action [31]. Estrogens exacerbate nociceptive pain via up-regulation of transient receptor potential vanilloid 1 (TRPV1) and anoctamin 1 (ANO1) expression in primary sensory neurons of female rats [32]. Overnight incubation with E2, TRPV1 mRNA expression increases in cultured mouse DRG neurons [33]. E2 also increases sensitivity to chronic neuropathic pain through up-regulating N-methyl-D-aspartate acid receptor 1 expression in the DRG of rats [34]. Exogenous E2 also modulates TRPV1 and P2X3 receptor expression in sensory neurons of OVX rats, and the lack of E2 effect in ERα KO and ERβ KO mice resulted in TRPV1 and P2X3 receptor downregulation [35]. Although E2 regulates the expression of pain-related ion
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channels through its receptors, the genomic mechanism remains unclear. Future research is needed to clarify the mechanism underlying the upregulation of ASIC3 expression by E2. For example, how does the estrogen-ER complex bind to estrogen-response element sequences in the promoter region of ASIC3 gene
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and modulates the levels of ASIC3 mRNA and protein?
ASIC3 is expressed in both DRG cell bodies and sensory terminals, which monitors extracellular pH fall
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and contributes to proton-evoked pain signaling [22, 36]. It has been shown that ASIC3 plays an important
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role in various pain conditions such as inflammatory pain, postoperative pain and migraine [19-21]. ASICs are members of the ENaC/DEG superfamily of amiloride-sensitive epithelial sodium channels. Amiloride, a
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nonspecific blocker of ASICs, abolishes the known sex difference in formalin test response and blocks late
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phase nociceptive behavior in female, but not male mice [37]. The present and our previous studies show that sex differences occur in acidosis-induced nociception in rats, with females being more sensitive than
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males to acetic acid. After ovariectomy in female rats, female's enhanced sensitivity to the acetic acidinduced nociceptive response disappear. We have reported previously that E2 rapidly increases the ASIC-
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mediated currents and membrane excitability in rat DRG neurons. E2 potentiates the functional activity of ASICs through ERα and ERK1/2 signaling pathway [14]. In the present study, E2 can also up-regulate ASIC3
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protein expression in 293T cells co-expressing ASIC3-GFP and ERα. Moreover, we find that sex differences occur in the ASIC3 expression in rat DRGs. ASIC3 protein expression in female rat DRG is higher than those in male rat DRG. And ASIC3 protein expression in DRG decreases significantly after ovariectomy, but not
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after orchiectomy. Thus, E2 may play a key role in the sex differences. Our present and previous results suggest a role of E2 on ASIC3 function via both non-genomic and genomic mechanisms [14]. In conclusion, we have revealed interactions between E2 and ASIC3 expression in DRG neurons and transfected cell lines. E2 up-regulation of ASIC3 protein expression could likely result in females to be more sensitive to acetic acid than males. Our results indicate a potential mechanism for the explanation of sex differences occur in the acetic acid-induced nociceptive responses. Therefore, ASIC3 may be promising target for new drugs in woman with the disorders related pain.
Conflicts of interest The authors declare no conflicts of interest. Funding The work described in this paper was supported by grants from the National Natural Science Foundation of China (No. 81671101 to WPH and No. 81502635 to RP), Natural Science Foundation of Hubei Province of China (No. 2015CFA145 to WPH), and the Education Departmental Science Foundation of
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Hubei Province of China (No. D20152803 to RP). Acknowledgments
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We are very grateful to Professor Jia-da Li from Middle and Southern University of China for his valuable technical guidance.
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[16] J. Wemmie, R. Taugher, C. Kreple, Acid-sensing ion channels in pain and disease, Nat. Rev. Neurosci., 14 (2013) 461-471. [17] M. Ikeuchi, S.J. Kolker, L.A. Burnes, R.Y. Walder, K.A. Sluka, Role of ASIC3 in the primary and secondary hyperalgesia produced by joint inflammation in mice, Pain, 137 (2008) 662-669. [18] J. Noel, M. Salinas, A. Baron, S. Diochot, E. Deval, E. Lingueglia, Current perspectives on acid-sensing ion channels: new advances and therapeutic implications, Expert Rev Clin Pharmacol, 3 (2010) 331-346. [19] E. Deval, E. Lingueglia, Acid-Sensing Ion Channels and nociception in the peripheral and central nervous systems, Neuropharmacology, 94 (2015) 49-57. [20] E. Deval, J. Noel, X. Gasull, A. Delaunay, A. Alloui, V. Friend, A. Eschalier, M. Lazdunski, E. Lingueglia, Acid-sensing ion channels in postoperative pain, J Neurosci, 31 (2011) 6059-6066. [21] G. Dussor, ASICs as therapeutic targets for migraine, Neuropharmacology, 94 (2015) 64-71. [22] E. Deval, J. Noël, N. Lay, A. Alloui, S. Diochot, V. Friend, M. Jodar, M. Lazdunski, E. Lingueglia, ASIC3, a sensor of acidic and primary inflammatory pain, Embo Journal, 27 (2008) 3047-3055. [23] F. Qiu, C.Y. Qiu, H. Cai, T.T. Liu, Z.W. Qu, Z. Yang, J.D. Li, Q.Y. Zhou, W.P. Hu, Oxytocin inhibits the activity of acidsensing ion channels through the vasopressin, V1A receptor in primary sensory neurons, Br J Pharmacol, 171 (2014) 3065-3076. [24] X. Liu, H. Shi, Regulation of Estrogen Receptor alpha Expression in the Hypothalamus by Sex Steroids: Implication in the Regulation of Energy Homeostasis, Int J Endocrinol, 2015 (2015) 949085. [25] C.J. Gruber, D.M. Gruber, I.M. Gruber, F. Wieser, J.C. Huber, Anatomy of the estrogen response element, Trends in Endocrinology & Metabolism Tem, 15 (2004) 73. [26] J.M. Hall, J.F. Couse, K.S. Korach, The multifaceted mechanisms of estradiol and estrogen receptor signaling, Journal of Biological Chemistry, 276 (2001) 36869-36872. [27] H.C. Evrard, Estrogen synthesis in the spinal dorsal horn: a new central mechanism for the hormonal regulation of pain, American Journal of Physiology, 291 (2006) R291. [28] S. Sarajari, M.M. Oblinger, Estrogen effects on pain sensitivity and neuropeptide expression in rat sensory neurons, Exp Neurol, 224 (2010) 163-169. [29] L.N. Schuhmacher, E.S.J. Smith, Expression of acid-sensing ion channels and selection of reference genes in mouse and naked mole rat, Molecular Brain, 9 (2016) 97. [30] H. Kobayashi, M. Yoshiyama, H. Zakoji, M. Takeda, I. Araki, Sex differences in the expression profile of acidsensing ion channels in the mouse urinary bladder: a possible involvement in irritative bladder symptoms, BJU Int., 104 (2009) 1746-1751. [31] R.Y. Bi, Z. Meng, P. Zhang, X.D. Wang, Y. Ding, Y.H. Gan, Estradiol upregulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed TMJ, PLoS One, 12 (2017) e0178589. [32] K. Yamagata, M. Sugimura, M. Yoshida, S. Sekine, A. Kawano, A. Oyamaguchi, H. Maegawa, H. Niwa, Estrogens Exacerbate Nociceptive Pain via Up-Regulation of TRPV1 and ANO1 in Trigeminal Primary Neurons of Female Rats, Endocrinology, 157 (2016) 4309-4317. [33] M. Payrits, E. Saghy, K. Cseko, K. Pohoczky, K. Bolcskei, D. Ernszt, K. Barabas, J. Szolcsanyi, I.M. Abraham, Z. Helyes, E. Szoke, Estradiol Sensitizes the Transient Receptor Potential Vanilloid 1 Receptor in Pain Responses, Endocrinology, 158 (2017) 3249-3258. [34] C. Deng, Y.J. Gu, H. Zhang, J. Zhang, Estrogen affects neuropathic pain through upregulating N-methyl-Daspartate acid receptor 1 expression in the dorsal root ganglion of rats, Neural Regen Res, 12 (2017) 464-469. [35] T. Cho, V.V. Chaban, Expression of P2X3 and TRPV1 receptors in primary sensory neurons from estrogen receptors-alpha and estrogen receptor-beta knockout mice, Neuroreport, 23 (2012) 530-534. [36] M. Price, S. McIlwrath, J. Xie, C. Cheng, J. Qiao, D. Tarr, K. Sluka, T. Brennan, G. Lewin, M. Welsh, The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice, Neuron, 32 (2001) 10711083. [37] M.L. Chanda, J.S. Mogil, Sex differences in the effects of amiloride on formalin test nociception in mice, Am J Physiol Regul Integr Comp Physiol, 291 (2006) R335-342.
Figure legends
Fig 1 E2 enhances the expression of ASIC3 in a concentration-dependent manner. A. E2 increases the ASIC3 expression in a dose-dependent manner (10-8-10-5 M) after the treatment for 12 h in 293T cells co-expressing ASIC3 and ERα. The effect of E2 (10-5 M) is blocked by co-treatment of MPP (3×10-5M), a specific ERα antagonist. B. the quantitative analysis ensures the effect of E2 on ASIC3 protein expression with statistical significances. Each column and vertical bar represents the mean ± S.E.M. n=5, *
P< 0.05, **P< 0.01 vs control group by Bonferroni’s post hoc test. ##P< 0.01 vs the E2 (10-5 M) group by
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unpaired t-test. Fig 2 E2 increases the expression of ASIC3 in the time-dependent manner (0-12 h) in 293T cells coexpressing ASIC3 and ERα.
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A. The level of ASCI3 expression obviously increases in the 293T cells co-expressing ASIC3 and ERα treated by E2 (10-5 M) for 2, 8, 12 h compared with 0 h group. B. The quantitative analysis ensures the time-
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dependent effect of E2 on ASIC3 protein expression with statistical significances. Each column and vertical
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bar represents the mean ± S.E.M. n=5, **P< 0.01 vs 0 h group by Bonferroni’s post hoc test.
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Fig 3 Sex differences occur in the ASIC3 expression in rat DRGs.
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A. The ASIC3 expression decreases significantly in the male group compared with that of female group. In addition, the ASIC3 expression is obviously decreased in OVX females than in sham females but was not
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significantly different in castrated and sham males after surgical treatment. B. ASIC3 quantitative analysis shows that the statistical significances exist to a varied extent between females and males, OVX females
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and sham females. Each column and vertical bar represents the mean ± S.E.M. n=8, ** P< 0.01.
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Fig 4 Sex differences occur in the acetic acid evoked nociceptive responses in rats. The bar graph shows that the nociceptive responses induced by subcutaneous injection of pH 6.0 acetic acid is less in OVX females than in sham females but is not significantly different in castrated and sham males. Furthermore, sham females exhibit greater nociceptive responses to injection of acetic acid into the
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hind paws, compared with sham males. Each bar represents the number of flinches that animals spent licking/lifting the injected paw during first 5-min observation period. Each column and vertical bar represents the mean ± S.E.M. of ten rats. n=10, ** P< 0.01 vs sham male group by unpaired t-test.
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S0304-3940(18)30542-1 https://doi.org/10.1016/j.neulet.2018.08.012 NSL 33747
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
15-6-2018 8-8-2018 11-8-2018
Please cite this article as: Ren P, Wang W-Bin, Pan H-Hua, Qiu C-Yu, Hu WPing, Up-regulation of ASIC3 expression by -estradiol, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.08.012 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 form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Up-regulation of ASIC3 expression by β-estradiol Ping Rena1, Wen-Bin Wang b1, Hai-Hua Pana, Chun-Yu Qiua, Wang-Ping Huc* a
Department of Pharmacology, School of Pharmacy, Hubei University of Science and Technology, 88
Xianning Road, Xianning 437100, Hubei, P R China Xianning Central Hospital, The First Affiliated Hospital of Hubei University of Science, 228 jingui Road,
Xianning 437100, Hubei, P R China c
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b
Research Center of Basic Medical Sciences, School of Basic Medical Sciences,Hubei University of
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Science and Technology, 88 Xianning Road, Xianning 437100, Hubei, P R China 1 The first two authors contributed equally to the work.
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* Corresponding author
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Correspondence should be addressed to:
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School of Basic Medical Sciences
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Research Center of Basic Medical Sciences
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Wang-Ping Hu
88 Xianning Road
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Hubei University of Science and Technology
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Xianning 437100, Hubei, PR China Phone: +86-715-8268210
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Fax: +86-715-82256221
Email: [email protected]
Highlights:
17β-estradiol (E2) up-regulates acid-sensing ion channel 3 (ASIC3) protein expression via estrogen receptor α.
There are sex differences in ASIC3 expression in rat dorsal root ganglia (DRG). And ASIC3 protein expression in female rat DRG is higher than those in male rat DRG.
ASIC3 protein expression in DRG decreases significantly after ovariectomy, but not after orchiectomy.
ABSTRACT
Sex differences occur in nociceptive pain, and estrogens are involved in the sex differences. Our previous study shows sex differences exist in acidosis-induced nociception in rats, with females being more sensitive than males to acetic acid. However, the mechanisms underlying the sex differences remain unclear. We report here17β-estradiol (E2) up-regulates expression of acid-sensing ion channel 3 (ASIC3), which can mediate the acidosis-induced events. The recombinant plasmid of pCDNA3.1-ASIC3-GFP and pCDNA3.1-estrogen receptor α (ERα) were cotransfected to 293T cells by lipid transfection method. And western blot assays showed expression of ASIC3. We found that E2 markedly increases ASIC3 protein
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expression in a dose- and time- dependent manner in 293T cells expressing ASIC3 and ERα. The upregulating effect of E2 on ASIC3 protein expression is almost completely blocked by the addition of MPP, a specific ERα antagonist. We also observed that sex differences occur in ASIC3 expression in rat dorsal root
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ganglia (DRG) and in acetic acid-induced nociceptive responses. ASIC3 protein expression in female rat DRG is higher than those in male rat DRG. And female rats are more sensitive to acetic acid-induced nociception than males. ASIC3 protein expression in DRG decreases significantly after ovariectomy, but not after
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orchiectomy. These results suggest that E2 up-regulates ASIC3 expression through ERα, which may
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contribute to sex differences in acetic acid-induced nociception.
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KeyWords: 17β-estradiol; acid-sensing ion channel 3; expression; dorsal root ganglia; sex difference 1. Introduction
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Sex differences occur in pain perception, and women are in general more sensitive to pain than men [1-6]. Although sex differences in pain have been linked to many factors, sex hormones are one of many
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important factors influencing pain sensitivity. Among all sex hormones, many studies implicate estrogen may be a key modulator of pain in adults [7, 8].
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Estrogens are the primary female sex hormones which are chiefly produced by the secretions of the ovaries and placentas in female animals. There are three major endogenous estrogens in females: estrone, estradiol (E2), and estriol, of which the activity of E2 is the strongest. It has been shown that E2 has a wide
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and important physiological function, for example, the development and regulation of the female reproductive system and secondary sex characteristics. Numerous studies have also demonstrated the potential effects of E2 on pain processing, although with considerable variability depending on the species, tissue, and the type of test employed [9]. Estrogen receptors, mainly consisting of two subtypes (ERα and ERβ), have been demonstrated to be distributed in peripheral nervous system including dorsal root ganglia (DRG) neurons which are involved in pain perception. ERα is selectively localized on small-diameter sensory neurons, which have been regarded as nociceptors [10]. Therefore, estrogens can potentially alter the
nociceptive process at the primary level. Acid-sensing ion channels (ASICs) are voltage-independent and proton-activated cation channels that have high permeability to Na＋ ions [11, 12]. To date, at least six ASIC subunits encoded by four genes have been identified in mammals [13]. Among the ASIC subunits, ASIC3 displays higher sensitivity to extracellular protons than other ASICs [13, 14]. Most of the ASIC subunits are expressed in both DRG cell bodies and sensory terminals [15, 16]. And ASIC3 is specifically localized in nociceptive fibers [17, 18] .
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Increasing evidences have shown that ASIC3 plays an important role in various pain conditions such as inflammatory pain, postoperative pain, and migraine [19-21]. Our previous studies have indicated that the acidosis-induced nociceptive responses are mediated by ASICs, especially by ASIC3, and there exist sex
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differences in the responses [14]. However, the exact mechanism of ASIC3 underling the sex differences has not been clarified.
In this study, we have investigated that E2 up-regulates ASIC3 expression in 293T cells co-transfected
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with ASIC3 and ERα, and there are sex differences in the ASIC3 expression from rat DRGs and in acidosis-
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evoked pain.
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2. Materials and Methods 2.1. Plasmid construction
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Acid-sensing ion channels 3 gene is cloned into pCDNA3.1-GFP vector, allowing GFP (green fluorescence protein) fusion at the C terminus of ASIC3 by the gene recombinant technology. Its chimeric
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protein has double bio-activities of ASIC3 and GFP. Meanwhile, a recombinant eukaryotic expression vector contained ERα (pCDNA3.1-ERα) is constructed. All plasmids are kindly provided by Dr Li at Middle
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and Southern University of China. 2.2. Cell culture and transfection
Human embryonic kidney (HEK) 293T cells are presented by Dr Li at Middle and Southern University of
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China and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 10% fetal bovine serum at 37℃with 5% CO2. The cells are plated in a 6-well plate at 50-60% confluency a day before transfection. The next day pCDNA3.1-ASIC3-GFP and pCDNA3.1- ERα plasmid are transiently transfected into 293T cells with Lipofectamine® 2000 transfection reagent (Lip2000, Invitrogen), and the DNA-to-Lip 2000 ratio is one to three. When the plasmid pCDNA3.1-ASIC3-GFP is co-transfected with pCDNA3.1- ERα plasmid, the ratio is kept at 1:1. After 12 hours of the transfection, E2 (10-8、10-7、10-6、10-5 M) is added to the cells and cultured continuously for 2, 8, 12 hours and then removed, with all cells waiting until the
same time point before ASIC3 levels were measured, while control group receives vehicle treatment. Meanwhile, E2 (10-5 M) and specific ERα antagonists methyl-piperidino-pyrazole (MPP, 3×10-5 M) are coadded to 293T cells co-transfected with ASIC3 and ERα for 12 hours. Finally, all the cells are collected to assay the level of ASIC3 expression by immunoblotting using anti-GFP antibody (1:1000, Cell Signaling Technology), because GFP can act as a reporter gene. 2.3. Animals and operation
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The experimental protocol is approved by the Animal Research Ethics Committee of Hubei University of Science and Technology. Adult Sprague-Dawley rats (8-10 wk old) are used in all experiments. All animals are kept on a 12-hour light/dark cycle with food and water available ad libitum. After anesthesia, female
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rats in the ovariectomized (OVX) group are treated with bilateral ovariectomy, and those in the shamoperated group are only given laparotomy during the proestrus stage. Meanwhile, bilateral testes of male rats are extirpated (castrated group) or are subjected to a sham surgical procedure. Later 4 weeks after the
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operation, all rats are sacrificed after anesthetized and the rat DRGs are taken out for the next experiment
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study [14].
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2.4. Isolation of DRGs and Western blotting
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The rat DRGs are taken out and transferred immediately into DMEM at pH 7.4. After the removal of the surrounding connective tissues, the rat DRGs are lysed using the RIPA lysis buffer (Beyotime, China) and
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then total protein is isolated. The content of protein is quantified by BCA Protein assay (Thermo Fisher) and then 20 µg protein is boiled with loading buffer. The protein samples are separated by SDS-PAGE using 10%
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gels. The separated proteins are transferred to the PVDF membranes (Merck Millipore). Polyclonal antibody against ASIC3 (1:2000, GeneTex) is used to probe the ASIC3 proteins. The loading control is
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polyclonal antibody against β-actin (1:1000, Cell Signaling Technology). After primary antibodies incubation for overnight, the PVDF membranes (Merck Millipore) are washed with TBS Tween-20 (Tris-buffered saline with Tween-20 detergent) buffers, and then incubated by horseradish peroxidase conjugated antirabbit
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IgG (1: 10,000, Santa Cruz) for 2 h. At last the proteins are visualized using an ECL detection system (Thermo Fisher). 2.5. Nociceptive behavior induced by acetic acid Rats are placed in a 30 × 30 × 30 cm3 Plexiglas chamber and allowed to habituate for at least 30 min before nociceptive behavior experiments. Separate groups of rats are pretreated with 20 μL capsazepine (100 μM, Sigma-Aldrich) in ipsilateral hind paw before injection of acetic acid. After 5 min, rats were restrained lightly and injected subcutaneously with acetic acid solution (0.6%, 20 μL, Sigma-Aldrich) into
the hind paw using a 30-gauge needle connected to a 100 μL Hamilton syringe. Nociceptive behavior (that is, number of flinches) is counted over a 5 min period starting immediately after the injection [22, 23]. 2.6. Drugs and chemicals Trypsin digestion solution contains 5 mL of DMEM in which 0.5 mg/mL trypsin (type Ⅱ-S; Sigma-Aldrich), 1.0 mg/mL collagenase (type Ⅰ-A; Sigma-Aldrich), 0.1 mg/mL deoxyribonuclease (type Ⅰ-A; Sigma-Aldrich) and 1.25 mg/mL soybean trypsin inhibitor (type II-S; Sigma-Aldrich). Capsaicin (Sigma-Aldrich) is dissolved
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in DMSO to obtain 10 mM stock solution. Further dilutions are made with normal saline (NS) to reach final concentrations of 100 μM working solution. MPP (Sigma-Aldrich) and E2(Sigma-Aldrich) are dissolved in ethanol solution to obtain 10 mM stocks of the drugs. Stocks of MPP and E2 are further diluted with cell
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culture medium to reach final concentrations of 3×10-5 M and 10-8 to 10-5 M respectively. Hydrochloric acid and DMEM are purchased from Sigma-Aldrich. Fetal bovine serum is purchased from Gibco (Grand Island, N.Y. USA). Lip2000 is purchased from Thermon fisher.
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2.7. Statistical analysis
Student's t-test or unpaired t-test is used for the analysis of differences in two groups (MPP and 10-5
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whereas statistical analysis of concentration- and time-dependent regulatory changes data was performed using a one-way analysis of variance followed by Bonferroni’s post hoc tests. Data are presented as mean ±
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S.E.M. P-values < 0.05 are considered statistically significant.
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3. Results
3.1. E2 increases the ASIC3 expression in a concentration-dependent manner.
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We firstly treat 293T cells co-expressing ASIC3-GFP and ERα with various concentrations of E2 (10-8、 10-7、10-6、10-5 M ) for 12 h. Figure 1A shows that the effect of different concentrations of E2 on ASIC3 protein expression. To analyze ASIC3 protein expression quantitatively, its expression is normalized to the
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inner reference (β-actin) in its respective group. Figure 1B shows that the level of ASIC3 protein expression gradually increases as concentration of treated E2 increased from 10-8 to 10-5 M. ASIC3 protein expression increases 30.5%±0.03% and 93.4%±0.13% after treatment of 10-8 M and 10-5 M E2, respectively, compared with the control group (n=5, P < 0.05). These results indicate that E2 increases the ASIC3 expression in a concentration-dependent manner. To further verify whether the E2 up-regulation of ASIC3 protein expression is mediated by ERα, we co-apply E2 with and MPP, a specific ERα antagonist, in 293T cells co-expressing ASIC3-GFP and ERα. After co-treatment of E2 (10-5 M) with and MPP (3×10-5M), the
ASIC3 protein expression is close to that of control group, unlike an increase of 93.4%±0.13% in treatment with E2 (10-5 M) alone (n=5, P < 0.01). Thus, MPP obviously blocks the up-regulating effect of E2 on ASIC3 protein expression. The results suggest that E2 concentration-dependently increases the expression of ASIC3 via ERα. The ASIC3-GFP fusion plasmid that we use here comes fully made from Dr Li at Middle and Southern University of China. 3.2. E2 enhances the ASIC3 expression in a time-dependent manner
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We then investigate whether up-regulation of ASIC3 protein expression is dependent upon E2 exposure time. As shown in figure 2, E2 (10-5 M) up-regulates the expression of ASIC3 in 293T cells co-expressing ASIC3 and ERα in time-dependent manner (0-12 h). ASIC3 expression increases 1.7-fold and 2.1-fold at 8
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and 12 h after E2 exposure, while ASIC3 expression has not significant change at 2 h after E2 exposure (figure 2A). The date of semi-quantitive western blotting shows that the ratio of GFP/β-actin increases with the increase of E2 exposure time (n=5, P < 0.01, figure 2B). These results indicate that E2 enhances the
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ASIC3 expression in a time-dependent manner. In addition, E2 does not induce any significant change of
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ASIC3 expression in 293T cells untransfected ERα (data not shown).
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3.3. Sex differences occur in the ASIC3 expression in rat DRGs.
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To verify whether there are sex differences in ASIC3 protein expression in vivo, we detect the level of ASIC3 protein in DRGs of OVX, castrated and sham-operated rats. As shown in figure 3, the expression of
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ASIC3 protein in sham male rats is significantly different compared to sham female rats (n=8, P < 0.01). Moreover, the expression of ASIC3 protein significantly decreases in female rats after removing ovary (n=8,
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P < 0.01）. In contrast, the change of ASIC3 expression in the castrated males group is not obvious compared with sham males group (n=8, P > 0.05). These results indicate that sex differences occur in the
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ASIC3 expression in rat DRGs, which may involve in estrogens. 3.4. Sex differences occur in the nociceptive responses evoked by acetic acid in rats.
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As previously reported, intense flinch/shaking responses are produced by injection of acetic acid on the dorsal surface of the rat hind paw [14]. To investigate whether there are gender differences in the nociceptive responses evoked by acetic acid in rats, we observe the nociceptive responses induced by intradermal injection of pH 6.0 acetic acid in OVX and sham females. As shown in figure 4, OVX females display less nociceptive responses compared with sham females (n =10, P < 0.01). In contrast, there is no significant difference between castrated and sham males (n =10, P > 0.05). Interestingly, compared with sham males, sham females exhibit greater nociceptive responses to intraplantar injection of acetic acid (n
=10, P < 0.01). These results suggest that female rats display an enhanced sensitivity to the acetic acidinduced nociceptive response compared with male and OVX female rats. 4. Discussion In the present study, we provide data for the first time to show that E2 concentration- and timedependently up-regulates ASIC3 protein expression via ERα. We also observe that sex differences occur in ASIC3 expression in rat DRGs and in acetic acid-induced nociceptive responses.
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Estrogens exert their biological effects through genomic and nongenomic mechanisms [24]. Estrogens can freely pass through the cell membrane and bind to ERs located in the cytoplasm and nucleus, and form an estrogen-ER complex. This complex binds to estrogen-response element sequences in the promoter
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region of target genes and modulates the levels of associated mRNAs and proteins [25, 26]. This genomic mechanism plays a regulatory role from hours to days after the initiation of hormonal manipulation [27]. In the present study, we find that E2 concentration-dependently increases the expression of ASIC3 protein.
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The up-regulation of ASIC3 expression is dependent upon E2 exposure time. If the exposure time of E2 is
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too short, less than 2 hours, E2 has no significant effect on ASIC3 expression. Only when the exposure time
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of E2 is longer than 8 hours, E2 significantly increases ASIC3 expression. Thus, we consider that E2 up-
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regulates ASIC3 expression through a genomic mechanism.
We find that MPP, a specific ERα antagonist, almost completely abrogates the up-regulation of ASIC3
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expression by E2 in 293T cells co-expressing ASIC3 and ERα. Moreover, E2 does not induce any significant change of ASIC3 expression in 293T cells untransfected ERα. These results indicate E2 up-regulation of
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ASIC3 expression is mediated by ERα. It has been shown that ERα is also expressed in the rat DRG neurons, and its activation alters the expression of certain mRNA and protein in primary sensory neurons [28] .
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Among ASICs, ASIC3 is highly expressed in both DRG cell bodies and sensory terminals[29] . The present study shows that sex differences occur in the ASIC3 expression in rat DRGs. The level of ASIC3 protein expression in female rat DRGs is higher than those in male rat DRGs. Our result is agreed with result of
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Kobayashi et al. who reported that ASIC3 mRNA expression in female mouse DRGs is more abundant than those in male DRGs [30]. We find that the expression of ASIC3 protein from DRGs decreases after ovariectomy in female rats, but not orchiectomy in male rats. The results suggest that ASIC3 expression in rat DRGs involves in circulating female sex steroids. Combined with the results from cell line expression in the present experiment, E2 may play a key role in sex differences in the ASIC3 protein expression from rat DRGs. It is reported recently that E2 up-regulates voltage-gated sodium channel 1.7 in trigeminal ganglion contributing to hyperalgesia of inflamed temporomandibular joint likely through the classical genomic
mechanism of ER action [31]. Estrogens exacerbate nociceptive pain via up-regulation of transient receptor potential vanilloid 1 (TRPV1) and anoctamin 1 (ANO1) expression in primary sensory neurons of female rats [32]. Overnight incubation with E2, TRPV1 mRNA expression increases in cultured mouse DRG neurons [33]. E2 also increases sensitivity to chronic neuropathic pain through up-regulating N-methyl-D-aspartate acid receptor 1 expression in the DRG of rats [34]. Exogenous E2 also modulates TRPV1 and P2X3 receptor expression in sensory neurons of OVX rats, and the lack of E2 effect in ERα KO and ERβ KO mice resulted in TRPV1 and P2X3 receptor downregulation [35]. Although E2 regulates the expression of pain-related ion
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channels through its receptors, the genomic mechanism remains unclear. Future research is needed to clarify the mechanism underlying the upregulation of ASIC3 expression by E2. For example, how does the estrogen-ER complex bind to estrogen-response element sequences in the promoter region of ASIC3 gene
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and modulates the levels of ASIC3 mRNA and protein?
ASIC3 is expressed in both DRG cell bodies and sensory terminals, which monitors extracellular pH fall
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and contributes to proton-evoked pain signaling [22, 36]. It has been shown that ASIC3 plays an important
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role in various pain conditions such as inflammatory pain, postoperative pain and migraine [19-21]. ASICs are members of the ENaC/DEG superfamily of amiloride-sensitive epithelial sodium channels. Amiloride, a
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nonspecific blocker of ASICs, abolishes the known sex difference in formalin test response and blocks late
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phase nociceptive behavior in female, but not male mice [37]. The present and our previous studies show that sex differences occur in acidosis-induced nociception in rats, with females being more sensitive than
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males to acetic acid. After ovariectomy in female rats, female's enhanced sensitivity to the acetic acidinduced nociceptive response disappear. We have reported previously that E2 rapidly increases the ASIC-
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mediated currents and membrane excitability in rat DRG neurons. E2 potentiates the functional activity of ASICs through ERα and ERK1/2 signaling pathway [14]. In the present study, E2 can also up-regulate ASIC3
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protein expression in 293T cells co-expressing ASIC3-GFP and ERα. Moreover, we find that sex differences occur in the ASIC3 expression in rat DRGs. ASIC3 protein expression in female rat DRG is higher than those in male rat DRG. And ASIC3 protein expression in DRG decreases significantly after ovariectomy, but not
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after orchiectomy. Thus, E2 may play a key role in the sex differences. Our present and previous results suggest a role of E2 on ASIC3 function via both non-genomic and genomic mechanisms [14]. In conclusion, we have revealed interactions between E2 and ASIC3 expression in DRG neurons and transfected cell lines. E2 up-regulation of ASIC3 protein expression could likely result in females to be more sensitive to acetic acid than males. Our results indicate a potential mechanism for the explanation of sex differences occur in the acetic acid-induced nociceptive responses. Therefore, ASIC3 may be promising target for new drugs in woman with the disorders related pain.
Conflicts of interest The authors declare no conflicts of interest. Funding The work described in this paper was supported by grants from the National Natural Science Foundation of China (No. 81671101 to WPH and No. 81502635 to RP), Natural Science Foundation of Hubei Province of China (No. 2015CFA145 to WPH), and the Education Departmental Science Foundation of
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Hubei Province of China (No. D20152803 to RP). Acknowledgments
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We are very grateful to Professor Jia-da Li from Middle and Southern University of China for his valuable technical guidance.
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Figure legends
Fig 1 E2 enhances the expression of ASIC3 in a concentration-dependent manner. A. E2 increases the ASIC3 expression in a dose-dependent manner (10-8-10-5 M) after the treatment for 12 h in 293T cells co-expressing ASIC3 and ERα. The effect of E2 (10-5 M) is blocked by co-treatment of MPP (3×10-5M), a specific ERα antagonist. B. the quantitative analysis ensures the effect of E2 on ASIC3 protein expression with statistical significances. Each column and vertical bar represents the mean ± S.E.M. n=5, *
P< 0.05, **P< 0.01 vs control group by Bonferroni’s post hoc test. ##P< 0.01 vs the E2 (10-5 M) group by
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unpaired t-test. Fig 2 E2 increases the expression of ASIC3 in the time-dependent manner (0-12 h) in 293T cells coexpressing ASIC3 and ERα.
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A. The level of ASCI3 expression obviously increases in the 293T cells co-expressing ASIC3 and ERα treated by E2 (10-5 M) for 2, 8, 12 h compared with 0 h group. B. The quantitative analysis ensures the time-
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dependent effect of E2 on ASIC3 protein expression with statistical significances. Each column and vertical
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bar represents the mean ± S.E.M. n=5, **P< 0.01 vs 0 h group by Bonferroni’s post hoc test.
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Fig 3 Sex differences occur in the ASIC3 expression in rat DRGs.
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A. The ASIC3 expression decreases significantly in the male group compared with that of female group. In addition, the ASIC3 expression is obviously decreased in OVX females than in sham females but was not
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significantly different in castrated and sham males after surgical treatment. B. ASIC3 quantitative analysis shows that the statistical significances exist to a varied extent between females and males, OVX females
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and sham females. Each column and vertical bar represents the mean ± S.E.M. n=8, ** P< 0.01.
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Fig 4 Sex differences occur in the acetic acid evoked nociceptive responses in rats. The bar graph shows that the nociceptive responses induced by subcutaneous injection of pH 6.0 acetic acid is less in OVX females than in sham females but is not significantly different in castrated and sham males. Furthermore, sham females exhibit greater nociceptive responses to injection of acetic acid into the
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hind paws, compared with sham males. Each bar represents the number of flinches that animals spent licking/lifting the injected paw during first 5-min observation period. Each column and vertical bar represents the mean ± S.E.M. of ten rats. n=10, ** P< 0.01 vs sham male group by unpaired t-test.
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