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. 2014 Jun 10;155(8):3112–3122. doi: 10.1210/en.2014-1152

BK Channels Regulate Myometrial Contraction by Modulating Nuclear Translocation of NF-κB

Youe Li 1, Ramón A Lorca 1, Xiaofeng Ma 1, Alexandra Rhodes 1, Sarah K England 1,✉
PMCID: PMC4098006  PMID: 24914944

Abstract

The large-conductance Ca2+-activated K+ (BK) channel plays an essential role in maintaining uterine quiescence during pregnancy. Growing evidence has shown a link between the BK channel and bacterial lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation in macrophages. In the uterus, NF-κB activation plays an important role in inflammatory processes that lead to parturition. Our objective was to determine whether the BK channel regulates uterine contraction, in part, by modulating NF-κB translocation into the nucleus. We compared the effects of BK channel modulation to those of LPS on NF-κB nuclear translocation and contraction in an immortalized human myometrial cell line (human telomerase reverse transcriptase [hTERT]) and uterine myocytes. Our results showed that BK channel inhibitors paxilline and penitrem A induced translocation of NF-κB into the nucleus in both hTERT cells and uterine myocytes to a similar extent as LPS treatment, and LPS and paxilline similarly reduced BK channel currents. Conversely, neither BK channel openers nor blockade of the small conductance Ca2+-activated K+ channel protein 3 had an effect on NF-κB translocation. Additionally, collagen-based assays showed that paxilline induced contraction of hTERT cells and uterine myocytes. This was dependent upon cyclooxygenase-2 activity. Moreover, paxilline-induced contractility and increased cyclooxygenase-2 expression both depended on availability of free NF-κB. This study suggests that BK channels regulate myometrial contraction, in part, by modulating nuclear translocation of NF-κB.

In humans, the uterus must remain noncontractile (quiescent) throughout 40 weeks of pregnancy and then become contractile to expel the fetus. This transition must be tightly controlled to ensure that labor and delivery are neither too early (preterm) nor too late (postterm). One important process that contributes to both term and preterm labor is intrauterine inflammation (1). The nuclear transcription factor κB (NF-κB), a key orchestrator of the inflammatory response, plays a pivotal role in parturition (2). Translocation of NF-κB into the nucleus induces the release of inflammatory mediators such as cytokines and chemokines (3–6). In most cell types, NF-κB is retained in the cytoplasm in an inactive form as a complex with the inhibitor IκB (7, 8). When cells are activated by a variety of stimuli, including lipopolysaccharide (LPS), an endotoxin of gram-negative bacteria that induces inflammation, and proinflammatory cytokines, IκB undergoes phosphorylation, ubiquitination, and degradation (8). This releases NF-κB and allows it to translocate into the nucleus, where it activates transcription of a number of target genes involved in the inflammatory response pathway. In the myometrium, one target of activated NF-κB is cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins, which play important roles in increasing myometrial contractility leading to parturition (9).

Another important aspect of the transition to uterine contractility is change in excitability of the myometrial smooth-muscle cells. The large-conductance calcium-activated K+ channel (BK) plays an important role in this transition. The BK channel maintains uterine quiescence by generating a potent repolarizing K+ current in response to depolarizing stimuli during pregnancy (10, 11); blocking the BK channel increases contractile activity in rat and human myometrium (12, 13). One well-supported model to explain how BK regulates contraction is that when the channel is blocked, the membrane becomes depolarized and L-type Ca2+ channels allow the intracellular concentration of Ca2+ to rise. This results in activation of myosin and muscle contraction. However, whether other mechanisms also contribute to regulation of contraction by BK remains unexplored.

Several studies have shown an association between BK channel activity and LPS. In macrophages, LPS increases the open probability of the BK channel. Conversely, blocking the BK channel inhibits LPS-induced activation of NF-κB, but how the BK channel regulates NF-κB activity, and how this might affect uterine contraction, is still unclear. We hypothesized that the BK channel regulates myometrial contraction, in part, by modulating NF-κB translocation into the nucleus. We tested this hypothesis by comparing the effects of the BK channel inhibitors paxilline and penitrem A and openers NS1619 and NS11021 to the effects of bacterial LPS in a human myometrial cell line (human telomerase reverse transcriptase [hTERT]) and uterine myocytes. We found that BK channel inhibitors induced NF-κB translocation into the nucleus in hTERT and uterine myocytes to a similar extent as LPS treatment; conversely, BK channel openers had no effect on NF-κB translocation. Blocking BK channel activity in the presence of LPS lengthened the period during which NF-κB was activated. Additionally, blocking BK activity resulted in myometrial cell contraction, and this response was inhibited when NF-κB was prevented from dissociating from IκB. Furthermore, we demonstrate that LPS inhibits BK channel current in myometrial cells. Our study may reveal a novel mechanism by which the BK channel regulates the initiation of uterine contraction.

Materials and Methods

Tissue collection

Human myometrial samples were collected from term nonlaboring women, who gave written informed consent, during elective cesarean. The study was approved by the Washington University in St Louis Institutional Review Board (IRB201108143).

Cell culture

hTERT (human telomerase reverse transcriptase) cells from nonpregnant human uterus were maintained in DMEM:F12 medium with 10% fetal bovine serum (FBS) and 25 μg/mL gentamicin at 37°C in 5% CO2. Primary cells were cultured from human nonlabor myometrial tissue. Myometrial tissue was cut into 2- to 3-mm pieces and cultured in DMEM:F12 medium with 5% FBS, 0.2% fibroblast growth factor-β, 0.1% epidermal growth factor, 0.05% insulin, 0.05% gentamicin, and 0.05% fungizone. After 2 weeks, colonies around the tissue were trypsinized and expanded into larger flasks in DMEM:F12 medium with 10% FBS and 25 μg/mL gentamicin. For experiments, hTERT cells and uterine myocytes were incubated in DMEM: F12 medium with 0.5% FBS and 25 μg/mL gentamicin for 12 hours. LPS from Escherichia coli (10 ng/mL, Sigma-Aldrich), paxilline (20 μM, Tocris Bioscience), penitrem A (500 nM, Tocris), NS1619 (100 nM, Sigma-Aldrich), and NS11021 (100 nM, Glixx Laboratories,) were used to stimulate the cells for 0, 15, 30, and 45 minutes, and 1, 2, 4, 8, and 16 hours. Bay 11–7082 (30 μm, Sigma-Aldrich), TAK-242 (1 μM, Millipore Corp), SC-236 (20 μM, Sigma-Aldrich) were applied 1 hour before the other reagents. Control experiments in the presence of polymyxin B sulfate (Sigma-Aldrich) confirmed that paxilline and penitrem A were LPS free.

Western blot analysis

hTERT cells were resuspended in NE1 buffer (10 mM HEPES, 10 mM MgCl2, 5 mM KCl, 0.1% Triton X-100, 0.1 mM EDTA, pH 8.0) and homogenized by passage through a 23-gauge needle; homogenates were centrifuged at 5000 × g for 10 minutes at 4°C. Retained supernatant was used as the cytoplasmic fraction. Pellets were resuspended in NE2 buffer (25% glycerol, 20 mM HEPES, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0), incubated on ice for 30 minutes, and then centrifuged at 10 000 × g for 10 minutes at 4°C. Retained supernatant was used as the nuclear fraction. Cytoplasmic and nuclear proteins (12.5 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Blots were blocked with 5% nonfat dry milk in PBS with 0.05% Tween 20 for 2 hours at room temperature and then incubated overnight at 4°C with primary antibodies: mouse monoclonal NF-κB-P65 (1:500; Santa Cruz Biotechnology [SCBT],), mouse monoclonal IgG1 β-actin (1:2000; SCBT), rabbit anti-histone H3 (1:2000; Abcam), mouse anti-COX-2 C-terminal (1:2000; Sigma-Aldrich), and mouse monoclonal anti-Slo 1/BK Alpha (1:500; NeuroMab). After 3 washes with PBS-Tween 20, blots were incubated with horseradish peroxidase-conjugated goat antimouse or goat antirabbit IgG (1:5000; Amersham Pharmacia). Signals were detected by using Clarity Western ECL (Bio-Rad Laboratories).

Immunofluorescence microscopy

hTERT and uterine myocytes were plated on chamber slides (Lab-Tek). After stimulation, cells were fixed and permeabilized in 2% paraformaldehyde/PBS with 0.01% Triton X-100 for 30 minutes at room temperature. After 3 washes with PBS, cells were blocked with 10% heat-inactivated FBS and 1% heat-inactivated goat serum in PBS at 37°C for 30 minutes, stained with mouse monoclonal NF-κB-p65 antibodies (1:100) or mouse monoclonal nuclear factor of activated T cells (NFAT)c3 antibodies (1:100; SCBT) in 1% heat-inactivated FBS and 0.1% heat-inactivated goat serum in PBS for 1 hour at 37°C, and then incubated with Alexa Fluor 488-conjugated goat antimouse IgG (1:2000; Jackson ImmunoResearch Laboratories) and TO-PRO-3 iodide (1:750; Invitrogen) for 30 minutes at 37°C. Cells were imaged by confocal microscopy (Nikon Eclipse E800 with a C1 confocal laser scanning head). Ten random areas were selected for counting.

Electrophysiology

Cell-attached patch-clamp recordings were performed on hTERT cells at room temperature in a bath solution containing the following (in mM): 135 NaCl, 4.7 KCl, 5 HEPES, 10 glucose, 1 MgCl2, and 2 CaCl2, pH 7.4. Cells were treated for at least 1 hour with LPS or paxilline as described in Cell Culture, and 10 ng/mL LPS or 20 μM paxilline was added to the bath solution during recordings. Pipette solution contained the following (in mM): 140 KCl, 20 KOH, 2 MgCl2, pH 7.4. Single-channel currents were recorded at a sampling rate of 100 kHz and filtered at 5 kHz by using an Axopatch 200B amplifier (Molecular Devices). Currents were evoked with 10 mV voltage steps (1-second duration) from 60 to 160 mV, from a holding potential of 0 mV by using pCLAMP software (version 10, Molecular Devices). This protocol was repeated at least 3 times on each cell. For analysis purposes, recordings on the same patch were concatenated, attaining at least a 3-second length recording from each voltage pulse. Mean open probability (Po) was calculated by using pCLAMP software. Patches containing 3 or fewer channels were used for Po analysis.

Collagen contractility assay

A standard kit assay was used (Cell Bio-Labs). Two parts of cells were mixed with 8 parts of collagen gel lattice mixture, and 1.6 × 105 cells per well were plated into 24-well plates for 1 hour at 37°C. After the gel was polymerized, DMEM:F12 medium with 10% FBS and 25 μg/mL gentamicin was added to each well and incubated at 37°C for 48 hours. Twelve hours before stimulation, cells in collagen gels were incubated in serum-free DMEM:F12 medium. Next, gels were released from the culture well sides, and stimulants were added at concentrations indicated above. Bay 11–7082, TAK-242, and SC-236 were applied to the cells 1 hour before the other reagents. NS1619 was applied 5 minutes before LPS. Gels were imaged at 1 hour, 4 hours, and 24 hours. The area of gel lattices was measured in Image J software (http://rsbweb.nih.gov/ij/) for each condition, and collagen contraction was determined in triplicate. Each experiment was performed at least 3 times.

Quantitative RT-PCR

Total RNA from hTERT cells treated as indicated in the figure was extracted by using Aurum Total RNA mini kit (Bio-Rad Laboratories). Reverse transcription and real-time PCR were performed by using the iScript cDNA Synthesis Kit (Bio-Rad) and the CFX96 Real-time System with IQ-SYBR Green Supermix (Bio-Rad), respectively. Primer sequences (IDT) were: human COX-2 (GenBank accession no: NM000963), 5′-TTCAAATGAGATTGTGGGAAAATT-3′ and 5′-ATATCATCTCTGCCTGAGTATTT-3′; human β-actin (GenBank accession no: NM001101), 5′-CAACTCCATGAAGTGTGAC-3′ and 5′-GTCAAGAAAGGGTGTAACGCA-3′. Thermal cycling conditions were as follows: 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. All samples were run in triplicate; mRNA levels are reported relative to β-actin.

Statistical analyses

Data were subjected to one-way ANOVA by using GraphPad software. A P value < .05 was considered significant. All data are presented as mean ± SEM.

Results

BK channel inhibition induces nuclear translocation of NF-κB-p65 in hTERT cells

Previous studies in macrophages showed that blocking the BK channel inhibited LPS-induced nuclear translocation of NF-κB. To determine whether the BK channel plays a role in NF-κB translocation in myometrial cells, hTERT cells were stimulated with LPS and treated with either the specific BK channel blockers paxilline or penitrem A, or openers NS1619 or NS11021. We assayed NF-κB-p65 translocation by 2 methods: immunofluorescence microscopy and Western blot analysis of nuclear and cytoplasmic fractions. We first counted the numbers of hTERT cells in which NF-κB-p65 was translocated into the nucleus (Figure 1A and Supplemental Figure 1 A). Within 30 minutes, neither the BK channel modulators nor LPS induced NF-κB-p65 translocation into the nucleus (Figure 1B). By 45 minutes, NF-κB-p65 was translocated into the nucleus in approximately 20% of hTERT cells treated with LPS, paxilline, or LPS plus paxilline. After 1 hour, treatment with LPS or paxilline led to nuclear translocation in about 70% of the cells (Figure 1, A and B); this effect was also observed with penitrem A (data not shown). In all 3 cases, NF-κB-p65 translocated back to the cytoplasm in 96% of cells by 16 hours. In cells treated with both paxilline and LPS, NF-κB-p65 remained nuclear for more than 16 hours (Figure 1, A and B). In contrast, the BK channel opener NS1619 had no effect on NF-κB-p65 translocation (Figure 1B); similar results were observed with the vehicle control (dimethylsulfoxide) and another BK channel opener, NS11021 (data not shown).

Figure 1.

Figure 1.

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LPS and the BK channel blocker paxilline induce NF-κB-p65 translocation into the nucleus in hTERT cells. A, Confocal microscopy of hTERT cells detecting NF-kB-p65 (green) after 0, 1, 8, and 16 hours treatment with the indicated compounds. Scale bar = 50 μm. B, Percentage of cells with nuclear NF-kB-p65 after the indicated times of treatment. Data are presented as mean ± SEM from 3 independent experiments. ‡, P < .05 compared with 0 hours of LPS and LPS + paxilline; #, P < .05 compared with 0 hours of LPS, paxilline, and LPS + paxilline; §, P < .05 compared with 0 hours of LPS + paxilline. C, Western blot of nuclear (left) or cytoplasmic (right) proteins in hTERT cells after the indicated treatments. Top 3 blots probed with NF-kB-p65 antibody, bottom left blot probed with histone H3 antibody, and bottom right blot probed with β-actin antibody. Histone H3 and β-actin expression were similar in all 3 treatments. D, Quantitation of NF-kB-p65 Western blots, including treatment with the BK channel opener NS1619, and LPS + NS1619, normalized to histone H3 (nuclear) and β-actin (cytoplasmic), and expressed as the ratio between nuclear and cytoplasmic levels. Data are presented as mean ± SEM from 3 experiments. *, P < .05 compared with the nuclear to cytoplasmic ratio at 0 hours for each treatment.

To confirm our immunofluorescence results, we extracted the nuclear and cytoplasmic fractions of hTERT cells and immunoblotted for NF-κB-p65. Consistent with the immunofluorescence data, NF-κB-p65 was significantly higher in the nuclear than the cytoplasmic fraction after 1 hour of treatment with LPS, paxilline, or LPS plus paxilline (Figure 1, C and D). In cells treated with LPS alone or paxilline alone, NF-κB-p65 was predominantly in the cytoplasmic fraction after 16 hours. Conversely, NF-κB-p65 remained in the nuclear fraction for an extended time in cells treated with LPS plus paxilline. Consistent with immunofluorescence results, the BK channel openers NS1619 (Figure 1D) and NS11021 (data not shown) had no effect on NF-κB-p65 localization.

LPS inhibits BK channel activity in hTERT cells

Given the similar effects of LPS and BK channel blockers on NF-κB-p65 translocation, we assessed whether LPS affected BK channel activity in hTERT cells. We first used the cell-attached patch-clamp configuration, which allows recording of single-channel currents without altering the cytoplasmic content, thus maintaining cellular signaling, to confirm that the channel blocker paxilline and the opener NS1619 acted as expected in hTERT cells. We observed that the single-channel open probability (Po) of the BK channel was highly variable between cells and patches in hTERT cells (Figure 2A). Incubating hTERT cells with 20 μM paxilline suppressed almost all BK channel current (Figure 2B). By contrast, superfusion of cells with 100 nM NS1619 for 5 minutes resulted in a marked increase in BK channel activity (Supplemental Figure 2). However, upon continuous 30-minute treatment with NS1619, channel activity was reduced to control levels.

Figure 2.

Figure 2.

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Effects of LPS and paxilline on BK channel currents. A, Representative cell-attached patch-clamp recordings from control and LPS- or paxilline-treated hTERT cells at a holding potential of 100 mV. Arrowheads indicate closed (c) and open (o) states of the channel. B, Voltage dependence of BK channel activation of control (open circles), LPS-treated (closed circles) and paxilline-treated (open squares) hTERT cells. Symbols are mean ± SEM. Repeated-measures ANOVA: *, P < .01; #, P < .001.

Having confirmed that the BK channel responded as expected in hTERT cells, we examined the effect of LPS. We found that treatment with 10 ng/mL LPS for 1 hour consistently reduced BK channel activity; at a potential of 160 mV, Po was significantly reduced from 0.309 ± 0.15 under control conditions to 0.061 ± 0.019 after treatment with LPS (Figure 2).

TLR4 inhibition does not prevent NF-κB-p65 translocation induced by BK channel inhibition

LPS is recognized by a complex array of extracellular “pattern recognition receptors,” which include the transmembrane receptor TLR4 and extracellular protein MD-2 (14–17). Furthermore, studies in macrophages have suggested that TLR4 and the BK channel are part of a cooperative complex (18). Thus, we wondered whether paxilline-induced NF-κB-p65 nuclear translocation depended on the TLR4 signaling pathway. Application of the TLR4 inhibitor TAK-242 abolished nuclear translocation of NF-κB-p65 in hTERT cells treated with LPS but had no effect on translocation in cells treated with paxilline or LPS plus paxilline (Figure 3). This indicates that TLR4 activity is required for LPS- but not paxilline-induced nuclear translocation of NF-κB-p65.

Figure 3.

Figure 3.

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TAK-242 prevents NF-κB-p65 translocation induced by LPS but not paxilline. A, Confocal microscopy of hTERT cells detecting NF-kB-p65 (green) after 0, 1, 8, and 16 hours treatment with the indicated compounds. Scale bar = 50 μm. B, Percentage of cells with nuclear NF-kB-p65 after 0, 1, 8, and 16 hours of each treatment. Data are presented as mean ± SEM from 3 independent experiments. #, P < .05 compared with 0 hours of TAK-242 + paxilline and TAK-242 + LPS + paxilline; §, P < .05 compared with 0 hours of TAK -242 + LPS + paxilline. TAK, TAK-242.

BK channel inhibition induces nuclear translocation of NF-κB-p65 in uterine myocytes

We next examined whether NF-κB-p65 translocation into the nucleus also occurs in uterine myocytes isolated from nonlabor uterine tissue. Similar to findings in hTERT cells, 1 hour of treatment with LPS alone, paxilline alone, penitrem A alone, or LPS plus paxilline or penitrem A induced NF-κB-p65 translocation into the nucleus in uterine myocytes. However, nuclear translocation occurred less frequently (45% of uterine myocytes vs 70% of hTERT cells), and NF-κB-p65 did not remain nuclear for as long in uterine myocytes as it did in hTERT cells (Figure 4). As in hTERT cells, cotreatment of uterine myocytes with LPS plus paxilline or penitrem A extended the length of time that NF-κB-p65 remained nuclear, but the length of time was shorter (8 hours vs 16 hours). As expected, the BK channel openers NS1619 (Figure 4) and NS11021 (data not shown) and the vehicle control (dimethylsulfoxide; data not shown) had no effect on NF-κB-p65 nuclear translocation in uterine myocytes. We conclude that uterine myocytes and hTERT cells behave similarly with respect to the effects of BK channel block and NF-κB-p65 translocation.

Figure 4.

Figure 4.

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LPS and paxilline induce NF-κB-p65 translocation in uterine myocytes. Percentage of nonlabor uterine myocytes with NF-κB-p65 translocation into the nucleus observed after the indicated treatments for the indicated times. Data are presented as mean ± SEM from 3 independent experiments. #, P < .05 compared with 0 hours of LPS, paxilline, and LPS + paxilline; §, P < .05 compared with 0 hours of LPS + paxilline.

Specificity of the NF-κB-p65 translocation effect

To determine whether inhibition of other K+ channels could induce NF-κB-p65 translocation, we blocked another prominent myometrial channel, the small conductance Ca2+-activated K+ channel protein 3 (SK3). SK3 channel expression in hTERT cells was confirmed by Western blot analysis (data not shown). In contrast to BK inhibition, treatment of hTERT cells with the SK3 blocker apamin had no effect on NF-κB-p65 nuclear translocation (Figure 5). The response to LPS was similar in the presence or absence of apamin, indicating that the SK3 channel does not mediate LPS-induced nuclear translocation of NF-κB-p65.

Figure 5.

Figure 5.

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Apamin, an SK3 channel blocker, has no effect on NF-kB-p65 translocation. A, Confocal microscopy of hTERT cells detecting NF-kB-p65 (green) after the indicated times of treatment with the indicated compounds. Scale bar = 50 μm. B, Percentage of cells with NF-kB-p65 translocation into the nucleus after these same treatments. Data are presented as mean ± SEM from 3 independent experiments. #, P < .05 compared with 0 hours of LPS and LPS + apamin.

We wondered whether other transcription factors translocate into the nucleus in response to BK channel inhibition. As a candidate, we chose the nuclear factor of activated T cells (NFAT), which has been reported to regulate the expression of smooth muscle contractile proteins and ion channels (19). We found that NFAT was not translocated into the nucleus of hTERT cells after treatment with LPS or BK channel blockers or openers (Supplemental Figure 1B). Together, these data indicate that the link between BK channel activity and NF-κB-p65 may be specific to both the channel and the transcription factor.

LPS and BK channel inhibition induce hTERT cells and uterine myocytes to contract

To evaluate whether LPS and paxilline could lead to contraction of hTERT cells and uterine myocytes, we used a collagen gel-based assay. hTERT cells or uterine myocytes were seeded into collagen gel plates, incubated at 37°C for 48 hours, and the extent of contraction of the collagen gel was measured after treatment with LPS or paxilline. After 4 hours, the gel area was decreased by approximately 30% in both hTERT cells and uterine myocytes treated with LPS or paxilline (Figure 6). To assess whether the paxilline-induced contraction depended on nuclear translocation of NF-κB-p65, NF-κB-p65 activation was blocked with Bay 11–7082, an inhibitor of IκB phosphorylation. We first used immunofluorescence microscopy to confirm that Bay11–7082 blocked LPS- and paxilline-induced NF-κB-p65 translocation in hTERT cells (Supplemental Figure 3A). We then tested Bay11–7082 in the contraction assay and found that it completely inhibited cell contraction induced by LPS or paxilline in both hTERT cells and uterine myocytes (Figure 6). The BK channel opener NS1619 had no effect on contraction of hTERT cells and did not inhibit LPS-induced contractions (Supplemental Figure 3, B and C). We conclude that paxilline-induced contraction of hTERT cells requires NF-κB translocation into the nucleus.

Figure 6.

Figure 6.

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LPS and paxilline induce contraction in hTERT cells and uterine myocytes. Contraction of hTERT (A) or uterine myocytes (B) in collagen-gel based contraction assay after 4 hours of the indicated treatments. Scale bar = 5 mm. Mean gel area (cm2) ± SEM of hTERT (C) and primary cells (D) treated as indicated. Data are presented from 4 independent experiments. *, P < .05 compared with nontreated control cells.

COX-2 plays a role in paxilline-induced contractility of hTERT cells

To understand how NF-κB-p65 translocation leads to contraction, we examined the transcript expression of a candidate gene, COX-2. COX-2 is a key enzyme in the synthesis of prostaglandins, which are pivotal to the parturition process, mediating cervical ripening and stimulating myometrial contractions (20). We found that COX-2 mRNA increased more than 5-fold after 8 hours in the presence of LPS, paxilline, or LPS plus paxilline (Figure 7, A and B). As expected, inhibition of IκB phosphorylation with Bay 11–7082 resulted in a 5- to 12-fold reduction in COX-2 mRNA levels in all conditions (Figure 7B). Consistent with the mRNA levels, COX-2 protein levels increased significantly after 1 hour of stimulation with LPS, paxilline, or LPS plus paxilline. By 24 hours, the COX-2 protein levels returned to baseline in both LPS and LPS + paxilline (Figure 7, C and D).

Figure 7.

Figure 7.

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COX-2 expression is increased in the presence of LPS or paxilline, is reduced by inhibition of IκB phosphorylation, and mediates contraction of hTERT cells. A, Levels of COX-2 mRNA transcripts were compared by quantitative RT-PCR after the indicated treatments and time periods (in hours). B, Levels of COX-2 mRNA transcripts were compared after treatment for 8 hours with the indicated compounds. C, Western blot of COX-2 proteins in hTERT cells after the indicated treatments at the indicated time points. D, Quantitation of COX-2 Western blots (C) normalized to β-actin. E, Collagen-gel based contraction assay after 4 hours of the indicated treatments. Scale bar = 5 mm. F. Mean gel area (cm2) of hTERT cells. Data are presented as mean ± SEM from 3 to 4 independent experiments. *, P < .05 compared with 0 hours (A and D), compared with the same treatment without Bay 11–7082 (B) or compared with control (F).

To assess whether COX-2 expression was required for contraction of hTERT cells, we applied the specific COX-2 inhibitor SC-256 to hTERT cells 1 hour prior to LPS and paxilline and measured contraction in the collagen gel-based assay. SC-236 prevented hTERT cell contractions induced by LPS and paxilline (Figure 7, E and F). Taken together, these results suggest that COX-2 expression depends on NF-κB-p65 translocation into the nucleus, and that contractions induced by LPS and paxilline are a result of increased COX-2 activity.

Discussion

Given the prevalence of the BK channel in uterine smooth muscle, and the fact that its expression has been reported to decrease in both the lower and upper uterine segments during late pregnancy and at the onset of labor (21), the BK channel is thought to be a key player in maintaining uterine quiescence throughout pregnancy. One likely scenario is that short-term loss of BK activity leads to a decrease of this buffering current, depolarization of the myometrial cells, an increase in intracellular Ca2+, and therefore an increase in actomyosin contractility. Our results suggest an additional model (Figure 8) in which longer-term decreases in channel activity cause NF-κB-p65 nuclear translocation and activation of inflammatory pathways. This model is supported by several key findings. First, blocking the channel with paxilline or penitrem A resulted in NF-κB translocation into the nucleus to a similar extent as observed in the presence of the bacterial endotoxin LPS. Second, inhibition of IκB kinase with Bay 11–7082 in the presence of LPS or paxilline resulted in reduced NF-κB translocation into the nucleus and abolished collagen-based cell contraction in hTERT and uterine myocytes. This strongly indicates that the action of BK block is through the IκB pathway. Finally, LPS and paxilline inhibit BK channel activity, resulting in NF-κB translocation, increased COX-2 expression, and uterine myocyte contractility (20).

Figure 8.

Figure 8.

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Model of regulation of NF-kB-p65 nuclear translocation by BK channel activity. LPS binds to the TLR4-MD2 complex, resulting in activation of IκB kinase, NF-κB translocation into the nucleus, and increased expression of COX-2. Blocking of the BK channel with paxilline or penitrem A might activate IκB kinase, inducing the release of NF-κB and allowing NF-κB to translocate into the nucleus. Alternatively, blocking the BK channel might directly induce NF-κB translocation into the nucleus or reduce the translocation of NF-κB back to the cytoplasm. Finally, the translocation of NF-κB into the nucleus leads to an increase in the expression of COX-2, resulting in cell contraction.

The mechanism by which inhibition of the BK channel induces NF-κB translocation and synergizes with LPS to retain NF-κB in the nucleus for an extended time period is unknown. We have considered 3 possible explanations and herein suggested that 2 are unlikely. First, our data argue against a model in which NF-κB translocation is a direct effect of the increased intracellular Ca2+ level that occurs as a result of blocking the BK channel and thereby depolarizing the membrane. This model was attractive because multiple signal transduction pathways that require activation of NF-κB are mediated by increases in intracellular Ca2+ (22, 23). For example, in both neurons and airway epithelial cells, NF-κB activity is regulated by a network of transduction cascades that are controlled by intracellular Ca2+ levels (24). However, blocking the SK3 channel, which is known to both depolarize myometrial cells and cause uterine contractions (25), had no effect on NF-κB translocation, indicating that this process may be specific for the BK channel. Additional experiments directly blocking Ca2+ channels in concert with BK channel inhibition are needed to completely rule out intracellular Ca2+ increases as a mechanism underlying NF-κB translocation.

A second possibility we ruled out is that the BK channel couples with the TLR4 receptor, which signals through the NF-κB pathway. Recent investigations have revealed that, upon stimulation with LPS, the TLR4 receptor complex moves to lipid raft domains on the plasma membrane to assemble with other signaling molecules, resulting in the formation of an active signaling complex (26–28). Consistent with the idea that BK couples with TLR4, data in human myometrial smooth muscle cells indicate that some BK channels reside in caveolar lipid rafts (29). In macrophages, studies suggest that the BK channel does not reside in the same cellular compartments as TLR4 in unstimulated cells, but after LPS treatment, translocation of BK channels and TLR4 to similar cellular compartments has been observed (30). However, our data demonstrated that TLR4 was not required for NF-κB-p65 translocation after BK channel inhibition.

The third model we have considered to explain our findings is that LPS and the BK channel function through MAPK or Rho-associated protein kinase (ROCK) pathways. For example, several studies have shown that LPS can activate ERK1/2, c-Jun NH2-terminal kinase, and P38 MAPK pathways in human placenta and fetal membranes (31). Additionally, one study indicated that LPS promotes myocyte contraction via increased ROCK activity (32). Thus, it is feasible that the LPS- and/or paxilline-induced increase in NF-κB translocation and subsequent increases in COX-2 expression are, in part, due to activation of MAPK or Rho/ROCK pathways. Further studies are necessary to elucidate the participation of these pathways in the LPS- and/or paxilline-induced contraction of uterine myocytes.

The role of the BK channel in inflammatory processes has been studied more comprehensively in macrophages, but the data have been conflicting. In 2 studies, the BK channel was shown to participate in LPS-induced macrophage activation by inducing NF-κB and TNF-α activation (4, 30). However, another study showed that TNF-α release and NF-κB signaling did not differ between macrophages derived from BK channel knockout mice and those from wild-type control mice (33), indicating that BK channel activity is not required for NF-κB activation in macrophages. Although the authors of the latter study suggested that the discrepancies may owe to species differences between mouse, human, and rat macrophages, they also urged caution in the use of pharmacologic compounds for studying these processes (33). Our study, of course, is open to this criticism as well, but we argue that the use of multiple channel inhibitors and openers, as well as direct evidence showing suppression of BK channel activity with both LPS and paxilline, alleviates this concern. Additionally, we have demonstrated that the effects are at least somewhat specific to the BK channel and NF-κB, because targeting the SK3 channel had no effect, and the transcription factor NFAT was not translocated into the nucleus upon block of the BK channel. Fully addressing this question will require examining the myometrium of BK-knockout mice with intrauterine infections and assessing whether the effects mimic what is seen in human myometrial cells.

The BK channel is prominent in myometrium, yet block of the channel does not always yield substantial changes in contractile activity (34). This has raised the important question of how activity of this channel is regulated. Several mechanisms have been suggested, including changes in transcript or protein expression (21, 35), alternative splicing (36), association with caveolar domains (37), association with accessory subunits (38), and functional coupling with ß-adrenergic receptors (39, 40). Interestingly, the gene that encodes the BK channel has an NF-κB binding site in the 5′-flanking sequence of the promoter (41); thus, we envisioned that we would see an increase in BK channel expression in the presence of LPS. However, we did not detect a change in BK protein expression in any of our experiments (data not shown), suggesting that NF-κB may not play a significant role in BK channel expression in myometrial cells.

It is clear that multiple signaling pathways that trigger both term and preterm labor interface with ion channels to dynamically regulate the status of uterine contraction. The inflammatory NF-κB pathway that is affected by blocking BK activity is important not only in the onset of term labor, but also in preterm labor. NF-κB is activated in response to infection and proinflammatory cytokines, and thus it is speculated that premature activation of NF-κB contributes to preterm birth (42). Understanding ionic mechanisms that regulate the transition from a quiescent to a contractile state during pregnancy will aid in establishing a biological basis for therapies capable of modulating uterine excitability.

Acknowledgments

We thank Dr Jennifer Condon for the hTERT cells and helpful comments on the manuscript and Drs Bin Cao and Deborah Frank for editorial comments.

S.E. is supported by the National Institutes of Health (Grant 5R01HD037831–11) and by the March of Dimes (Grant 21-FY12–133).

Disclosure Summary: None of the authors have a conflict of interest.

Footnotes

Abbreviations:
COX-2
cyclooxygenase-2
FBS
fetal bovine serum
hTERT
human telomerase reverse transcriptase
LPS
lipopolysaccharide
NFAT
nuclear factor of activated T cells
NF-κB
nuclear transcription factor-κB
ROCK
Rho-associated protein kinase.

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