Prostacyclin primes pregnant human myometrium for an enhanced contractile response in parturition

Abstract

An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI2), a smooth muscle relaxant. However, here we have shown that activation of PGI2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI2-mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor.

Figure 1. Iloprost enhances oxytocin-induced contractility in human myometrial tissue.

Human myometrial tissue strips were suspended vertically under 400 mg tension in DMEM plus 10% FBS. Tissue was treated with vehicle (control) or 25 nmol/l iloprost for 48 h, then transferred to an isometric muscle bath and treated with 5 nmol/l oxytocin as described in Methods. Average peak contraction force in response to oxytocin in paired tissue strips is plotted from 7 independent experiments. Arithmetic mean values are represented by bars, and individual measurements are plotted for the 7 patient samples. *P < 0.05 versus control, Mann-Whitney U test.

Figure 2. Iloprost upregulates contractile apparatus protein and mRNA expression in human myometrial tissue.

(A and B) Human myometrial tissue strips in organ culture were pretreated with 25 nmol/l iloprost for (A) 48 h or (B) 6 h. After contraction assay with 5 nmol/l oxytocin, protein was isolated from the tissue as described in Methods and subjected to Western blot analysis with antibodies to SM2-MHC (A and B) and calponin (A). GAPDH served as a loading control. (C and D) Protein was isolated from human myometrial tissue in organ culture treated with vehicle or 25 nmol/l iloprost for (C) 48 h or (D) 6 h. Cell lysates were subjected to Western blot analysis with antibodies to SM2-MHC and calponin (C and D) as well as α-SMA and h-caldesmon (C). (E) Human myometrial tissue strips in organ culture were treated with vehicle or 25 nmol/l iloprost for 48 h. Total RNA was isolated as described in Methods and subjected to semiquantitative RT-PCR using primers to basic calponin, SM-MHC, and PDH housekeeping gene. Lanes were run on the same gel but were noncontiguous (lines). Data in A–E are from 5 individual patients.

Figure 3. Iloprost increases connexin 43 expression.

Protein was isolated from human myometrial tissue in organ culture treated with vehicle or 25 nmol/l iloprost for 48 h. Left: Cell lysates were subjected to Western blot analysis with antibodies to connexin 43 (Cx43) and GAPDH. A representative blot is shown. Right: Densitometric quantitation from 5 independent experiments on samples from 5 different patients. Because connexin 43 is known to migrate as a doublet or triplet spanning the 39- to 44-kDa range, we summed the band intensities for both bands for each sample and corrected the values to the corresponding GAPDH. Results are mean ± SEM. *P < 0.05, paired 1-tailed Student’s t test.

Figure 4. An IP antagonist opposes iloprost-enhanced oxytocin-induced contractions and iloprost-induced contractile protein expression.

(A) Human myometrial tissue in organ culture was pretreated with 90 μmol/l RO3244794 (RO) for 30 min and then stimulated with or without 25 nmol/l iloprost for 48 h, after which tissue was transferred to an isometric muscle bath and treated with 5 nmol/l oxytocin as described in Methods. Average peak contraction data are represented as fold change relative to vehicle control. Arithmetic mean values are represented by bars, and individual measurements are plotted for the 3 patients. (B) Representative tracings for 1 patient in A. (C) Homogenates of uterine tissue isolated from A were subjected to Western blot analysis with antibodies to h-caldesmon, connexin 43, GAPDH, or β-tubulin. (D) Human myometrial tissue in organ culture was pretreated with 90 μmol/l RO3244794 for 30 min and then treated with vehicle or 25 nmol/l iloprost for 48 h, followed by treatment with 5 nmol/l oxytocin for 8 h, as indicated. Protein was isolated from the tissue and subjected to Western blot analysis with antibodies to SM2-MHC or GAPDH. Lanes were run on the same gel but were noncontiguous (lines).

Figure 5. Iloprost induces SM-MHC and calponin message in uSMC culture.

(A) Human uSMCs were treated with the indicated concentrations of iloprost for 6 h. Total RNA was isolated and subjected to RT-PCR using primers to SM-MHC and PDH. (B and C) Total RNA was isolated from cells treated with vehicle (Veh) or 2.5 nmol/l iloprost for the indicated times, and RT-PCR was performed as in A. (D) Human uSMCs were treated with vehicle or 2.5 nmol/l iloprost for 4 h, followed by treatment with 1 μg/ml actinomycin D for an additional 4 h. Total RNA was isolated and subjected to RT-PCR using primers to SM-MHC or PDH. Representative gels are shown, as well as densitometric quantitation of 2 separate experiments below. Arithmetic mean values represent fold induction corrected to PDH. Data in A–D were from cell cultures derived from 3 different patients. Lanes in C and D were run on the same gel but were noncontiguous (lines).

Figure 6. Iloprost induces expression of contractile apparatus proteins in human uSMCs.

(A) Cell lysates from human uSMCs treated with the indicated concentrations of iloprost for 8 h were subjected to Western blot analysis with antibodies to α-SMA, calponin, h-caldesmon, and SM2-MHC, or β-tubulin as a loading control. (B–D) Human uSMCs were treated with vehicle or 2.5 nmol/l iloprost for the indicated times. Cell lysates were subjected to Western blot analysis with antibodies to (B) calponin, (C) h-caldesmon, and (D) SM2-MHC; shown is 1 representative immunoblot for each antibody and quantified protein expression, corrected to β-tubulin and presented as fold induction in order to average data from multiple experiments. n is indicated for each time point. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle, 1-way ANOVA with Newman-Keuls post-hoc test. Data are mean ± SEM (where n ≥ 3) and were generated from cell cultures from 2 patients.

Figure 7. Iloprost induces expression of connexin 43 in human uSMCs.

(A) Human uSMCs were treated with vehicle or 2.5 nmol/l iloprost for the indicated times. Total RNA was isolated and subjected to RT-PCR using primers against connexin 43 and PDH. Lanes were run on the same gel but were noncontiguous (lines). (B) Cell lysates from human uSMCs treated with 2.5 nmol/l iloprost for the indicated times were subjected to Western blot analysis with antibodies to connexin 43 and GAPDH.

Figure 8. Iloprost induces cAMP/PKA activity in human uSMCs.

(A) Concentration-response curves were determined by treating uSMCs with concentrations of iloprost ranging from 10 pmol/l to 1 μmol/l. After 20 min, cells were harvested, and cAMP was measured as described in Methods. Results are mean ± SEM. EC₅₀ was determined from best-fit curve with nonlinear regression. (B) Human uSMCs were treated with vehicle or the indicated concentrations of iloprost for 20 min, and cell lysates were analyzed for PKA activity as described in Methods. A representative gel is shown. Fluorescence units of phosphorylated kemptide (p-Kemptide) were quantified by densitometry and expressed as fold induction. n = 5 separate experiments. (C) Human uSMCs were treated with 2.5 nmol/l iloprost for the indicated times, and cells were harvested and analyzed for PKA activity.

Figure 9. cAMP is sufficient to induce contractile protein expression.

(A) Human uSMCs were treated with vehicle, 2.5 nmol/l iloprost, or 0.5 μmol/l 8-Br-cAMP for 20 min, and cells were harvested and analyzed for PKA activity. Shown are a representative gel showing phosphorylated kemptide and densitometric quantitation of 2 independent experiments expressed as fold induction. Arithmetic mean values are shown. (B) Total RNA was isolated from human uSMCs at time point 0, treated with vehicle for 7.5 h, or treated with 0.5 μmol/l 8-Br-cAMP for the indicated times. RNA was subjected to RT-PCR using primers to the basic calponin, SM-MHC, or PDH genes. (C) Human uSMCs were treated with the indicated concentrations of 8-Br-cAMP for 8 h, and cell lysates were subjected to Western blot analysis with antibodies to α-SMA, calponin, h-caldesmon, SM2-MHC, and GAPDH. (D) Human uSMCs treated with vehicle or 0.5 μmol/l 8-Br-cAMP for the indicated times were harvested and subjected to Western blot analysis with antibodies to calponin, h-caldesmon, SM2-MHC, and GAPDH.

Figure 10. PKA is required for iloprost-induced contractile protein and connexin 43 expression.

(A) Human uSMCs were transiently transfected with siRNA to PKA Cα and PKA Cβ (siPKA) or negative control siRNA, siControl (siCont). At 40 h after transfection, cells were treated with vehicle or 2.5 nmol/l iloprost for 8 h, harvested, and subjected to Western blot analysis for PKA Cα and PKA Cβ. Representative blots are shown. Amounts of PKA Cα and PKA Cβ proteins relative to GAPDH were quantified by densitometry (n = 7, including cell cultures derived from 2 different patient samples). ***P < 0.001 versus vehicle- and iloprost-treated siControl. (B) Human uSMCs were transfected with PKA Cα and PKA Cβ (0.5 μg each) or siControl for 48 h followed by treatment with vehicle or 2.5 nmol/l iloprost (ilo) for 20 min. Cell lysates were analyzed for PKA activity as described in Methods. A representative gel is shown. Densitometric quantitation of fluorescence units of phosphorylated kemptide is expressed as fold induction relative to siControl treated with vehicle (n = 3). (C–F) Human uSMCs were transfected as in A and treated with vehicle or 2.5 nmol/l iloprost for 8 h. Western blots for (C) calponin, (D) h-caldesmon, (E) SM2-MHC, and (F) connexin 43 are shown with β-tubulin or GAPDH loading controls. Lanes in C were run on the same gel but were noncontiguous (lines). Densitometric quantitation of at least 3 independent experiments, including cell cultures derived from 2 different patient samples, are expressed as fold induction corrected to the loading control. All data are mean ± SEM. Significance of differences were determined using 1-way ANOVA with Newman-Keuls post-hoc test.

Figure 11. Cicaprost increases PKA activity as well as contractile protein and connexin 43 expression.

(A) Human uSMCs were treated with vehicle or with the indicated concentrations of cicaprost for 20 min. Cell lysates were analyzed for PKA activity as described in Methods. Densitometric quantitation of fluorescence units of phosphorylated kemptide from the gel shown are expressed as fold induction. (B) Human uSMCs were treated with 2.5 nmol/l cicaprost or iloprost for the indicated times, and cells were harvested and analyzed for PKA activity. The phosphorylated kemptide gel is shown. (C) Cell lysates from human uSMCs were treated with the indicated concentrations of cicaprost for 8 h and were subjected to Western blot analysis with antibodies to α-SMA, h-caldesmon, SM2-MHC, and β-tubulin as a loading control. (D and E) Cells were treated with 2.5 nmol/l cicaprost for the indicated times or 2.5 nmol/l iloprost for 8 h, and Western blots were performed using antibodies against (D) α-SMA and calponin or (E) SM2-MHC. GAPDH and β-tubulin served as loading controls. (F) uSMCs were treated with 2.5 nmol/l cicaprost, and Western blot analysis was performed for connexin 43 and β-tubulin as a loading control.

Figure 12. RO3244794 inhibits contractile protein expression induced by iloprost, but not by 8-Br-cAMP.

(A) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with vehicle or 2.5 nmol/l iloprost for 20 min. Cell lysates were analyzed for PKA activity using a nonradioactive in vitro PKA assay. A representative experiment is shown. (B and C) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with or without 2.5 nmol/l iloprost for 8 h. Cell lysates were prepared and subjected to Western blot analysis with antibodies against (B) h-caldesmon and (C) SM2-MHC. Lanes were run on the same gel but were noncontiguous (lines). (D) Human uSMCs were pretreated with 1 μM RO3244794 for 30 min followed by treatment with 0.5 μM 8-Br-cAMP for 8 h. Cell lysates were subjected to Western blot analysis with antibodies against h-caldesmon and β-tubulin.

Figure 13. PGI₂ mimics iloprost-induced contractile apparatus and connexin 43 protein expression.

(A) Effect of PGI₂ on PKA activity. Human uSMCs were treated for 20 min with PGI₂ or iloprost at the concentrations indicated and subjected to in vitro PKA assay. The phosphorylated kemptide gel is shown. (B and C) Effect of PGI₂ on contractile apparatus protein and connexin 43 expression. (B) Human uSMCs were treated with PGI₂ every 1.5 h up to 6 h or with a single treatment of iloprost for 6 h at the concentrations indicated. Western blots were performed with antibodies against SM2-MHC, calponin, β-tubulin, and GAPDH. (C) uSMCs were subjected to a single treatment of 0.6 μmol/l PGI₂ for the indicated times. Western blot analysis was performed with antibodies against SM2-MHC, h-caldesmon, connexin 43, and β-tubulin as a loading control.