Mol. Hum. Reprod. Advance Access originally published online on January 16, 2007
Molecular Human Reproduction 2007 13(3):181-187*; doi:10.1093/molehr/gal113
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Involvement of nuclear factor-kB activation through RhoA/Rho-kinase pathway in LPS-induced IL-8 production in human cervical stromal cells
Osaka University Graduate School of Medicine, Department of Obstetrics and Gynecology, Osaka, Japan
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: taharam{at}gyne.med.osaka-u.ac.jp
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Abstract |
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Interleukin-8 (IL-8) is a chemokine that recruits and activates neutrophils in stromal tissue and plays an essential role in cervical ripening. Nuclear factor-kB (NF-kB) is known to be important for the up-regulation of IL-8 gene expression. We examined the molecular mechanisms responsible for NF-kB activation in IL-8 production in cervical stromal cells. Lipopolysaccharide (LPS) and IL-1ß stimulated IL-8 production by cervical stromal cells in a dose-dependent manner. Pretreatment of cervical stromal cells with inhibitors of RhoA (C3 transferase exoenzyme), Rho-kinase (Y-27632) or NF-kB (BAY11-7082) effectively blocked LPS-induced IL-8 release. In contrast, IL-1ß-induced IL-8 production was significantly blocked by BAY11-7082, but not by C3 transferase exoenzyme or Y-27632. Pull-down assays showed that LPS activated RhoA, but IL-1ß caused only a lower level of activation. Transfection of the cervical stromal cells with RhoA small interfering RNA (siRNA) inhibited LPS-stimulated IL-8 production, whereas IL-1ß-induced IL-8 production was not significantly inhibited by knockdown of RhoA with siRNA. Using an NF-kB transcription reporter vector, luciferase assays demonstrated that incubation with LPS or IL-1ß induced the activation of NF-kB in cervical stromal cells. Activation of NF-kB by LPS was inhibited by treatment with C3 exoenzyme, Y-27632 or RhoA siRNA. However, inhibition of the RhoA/Rho-kinase pathway did not attenuate the activation of NF-kB by IL-1ß. These results suggest that LPS-induced IL-8 production is accompanied by enhanced NF-kB activation through the RhoA/Rho-kinase pathway in human cervical cells.
Key words: cervix/IL-8/RhoA/Rho-kinase/lipopolysaccharide/IL-1ß
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Introduction |
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The uterine cervix becomes softer and dilates as the uterine contractions increase during labour. This cervical change is called ‘cervical ripening’ and is considered to be an inflammatory-like phenomenon (Junqueira et al., 1980). Interleukin-8 (IL-8) belongs to the CXC chemokine family (Baggiolini et al., 1995; Mukaida et al., 1998) and plays an essential role in the mechanisms controlling the cervical ripening process (Osmers et al., 1995; Murphy, 1997; Winkler et al., 1998). Although cervical ripening is required for appropriate progress of delivery of the fetus, the cervix often dilates prematurely in the case of preterm labour.
Recent evidence suggests that intrauterine infection with gram-negative organisms plays a key role in the pathogenesis of preterm delivery (Romero et al., 1987). One cell-surface component from gram-negative organisms is lipopolysaccharide (LPS), which stimulates the synthesis and release of proinflammatory cytokines such as IL-6 and IL-8 from monocytes and macrophages (Beutler and Rietschel, 2003). There is increasing evidence that IL-1ß also regulates the cervical secretion of IL-8 (El-Maradny et al., 1995; Winkler et al., 1998). Inflammatory responses are now thought to be mediated by the activation of the transcription factor nuclear factor-kB (NF-kB), which is a key regulator of inflammatory cytokine gene transcription (Barnes and Karin, 1997). LPS and IL-1ß are known to activate NF-kB in various cells (Muller et al., 1993; Hayden and Ghosh, 2004). However, the molecular mechanisms responsible for IL-8 production in cervical cells remain unknown.
Monomeric GTP-binding proteins of the Rho family, including RhoA, have been shown to regulate the actin cytoskeleton, cell motility and smooth muscle contraction (Ridley and Hall, 1992; Mackay and Hall, 1998). These cellular functions of RhoA are largely dependent on the activation of its downstream effector, Rho-kinase (Fukuta et al., 2001). The RhoA/Rho-kinase cascade has been reported to be involved in uterine smooth muscle contraction (Moran et al., 2002; Tahara et al., 2002). We previously reported that a Rho-kinase inhibitor prevents preterm delivery in a mouse model (Tahara et al., 2005). Besides their roles in cytoskeletal regulation, RhoGTPases may also have potentially important roles in the cytokine production (Hippenstiel et al., 2000; Zhao et al., 2002; Zhao and Pothoulakis, 2003; Zhao et al., 2003). Moreover, it has been demonstrated that RhoA/Rho-kinase can activate NF-kB (Perona et al., 1997; Van Aelst and D'Souza-Schorey, 1997; Montaner et al., 1998), suggesting a potential role of the RhoA/Rho-kinase cascade in inflammation. Therefore, we speculated that, in addition to its effect on uterine contraction, the Rho/Rho-kinase pathway may exert its effect on preterm delivery through regulating IL-8 production in the cervix. However, the function of RhoGTPases in cytokine production in the cervix has not been previously addressed.
The aim of this study was to determine the role of RhoGTPase in the regulation of NF-kB activation and IL-8 production in primary human cervical cell culture. In this study, we performed western blot analysis and luciferase assays to examine the molecular mechanisms responsible for the NF-kB activation during the induction of IL-8. Our findings indicate that NF-kB activation through RhoA activity is necessary for LPS-induced IL-8 production in cervical stromal cells.
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Materials and methods |
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Reagents
The following reagents were obtained commercially: Dulbecco's modified Eagle medium, penicillin and streptomycin (GIBCO BRL, Rockville, MD, USA); fetal bovine serum (CANSERA Interenational, Inc., Ontario, Canada); recombinant human IL-1ß (R&D Systems, Inc., Minneapolis, MN, USA); Y-27632 (Calbiochem-Novabiochem Corp., San Diego, CA, USA); BAY11-7082 (BIOMOL International, L.P., Plymouth Meeting, PA, USA); LPS (Sigma-Aldrich, St Louis, MO, USA); Clostridium botulinum C3 transferase exoenzyme, monoclonal antibody against RhoA and Rhotekin Rho binding domain (RBD) agarose (Upstate Biotechnology, Lake Placid, NY, USA); anti-IkB
(sc-371; epitope C terminus) and anti-phospho-IkB (sc-8404; epitope, p-Ser-32) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); goat anti-mouse IgG, Horse radish peroxidase-conjugated affinity purified (CHEMICON International, Inc., Temecula, CA, USA); Enhanced chemiluminescent (ECL) detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA); Lipofectamine plus reagent, Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA); Pica Gene (Toyo B-Net, Tokyo, Japan); NF-kB transcription reporter vector (Clontech Laboratories, Inc., Mountain View, CA, USA); PSV-ß-galactosidase control vector (Promega Corporation, Madison, WI, USA); human IL-8 enzyme-linked immunosorbent kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Other reagents were of analytic reagent grade.
Preparation of cultured human uterine cervical stromal cells
Cervical stromal tissues were obtained from gynaecologic surgical specimens from non-pregnant women (n = 7, age 33–44 years). Hysterectomy was performed for benign indications: uterine myoma (n = 7). Ethical approval for the collection of the tissues was obtained from the Osaka University Hospital ethics committee. Primary cultures of the cervical stromal fibroblasts were established as previously described (Ogawa et al., 1998) with slight modification. Briefly, the cervical stromal tissues were rinsed several times in 10 mM phosphate-buffered saline and minced into 
2 mm pieces. Then, the cervical tissues were digested with 0.25% trypsin at 37°C for 20 min, and the supernatant was filtered through nylon mesh (NBC Industries Co., Tokyo, Japan). The dispersed fibroblasts were cultured in a flask in phenol red free-DMEM containing 10% fetal bovine serum (charcoal stripped), penicillin and streptomycin at 37°C in an atmosphere of 5% CO2 and 95% air, and the culture medium was changed every 2 days. The immunofluorescence study showed 99% positive staining for vimentin in cervical fibroblast cells at the sixth passage and <1% positive staining for cytokeratin (data not shown), indicating a high purity of cervical fibroblast cells. Thus, the cervical stromal cells at the fifth–sixth passage were confirmed to be of high purity and used in the further experiments in this study.
Enzyme-linked immunosorbent assay for IL-8 measurements
IL-8 levels in conditioned medium from cervical stromal cells were measured with an enzyme-linked immunosorbent assay. After the cells had grown to confluence, cells (1x 106) were cultured in six-well plates with 1 ml of serum-free medium. Then, various concentrations of LPS (1–100 µg ml–1) and recombinant human IL-1ß (1–10 ng ml–1) were added to the culture medium in each well. After culturing in the presence of these agents, the media were collected and used for ELISA. For the control experiments, the same volume of phosphate-buffered saline solution was added to each well. To inhibit RhoA activity, cells were pretreated with C3 exoenzyme at various concentrations for 24 h before stimulation with LPS or IL-1ß. To inhibit Rho-kinase or NF-kB activity, cells were pretreated with Y-27632 or BAY11-7082 at various concentrations for 1 h before stimulation with LPS or IL-1ß. The conditioned media were collected and centrifuged at 18 000 g for 5 min. Then, secreted IL-8 was measured by ELISA using a commercial kit according to the manufacturer's protocol. The quantities of secreted IL-8 in the test samples were determined using a standard curve generated with purified recombinant human IL-8 provided with the kit. The sensitivity of the IL-8 assay was 2–1000 pg mL–1
RhoA activation assay
RhoA activity was determined as described previously (Sawada et al., 2002). This pull-down assay uses the RBD from the effector protein rhotekin as a probe to specifically isolate the active forms of RhoA. Briefly, cervical stromal cells (1 x 106) were cultured in 100 mm culture dishes with complete medium (DMEM with 10% FBS and antibiotics) until they reached 90% confluence. After 24 h of starvation, cells were stimulated with LPS (10 µg ml–1) or IL-1ß (1 ng ml–1) for 5 min and then lysed in lysis buffer: 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol and 2 µg ml–1 each of leupeptin and aprotinin, 1 µl ml–1 phenylmethylsulphonyl fluoride and 0.5 µl ml–1 DTT. The cellular protein was harvested by scraping with a rubber policeman. Lysates were centrifuged at 4°C at x 14 000 g for 5 min to remove particulate material, and the protein concentrations were determined by the Bradford assay. Twenty-five micrograms of each protein extract were incubated while rotating at 4°C for 3 h with an equal volume of Rhotekin-RBD (RhoA-binding domain of the RhoA effector Rhotekin) bound to glutathione-agarose beads. The beads were washed three times with washing buffer (Sawada et al., 2002), and activated RhoA bound to the beads or total RhoA in cell extracts was detected by western blot using a monoclonal antibody against RhoA.
IkB phosphorylation
Cervical stromal cells were plated in a 100 mm dish, and cultures were allowed to proliferate until they reached confluence, with an exchange of the medium containing 10% FBS every 48 h. The cells were starved for 24 h in serum-free medium. Then, LPS (10 µg ml–1) or IL-1ß (1 ng ml–1) was added to the medium, followed by incubation for 20 min. The incubation was terminated by aspiration of the medium, two washes with ice-cold PBS, and the addition of 80 µl of lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol and 2 µg ml–1 each of leupeptin and aprotinin, 1 µl ml–1 phenylmethylsulphonyl fluoride and 0.5 µl ml–1 DTT). Equal quantities of cell lysates (50 µg of protein) were separated by electrophoresis on a 10% gradient polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, USA). Western blot was performed with mouse polyclonal antibodies (Abs) against IkB and phosphorylated-IkB (p-IkB). Immunoreactive bands were visualized with horseradish peroxidase-conjugated secondary Ab and subsequent ECL detection. To examine the effects of Rho/Rho-kinase inhibitor on IkB phosphorylation, cells were pretreated or not with Y-27632 (10 µM) for 10 min at 37°C before stimulation with LPS or IL-1ß.
Transfection and NF-kB activity luciferase reporter assay
To assay the level of NF-kB-dependent transcriptional activity in the cervical stromal cells, the luciferase reporter assay was used as described previously (Hodge et al., 2003). The pNF-kB-Luc reporter plasmid and the PSV-ß-galactosidase control vector transfections were performed using the lipofectamine plus reagent-mediated transfection procedure as recommended by the manufacturer. Transfected cells were serum-starved for 24 h, followed by stimulation with LPS or IL-1ß with or without Y-27632 or BAY11-7082 pretreatment. Cytoplasmic extracts of the cells were prepared and analysed for luciferase activity using Pica Gene. All experiments were repeated five times. Each result was expressed as the luciferase activity (luciferase activity/ß-galactosidase activity) relative to that in the unstimulated state.
siRNA transfection
The small interfering RNA (siRNA) oligonucleotides were custom constructed by QIAGEN Corp. The sequences of the siRNA templates were: RhoA-siRNA, 5-TAC CCA GAT ACC GAT GTT ATA-3; and for scrambled siRNA, 5-AAC TGC ATT GAA AGG CAG TCG-3. Cells were grown on 60 mm dishes to 50% confluency with complete medium and transfected with 4 µg of siRNA using Lipofectamine 2000 according to the manufacturer's recommended procedure. Efficiency of knockdown by siRNA was assessed by real-time PCR and western blot analysis. After 24 h, the culture medium was changed to serum-free DMEM and the cells were incubated for 24 h and then stimulated with LPS or IL-1ß for 8 h. Then, the media were collected and used for ELISA, and cell extracts were prepared and assayed for luciferase activity using PicaGene. All experiments were repeated five times.
Statistical analysis
The data are expressed as the mean ± SE. Results were evaluated by one-way ANOVA, followed by Fisher's protected least significant difference test. A P-value of <0.05 was considered statistically significant.
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Results |
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Effects of LPS and IL-1ß on IL-8 production in cervical stromal cells
We first investigated the effects of LPS and IL-1ß on IL-8 production by cervical stromal cells. The cells were treated with LPS or IL-1ß for various times, and IL-8 was measured in the conditioned media by ELISA. The results showed that LPS stimulated IL-8 production in cervical stromal cells in a time- and dose-dependent manner (Figure 1A). Incubation with IL-1ß also significantly elevated IL-8 production in cultured human uterine cervical stromal cells (Figure 1B). Because the average level of IL-8 production induced by IL-1ß at 1 ng ml–1 was similar to that induced by LPS at 10 µg ml–1 in the cells, we used 10 µg ml–1 of LPS and 1 ng ml–1 of IL-1ß in the following experiments. As shown in Figure 1, IL-8 production in response to LPS or IL-1ß was significantly enhanced after 6 h. The level of IL-8 increased up to 12 h, then seems to reach a plateau phase. Therefore, we chose 8 h after stimulation to evaluate the effects of inhibitors in the following experiments.
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Effects of Rho/Rho-kinase and NF-kB inhibitor on IL-8 release
C3 transferase exoenzyme is an exotoxin produced by C. botulinum that specifically inhibits the Rho small GTP binding proteins (RhoA, RhoB, and RhoC) (Aktorirs, 1997), and Y-27632 specifically inhibits Rho-kinase (Uehata et al., 1997). We pretreated human cervical stromal cells with C3 transferase exoenzyme or Y-27632 to determine whether the Rho/Rho-kinase pathway is involved in LPS-induced IL-8 production. Preincubation of cervical stromal cells with 5 µg ml–1 C3 transferase exoenzyme significantly attenuated LPS-stimulated IL-8 production (Figure 2). This concentration was selected from a dose range as a concentration that reproducibly inhibited IL-8 production (data not shown). A significant decrease in LPS-stimulated IL-8 production was also observed in the Y-27632-pretreated cells (Figure 2). IL-8 production was reduced dose-dependently by Y-27632 (1–100 µM; data not shown) with the maximal inhibitory action obtained at 10 µM Y-27632. Pretreatment with C3 transferase exoenzyme or Y-27632 did not affect cell viability. These results suggest that the RhoA/Rho-kinase cascade is involved in LPS-induced IL-8 production.
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Next, we examined the effects of these inbibitors on IL-1ß-induced IL-8 production. Rho inhibition with C3 transferase exoenzyme did not decrease the induction of IL-8 production by IL-1ß (Figure 3). In addition, although we detected a small decrease of the induction of IL-8 production by IL-1ß after preincubation with Y-27632, there was no significant reduction even with 10 µM Y-27632.
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We also investigated the involvement of NF-kB in IL-8 production by cervical stromal cells. As shown in Figure 2, the LPS-induced augmentation of IL-8 production was significantly suppressed by an inhibitor of NF-kB, BAY11-7082 (Mori et al., 2002). Similarly, treatment with BAY11-7082 suppressed the IL-1ß-induced augmentation of IL-8 release (Figure 3). Pretreatment with BAY11-7082 did not affect cell viability (data not shown). These results confirm that NF-kB activation is involved in LPS- and IL-1ß-induced IL-8 production.
Rho activation by LPS and IL-1ß
To assess the ability of LPS to activate Rho GTPase in cervical stromal cells, RhoA activity in the cells was assessed by a pull-down assay (Ren et al., 1999). Cells were stimulated with LPS (10 µg ml–1), total cell lysates were incubated with RBD-glutathione agarose beads, and western blot of the bound proteins was performed as described in Materials and Methods. As shown in Figure 4, the level of active GTP-bound RhoA was higher in cells treated with LPS compared with control cells. Western blot analysis of unstimulated and LPS-stimulated cervical stromal cell lysates showed that total RhoA levels were equivalent between these two samples (Figure 4). LPS induced RhoA activation within 5 min, and RhoA activity peaked at 10 min (data not shown). In contrast, no RhoA was detected bound to the beads alone. When the cells were treated with IL-1ß (1 ng ml–1), we observed a much smaller increase in RhoA activation compared with control cells (Figure 4).
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Effect of RhoA knockdown by siRNA on IL-8 production
C3 exoenzyme inhibits all three isoforms of Rho (RhoA, RhoB and RhoC). In order to specifically inhibit RhoA and thereby to clarify its function in IL-8 induction, we employed RNA interference for selective down-modulation of RhoA. The reduction of RhoA mRNA was confirmed by quantitative real-time PCR (84% reduction compared to control; data not shown). As shown in Figure 5, western blot analysis showed a marked and specific reduction of RhoA protein expression in cells transfected with RhoA siRNA. When cells were transfected with RhoA siRNA, IL-8 production in response to LPS was significantly reduced compared with that in cells treated with a scrambled siRNA sequence (Figure 6). In contrast, we observed only a small, but not significant, reduction in IL-8 production when cells were transfected with the RhoA-specific siRNA and stimulated with IL-1ß (Figure 6).
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Effects of LPS and IL-1ß on IkB
phosphorylationNF-kB is normally found in its inactive form bound to an inhibitory protein known as IkB
in the cytoplasm (Henkel et al., 1993). NF-kB activation occurs through the phosphorylation of IkB, followed by the degradation of IkB by the proteasome and NF-kB nuclear translocation (Zingarelli et al., 2003; Hayden and Ghosh, 2004). To examine the activation of NF-kB by LPS in cervical stromal cells, western blot analysis was performed using an antibody for p-IkB. Within 10 min of stimulation with LPS, p-IkB was clearly observed (Figure 7). We also examined the effect of IL-1ß on the phosphorylation of IkB. As shown in Figure 7, IL-1ß increased the level of p-IkB.
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Effect of LPS and IL-1ß on NF-kB-dependent transcriptional activity
To further examine the level of NF-kB-dependent transcriptional activity in the cervical stromal cells, the luciferase reporter assay was used as described previously (Lindholm et al., 2001). After the cells were transfected with the pNF-kB-Luc reporter plasmid, they were stimulated with LPS or IL-1ß. As shown in Figure 8A and B, the luciferase activity in the cell extracts showed that both LPS and IL-1ß significantly stimulated NF-kB-dependent transcriptional activity.
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To elucidate the intracellular mechanisms of NF-kB activation, we examined the effect of C3 exoenzyme or Y-27632 on the activation of NF-kB induced by LPS or IL-1ß in cervical stromal cells. After a 48 h incubation in normal culture medium, the cells were stimulated and then harvested. When C3 exoenzyme- or Y-27632-treated cell cultures were stimulated with LPS (10 µg ml–1), we observed a significant decrease of NF-kB-dependent transcriptional activity (Figure 8A). In contrast, when C3 exoenzyme- or Y-27632-treated cells were stimulated with IL-1ß (1 ng ml–1), we observed only a small and insignificant decrease of NF-kB-dependent transcriptional activity (Figure 8B).
To confirm the involvement of RhoA in NF-kB activation by LPS, the cells were co-transfected with siRNA. When cells were co-transfected with scrambled siRNA, NF-kB activity was significantly enhanced by LPS or IL-1ß. However, LPS-induced NF-kB activity was significantly reduced when cells were co-transfected with RhoA siRNA (Figure 9). By contrast, the activation of NF-kB by IL-1ß was not significantly inhibited by knockdown of RhoA with specific siRNA (Figure 9).
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Discussion |
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IL-8 plays an essential role in the mechanisms controlling the cervical ripening process (Osmers et al., 1995; Murphy, 1997; Winkler et al., 1998). In the present study, we showed that RhoA is activated by LPS in cervical stromal cells, and that inhibition of RhoA or Rho-kinase inhibits LPS-induced IL-8 production. This study provides the first evidence for a role of a RhoA-dependent signalling pathway in the IL-8 production of uterine cervical cells.
NF-kB is known to be important for the up-regulation of IL-8 gene expression, and the promoter region of the IL-8 gene contains binding sites for NF-kB (Roebuck, 1999). Using an NF-kB inhibitor and luciferase activity assay, we demonstrated here that NF-kB is involved in IL-8 up-regulation by LPS and IL-1ß (Figures 2 and 3), and that both LPS and IL-1ß activate NF-kB in cervical stromal cells (Figure 7). As the molecular mechanisms responsible for the activation of NF-kB leading to enhanced IL-8 production in cervical stromal cells remain unknown, we tried to clarify whether RhoGTPase is involved in IL-8 production in the cervix. Here, we showed that inhibition of RhoA/Rho-kinase can attenuate the LPS-induced NF-kB activity (Figures 8A and 9), suggesting that LPS-mediated IL-8 synthesis results, at least in part, from RhoA/Rho-kinase pathway-dependent NF-kB activation.
As for the involvement of RhoA in IL-1ß signal transduction, Singh et al. (1999) demonstrated that IL-1ß stimulated actin stress fiber formation in HeLa cells in a Rho-dependent manner. However, we showed here that IL-1ß only slightly induced RhoA activation in cervical stromal cells (Figure 4). As shown in Figure 3, the IL-1ß-induced IL-8 production was not significantly inhibited by C3 exoenzyme or Y-27632. Thus, RhoA may not play a major role in downstream signalling of IL-1ß-induced IL-8 production. It is possible that other signal transduction factors, such as MAPK, could be involved because MAPK inhibitor attenuated the IL-1ß-induced IL-8 production in cervical stromal cells (unpublished data). Future studies should be performed to address this possibility.
The modulation of cellular functions by RhoA is largely dependent on the activation of its downstream effector, Rho-kinase (Ridley and Hall, 1992; Fukuta et al., 2001). Our data provide the first evidence that Rho-kinase plays an important role in IL-8 production in the cervix. The inhibitory effect of Y-27632 on NF-kB activation was associated with the reduction of IL-8 production. Considering that the role of NF-kB activation in cervical ripening is well documented (Stjernholm-Vladic et al., 2004), inhibition of the NF-kB signalling pathway could be an effective strategy for blocking the inflammatory process. The present results suggest that the specific Rho-kinase inhibitor could inhibit cervical IL-8 production through inhibition of the NF-kB signalling pathway. Rho-kinase inhibitors are very promising clinically, and clinical application for the treatment of asthma, glaucoma, hypertension, and cancer metastasis is now under investigation (Zhao and Pothoulakis, 2003). In addition to being useful for inhibiting uterine contractility (Moran et al., 2002; Tahara et al., 2002), Rho-kinase inhibitors may thus be useful for preventing cervical ripening.
Labour is the physiological process for delivering a fetus and is defined as regular uterine contractions accompanied by cervical effacement and dilation (Norwitz et al., 1999). Cervical ripening involves biochemical changes in the cervical connective tissue and usually precedes uterine contractions and cervical dilation for normal term labour (Norwitz et al., 1999). However, the cervix often dilates prematurely, and premature ripening occurs as part of preterm delivery (Olah and Gee, 1992). Preterm birth is defined as delivery before 37-week gestation and is the most common obstetric complication (Norwitz et al., 1999; Newton, 2005). Accumulating evidence clearly indicates that we do not have effective methods for prevention of preterm birth (King, 2004); current therapy to use antibiotics or suppress uterine contractions with tocolytics has not decreased the rate of preterm delivery or perinatal mortality (Huddleston et al., 2003; Newton, 2005). As the cervix may be an important barrier against ascending infection (Shennan and Jones, 2004), prevention of cervical ripening has the potential to contribute to the integrity of the fetal membranes and decrease the risk of premature rupture of the membranes. Therefore, one of the keys to treating preterm labour would be regulation of cervical ripening. Whether application of Rho-kinase inhibitors in vivo may delay cervical ripening and prevent preterm delivery should be determined.
As cervical cells used in the present study were obtained from non-pregnant women, it remains possible that cervical cells in the pregnant uterus may respond differently. For example, there are dramatic changes in the expression of a range of genes within the uterus during pregnancy (Rehman et al., 2003, Havelock et al., 2005). Future studies designed to assess the cellular response using cervical cells from pregnant cervixes are indicated. Another possibility is that cultured cervical cells may alter their signal transduction system during culture in vitro. Thus, it should be noted that the present study does not provide direct evidence of IL-8 production in the uterine cervix at term. These issues should be addressed in future studies.
In summary, we have shown that LPS activates RhoA and consequently increases NF-kB activity in cervical stromal cells. Our findings suggest that NF-kB activation through RhoA/Rho-kinase cascade may play an important role in cervical ripening through modulation of IL-8 production.
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Acknowledgement |
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This work was supported in part by Grants-in-Aid for Scientific Research (No. 14571559 and 16591657 to M.T.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan).
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Footnotes |
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* N.B. An error was made in the initial online pagination of Molecular Human Reproduction 13/3. The page span of this article was originally shown as 41–47. The publisher wishes to apologise for this error.
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Submitted on October 24, 2006; resubmitted on November 20, 2006; accepted on November 30, 2006.
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