Activation of the epithelial sodium channel ( EN aC) leads to cytokine profile shift to pro‐inflammatory in labor
2018; Springer Nature; Volume: 10; Issue: 10 Linguagem: Inglês
10.15252/emmm.201808868
ISSN1757-4684
AutoresXiao Sun, Jinghui Guo, Dan Zhang, Jun-Jiang Chen, Wei Lin, Yun Huang, Hui Chen, Wen Huang, Yifeng Liu, Lai Ling Tsang, Mei Yu, Yiu Wa Chung, Xiaohua Jiang, Hefeng Huang, Hsiao Chang Chan, Ye Chun Ruan,
Tópico(s)Pregnancy and Medication Impact
ResumoResearch Article28 August 2018Open Access Source DataTransparent process Activation of the epithelial sodium channel (ENaC) leads to cytokine profile shift to pro-inflammatory in labor Xiao Sun Xiao Sun Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Jing Hui Guo Jing Hui Guo Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Department of Physiology, School of Medicine, Jinan University, Guangzhou, China Search for more papers by this author Dan Zhang Dan Zhang Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Jun-jiang Chen Jun-jiang Chen Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Department of Physiology, School of Medicine, Jinan University, Guangzhou, China Search for more papers by this author Wei Yin Lin Wei Yin Lin Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Yun Huang Yun Huang Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Hui Chen Hui Chen Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Wen Qing Huang Wen Qing Huang Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Yifeng Liu Yifeng Liu Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Lai Ling Tsang Lai Ling Tsang Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Mei Kuen Yu Mei Kuen Yu Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Search for more papers by this author Yiu Wa Chung Yiu Wa Chung Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Xiaohua Jiang Xiaohua Jiang Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Hefeng Huang Hefeng Huang Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China International Peace Maternal and Child Health Hospital, Shanghai Jiao Tong University, Shanghai, China Search for more papers by this author Hsiao Chang Chan Corresponding Author Hsiao Chang Chan [email protected] orcid.org/0000-0002-5888-0595 Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Ye Chun Ruan Corresponding Author Ye Chun Ruan [email protected] orcid.org/0000-0002-4266-6789 Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Search for more papers by this author Xiao Sun Xiao Sun Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Jing Hui Guo Jing Hui Guo Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Department of Physiology, School of Medicine, Jinan University, Guangzhou, China Search for more papers by this author Dan Zhang Dan Zhang Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Jun-jiang Chen Jun-jiang Chen Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Department of Physiology, School of Medicine, Jinan University, Guangzhou, China Search for more papers by this author Wei Yin Lin Wei Yin Lin Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Yun Huang Yun Huang Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Hui Chen Hui Chen Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Wen Qing Huang Wen Qing Huang Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Yifeng Liu Yifeng Liu Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Lai Ling Tsang Lai Ling Tsang Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Mei Kuen Yu Mei Kuen Yu Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Search for more papers by this author Yiu Wa Chung Yiu Wa Chung Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Xiaohua Jiang Xiaohua Jiang Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Hefeng Huang Hefeng Huang Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China International Peace Maternal and Child Health Hospital, Shanghai Jiao Tong University, Shanghai, China Search for more papers by this author Hsiao Chang Chan Corresponding Author Hsiao Chang Chan [email protected] orcid.org/0000-0002-5888-0595 Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China Search for more papers by this author Ye Chun Ruan Corresponding Author Ye Chun Ruan [email protected] orcid.org/0000-0002-4266-6789 Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China Search for more papers by this author Author Information Xiao Sun1,‡, Jing Hui Guo1,2,3,‡, Dan Zhang4, Jun-jiang Chen1,2,3, Wei Yin Lin1, Yun Huang4, Hui Chen1, Wen Qing Huang1, Yifeng Liu4, Lai Ling Tsang1, Mei Kuen Yu1,2, Yiu Wa Chung1, Xiaohua Jiang1, Hefeng Huang4,5, Hsiao Chang Chan *,1 and Ye Chun Ruan *,2 1Epithelial Cell Biology Research Centre, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China 2Department of Biomedical Engineering, Faculty of Engineering, The Hong Kong Polytechnic University, Hong Kong, China 3Department of Physiology, School of Medicine, Jinan University, Guangzhou, China 4Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China 5International Peace Maternal and Child Health Hospital, Shanghai Jiao Tong University, Shanghai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +852 39436839; E-mail: [email protected] *Corresponding author. Tel: +852 34008084; E-mail: [email protected] EMBO Mol Med (2018)10:e8868https://doi.org/10.15252/emmm.201808868 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The shift of cytokine profile from anti- to pro-inflammatory is the most recognizable sign of labor, although the underlying mechanism remains elusive. Here, we report that the epithelial sodium channel (ENaC) is upregulated and activated in the uterus at labor in mice. Mechanical activation of ENaC results in phosphorylation of CREB and upregulation of pro-inflammatory cytokines as well as COX-2/PGE2 in uterine epithelial cells. ENaC expression is also upregulated in mice with RU486-induced preterm labor as well as in women with preterm labor. Interference with ENaC attenuates mechanically stimulated uterine contractions and significantly delays the RU486-induced preterm labor in mice. Analysis of a human transcriptome database for maternal–fetus tissue/blood collected at onset of human term and preterm births reveals significant and positive correlation of ENaC with labor-associated pro-inflammatory factors in labored birth groups (both term and preterm), but not in non-labored birth groups. Taken together, the present finding reveals a pro-inflammatory role of ENaC in labor at term and preterm, suggesting it as a potential target for the prevention and treatment of preterm labor. Synopsis This study reveals a novel role for the epithelial sodium channel (ENaC) in transducing the mechanical signal from uterine contractions that results in production of pro-inflammatory cytokines during labor. ENaC has the potential to be targeted for the prevention and treatment of preterm labor. ENaC expression is increased and its channel function is activated in the uterus during labor in mice. Mechanical stimulation activates ENaC, which leads to activation of the transcription factor CREB, and subsequent production of pro-inflammatory cytokines/factors in uterine epithelial cells. ENaC expression is also increased in mice with RU486-induced preterm labor as well as in women with preterm labor. Interference with ENaC attenuates mechanically stimulated uterine contractions and significantly delays the RU486-induced preterm labor in mice. ENaC is significantly and positively correlated with labor-associated pro-inflammatory factors in human maternal-fetus tissue/blood collected at onset of birth in women in labor only. Introduction Labor or parturition, the end step of pregnancy, remains poorly understood (Muglia & Katz, 2010; Rubens et al, 2014), which accounts for the lack of effective method to prevent or predict preterm labor, a leading cause of neonatal death and disability (Liu et al, 2012; Iams, 2014b; Romero et al, 2014). Given the similar clinical events involved, both term and preterm labor are considered to share common pathways of labor (Romero et al, 2014). The shift in cytokine profile from anti-inflammatory to pro-inflammatory and uterine activity from quiescent to contractile is the most recognizable sign of labor (Simhan & Caritis, 2007; Renthal et al, 2010, 2013; Tan et al, 2012; Adams Waldorf & McAdams, 2013; Romero et al, 2014). However, the molecular mechanism underlying the "shift" of these events remains elusive. Among the recognized maternal or fetal derived signals associated with the initiation of labor, mechanical forces generated by either the growing fetus or initial uterine contractions are believed to be important (Lye et al, 2001). Mechanical stimulations are observed to induce uterine production of pro-inflammatory cytokines (e.g., IL-8; Maehara et al, 1996; Loudon et al, 2004; Shynlova et al, 2008) and prostaglandins (PGs; Challis et al, 2000; Sooranna et al, 2004), which in turn potently evoke uterine contractions (Wray, 1993) and therefore may underlie a positive-feedback loop resulting in increasingly powerful expelling forces required for labor (Wray, 1993; Lye et al, 2001). A large number of studies are dedicated to myometrium muscle cells and suggest their essential involvement in the positive-feedback loop at labor (Wray, 1993; Lye et al, 2001). Interestingly, epithelial cells lining the uterine cavity as part of the decidua are also believed to be a source of PGs at term (Dong et al, 1996; Olson, 2003; Satoh et al, 2013), although whether and how these epithelial cells play a role in the process of labor is not well studied. We have previously demonstrated that ENaC in the endometrial epithelium plays an essential role in embryo implantation by activating Ca2+/cAMP response element binding protein (CREB) and cyclooxygenase 2 (COX-2) to produce prostaglandins E 2 (PGE2), which acts as a paracrine factor for stromal cell decidualization, the prerequisite of embryo implantation (Ruan et al, 2012). Interestingly, many of the labor-associated inflammatory cytokines, such as IL-6, IL-8, and TNFα, are known to be downstream targets of either CREB or PGE2 (Wen et al, 2010; Srivastava et al, 2012). Moreover, the open probability of ENaC has been shown to be increased by mechanical stimuli (Fronius & Clauss, 2008; Shi et al, 2013), although the physiological significance of this mechano-sensitivity has not been well demonstrated. The mechano-sensitivity of ENaC and its ability to regulate the CREB/COX-2/PGE2 axis led us to hypothesize that ENaC might play a role in mechano-sensing and signal transduction leading to the shift in the cytokine profile and uterine contractile state during labor process, at term or preterm. We undertook the present study to test this hypothesis and explore treatment strategy for preterm labor. Results ENaC is upregulated and activated at labor in mice To establish a possible role of ENaC in labor, we first examined the expression level of ENaC in the uterus during late gestation approaching parturition in mice. ENaCα, the rate-limiting subunit of ENaC, showed a gradual increase at the mRNA level from 15 d.p.c. to the labor day (19 d.p.c.) in fetus/placenta-removed uterine tissues (Fig 1A). Western blot analysis showed that ENaCα and its cleaved form, indicative of ENaC activation (Kleyman et al, 2009), were substantially increased at 18 and 19 d.p.c. as compared to 15 d.p.c. (Fig 1B). Immuno-staining for ENaCα in mouse uterus also showed stronger signal intensity detected at 19 d.p.c. compared to that at 15 d.p.c. in decidua epithelial cells (higher level detected at the apical membrane) lining the uterine cavity and placental regions (Fig 1C), suggesting a potential role of ENaC in the process of labor. Figure 1. ENaC is upregulated at labor in mice Quantitative PCR (qPCR) analysis of ENaCα in fetus/placenta-removed uterine tissues from pregnant mice at 15–19 days post-coitum (d.p.c.) and 1–2 day(s) post-partum (d.p.p.). Data are shown as mean ± SEM. n = 4. *P < 0.05, ***P < 0.001, one-way ANOVA with Tukey's multiple comparisons test, compared to 15 d.p.c. 18S rRNA was used as a housekeeping gene for normalization. Western blot (with quantification shown on the right) analysis of ENaCα in fetus/placenta-removed uterine tissues from pregnant mice at 15–19 d.p.c. and 1 d.p.p. Data are shown as mean ± SEM. n = 4. Two-tailed unpaired Student's t-test with unequal variance corrected by Welch's correction. **P < 0.01. β-tubulin was blotted as a loading control. Immuno-histochemical labeling for ENaCα in uterine tissues from mice at 15 or 19 d.p.c. E: epithelial tissues, P: placenta, and M: myometrium. NC: negative control done with the absence of primary antibody. Nuclei are labeled with hematoxylin. Scale bar, 20 μm. Data information: Exact P-values are listed in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 1 [emmm201808868-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Mechano-activation of ENaC-dependent signaling and pro-inflammatory cytokines in the uterine epithelial cells We had previously shown that ENaC activation could lead to phosphorylation/activation of CREB (Ruan et al, 2012). CREB is a key transcription factor known to promote the expression of pro-inflammatory cytokines (Wen et al, 2010). Given the known mechano-sensitivity of ENaC (Shi et al, 2013), we next investigated whether ENaC-dependent CREB activation and subsequent upregulation of pro-inflammatory cytokines could be elicited mechanically during labor. We adopted ex vivo stretch stimulation to uterine preparations collected from mice at 19 d.p.c. (See Materials and Methods). After 1-h stretch, immunofluorescence labeling for phosphorylated CREB (pCREB) in the uterine tissues showed abundant pCREB expression in the nuclei of the uterine epithelial cells (Fig 2A), whereas other cell types (e.g., muscle cells) showed much weaker or absence of fluorescence. Quantification of the fluorescence intensity revealed that the epithelial nuclear pCREB expression was significantly stronger in the stretched uterine preparations as compared to that in non-stretched ones (Fig 2B). Moreover, the stretch-induced nuclear expression of pCREB was abolished in the presence of amiloride (10 μM, Fig 2A and B), a selective blocker of ENaC, indicating ENaC-dependent epithelial CREB activation by mechanical stimulations. This was also tested in a human endometrial epithelial cell line, Ishikawa (ISK) cells. Results showed that stretch (15% elongation) of the cells for 30 min increased phosphorylation of CREB, which was blocked by pretreatment with amiloride (10 μM, Fig EV1). Figure 2. Mechano-activation of ENaC-dependent signaling and pro-inflammatory cytokines in uterine epithelial cells A, B. Immunofluorescence labeling for phosphorylated CREB (pCREB, A) with quantification of fluorescence intensity in nuclei of epithelial cells (B) in mouse uterine tissues (19 d.p.c.), either kept slack (Ctrl) or stretched and incubated for 1 h (see Materials and Methods), in the presence or absence of amiloride (Ami, 10 μM), a selective blocker of ENaC. Data are shown as mean ± SEM. ***P < 0.001, one-way ANOVA with Tukey's multiple comparisons test, n is shown in each column. Scale bars, 10 μm. C. qPCR analysis of IL-6 and TNFα mRNA levels in mouse uterine tissues with or without 1-h stretch in the absence or presence of Ami (10 μM). Data are shown as mean ± SEM. n = 16. ***P < 0.001, one-way ANOVA with Tukey's multiple comparisons test. Data are relative mRNA levels normalized to GAPDH levels. D. qPCR of ENaCα in ISK cells transfected with shRNAs against ENaCα (shENaCα) or non-silencing control (shNC). Data are shown as mean ± SEM. **P < 0.01, two-tailed unpaired Student's t-test, n = 4. Data are relative mRNA levels normalized to GAPDH levels. E. qPCR of IL-6, IL-8, TNFα, and COX-2 in ISK cells transfected with shENaC or shNC with (+) or without (−) 1-h stretch. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey's multiple comparisons test, n = 3 (IL-6), 6 (IL-8), or 4 (TNFα and COX-2). Data are relative mRNA levels normalized to GAPDH levels. F, G. Western blotting with quantification for COX-2 in mouse uterine preparations (F) and ELISA analysis of PGE2 levels in uterine preparation-incubated media (G) with (+) or without (−) overnight stretch, in the absence (−) or presence (+) of Ami (10–50 μM). Data are shown as mean ± SEM. *P < 0.05, ***P < 0.001, one-way ANOVA with Tukey's multiple comparisons test, n is shown in each column in (F) and n = 3 in (G). H. Western blotting with quantification for ENaCα, β, γ, and COX-2 expression in mouse uterine preparations (19 d.p.c.), either kept slack (Ctrl) or stretched and incubated overnight. β-tubulin was blotted as a loading control. Data are shown as mean ± SEM. n = 3. *P < 0.05, two-tailed unpaired Student's t-test. Data information: Exact P-values are listed in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 2 [emmm201808868-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Stretch induces ENaC-dependent CREB activation in human endometrial epithelial cellsWestern blotting with quantification for pCREB in ISK cells with (+) or without (−) stretch (30 min, 15% elongation), in the absence (−) or presence (+) of Ami (10 μM). Data are shown as mean ± SEM. n is indicated in each column. *P < 0.05, one-way ANOVA with Tukey's multiple comparisons test. Exact P-values are listed in Appendix Table S1. Download figure Download PowerPoint We further investigated whether the stretch-induced ENaC/CREB activation could lead to a shift in cytokine profile, switching on the pro-inflammatory cytokines involved in labor. Indeed, the stretch of mouse uterine tissues upregulated IL-6 and TNFα, which were both significantly inhibited by amiloride (10 μM, Fig 2C), suggesting the involvement of ENaC in the upregulation of pro-inflammatory cytokines. To confirm this, we next used ISK cells in conjunction with ENaC knockdown by shRNAs. To better mimic mechanical stimuli in the uterus during labor, which are mostly rhythmic contractile forces, we applied a cyclic stimulation of stretch (15% elongation) at frequency of 1 Hz as previously reported (Korita et al, 2002; Yoshida et al, 2002) to ISK cells grown on flexible culture supports. shRNAs against ENaCα (shENaCα), the rate-limiting subunit of ENaC, were used to knockdown ENaC. Quantitative PCR showed ~ 50% reduction in ENaCα mRNA level in ISK cells treated with the shENaCα as compared to the cells treated with non-silencing control shRNAs (shNC; Fig 2D). The cyclic stretch-induced increases in mRNA levels of IL-6, IL-8, TNFα, and COX-2 were all significantly inhibited by ENaC knockdown (Fig 2E), confirming the involvement of ENaC. These results suggest that the stretch-induced upregulation of pro-inflammatory cytokines may be a subsequence to the mechano-activation of ENaC. To demonstrate mechano-activation of ENaC directly, we also examined ENaC current activation in response to varied mechanical force in the human endometrial epithelial cells using an automatic patch-clamp system (See Materials and Methods). Negative pressures (40, 80, and 120 mPa) were used to stretch the cells during whole-cell recording. At holding voltage of −80 mV, an inward whole-cell current was observed as the stretching force was increased to 120 mPa, which was subsequently abolished by amiloride (10–20 μM, Fig EV2), indicating stretch-induced activation of ENaC. Click here to expand this figure. Figure EV2. Mechano-activation of ENaC in human endometrial epithelial cellsWhole-cell patch-clamp recording of human endometrial epithelial cells at holding voltage of −80 mV when stretches (40–120 mPa) were applied before and after addition of amiloride (Ami, 20 μM). Download figure Download PowerPoint Since PGE2 is implicated in the positive-feedback loop of uterine contractions during labor process, we also examined COX-2 level in the stretched uterine tissues and the PGE2 concentration in the incubating media. The results showed that the levels of both COX-2 (Fig 2F) and PGE2 (Fig 2G) were significantly elevated by overnight uterine stretch, which were abrogated by the treatment with amiloride (10–50 μM, Fig 2F and G). Of interest, overnight stretch also induced upregulation of all three ENaC subunits, as compared to non-stretched ones (Fig 2H). The cleaved/activated form of ENaCα was also enhanced after the stretch (Fig 2H), suggesting activation of ENaC by mechanical forces. Taken together, these results suggest the involvement of ENaC in mechanically stimulated upregulation of the pro-inflammatory cytokines/factors. Epithelial cells and ENaC are involved in stretch-facilitated uterine contractions The suggested role of ENaC in mechanically stimulated increases in COX-2 and PGE2, a potent elicitor of uterine contractions, in epithelial cells, prompted us to examine possible involvement of uterine epithelium and ENaC in regulating uterine contractions in response to mechanical stimulation. The contractility of mouse uterine tissue preparations (19 d.p.c.) was measured ex vivo, which exhibited rhythmic spontaneous contractions. Stretches by 10–40% elongation of in situ length (L0) of the preparations were applied at the end of a rhythmic spontaneous contractive phase, which substantially advanced the following contractive phase (Fig 3A). The stretch-induced phase-advancing effect was calculated, according to a previously reported method (Kasai et al, 1995), as (T0−T1)/(T0−Ts) (See Materials and Methods). As shown in Fig 2B, the maximal phase-advancing effect on 19 d.p.c. uterine preparations was achieved by 20% L0 stretch, which was then used in the following experiments. Figure 3. Epithelial cells and ENaC are involved in mechano-facilitated uterine contractions in mice A, B. Representative mechanogram (A) with quantification (B) showing stretch-induced advancement of spontaneous rhythmic contractile phase in uterine preparations collected from mice at 19 d.p.c. Stretch varied by 10–40% elongation of original uterine length, L0. The advancing effect is calculated by (T0−T1)/(T0−Ts), where T0 is the time between onsets of two consecutive contractions prior to the stretch; T1, period between two consecutive contractile onsets before and after the stretch; and Ts, the time when the stretch is initiated as measured from the onset of the previous contraction. Data are shown as mean ± SEM. n is shown in each column. C. H&E staining of uterine tissues with intact or denudated (de-epi) epithelium. E: epithelium. M: myometrium. Scale bars, 100 μm. D. Representative mechanogram with quantification showing the effect of removal of the epithelium (de-epi) on stretch (20% L0)-facilitated uterine contractions. Data are shown as mean ± SEM. *P < 0.05, two-tailed paired Student's t-test. n = 5. E. Representative mechanograms (left) of uterine contractions before and after the stretch (20% L0) are applied in the absence (DMSO) or presence of amiloride (Ami, 200 μM) with quantification (right) showing the effect of Ami (10–200 μM) on stretch-facilitated uterine contractions. Data are shown as mean ± SEM. n is shown in each column. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the group treated with DMSO as the control, one-way ANOVA with Tukey's multiple comparisons test. F. Stretch-induced contractile phase-advancing effect in mouse uterus at 15 or 19 d.p.c. Data are shown as mean ± SEM. ***P < 0.001, two-tailed unpaired Student's t-test. n is shown in each column. Data information: Exact P-values are listed in Appendix Table S1. Download figure Download PowerPoint To explore a possible role of the epithelium, we denudated the epithelium from the uterine preparations (Fig 3C) and this resulted in significant inhibition of the stretch (20% L0)-induced phase advancement as compared to the intact tissues (Fig 3D), suggesting a role of the epithelium in regulating uterine contractions. Similarly, inhibition of ENaC by amiloride (10–200 μM), starting from a low dose (10 μM), significantly inhibited the stretch (20% L0)-induced phase advancement in a concentration-dependent manner (Fig 3E), indicating the involvement of ENaC in stretch-facilitated contractions. Of note, in contrast to the time of parturition (19 d.p.c.), the stretch (20% L0)-induced phase-advancing effect was found significantly less in uterine preparations from mice at 15 d.p.c. (Fig 3F). This is consistent with the time course expression profile of ENaC (Fig 1), with stronger uterine contractions observed when ENaC expression level is maximal, further suggesting the involvement of ENaC in regulating uterine contractions during labor process. Interference with ENaC delays RU486-induced preterm labor in mice We further explored the role of ENaC in preterm labor and possible prevention in two well-established preterm labor mouse models, induced by RU486 (a progesterone receptor antagonist; Dudley et al, 1996) and lipopolysaccharide (LPS; Gross et al, 2000), respectively. We subcutaneously injected RU486 (200 μg per mouse) into pregnant mice at 15 d.p.c., which resulted in preterm labor in ~ 20 h. Uterine tissues were collected either at preterm labor from RU486-treated mice or time-match
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