The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression

Abstract

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1β to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1β-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1β-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1β-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1β alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.

Fig 1. The effect of forskolin on the IL-1β-induced increase in OTR mRNA and protein levels and the impact of PKA knockdown.

Myometrial cells were isolated as described above in Materials and Methods, and treated with IL-1β (1ng/mL) and/or forskolin (100μM) either alone or in combination for 6 hours. mRNA and protein were extracted, and the levels of OTR (A) mRNA were measured using quantitative rt-PCR. The levels of OTR (B) protein were measured using Western blotting. A representative western blot is shown next to the graph displaying the densitometry of the protein levels. PKAC-α was knocked down using siRNA (siPKAC-α) controlled with non-targeted siRNA [siNT]. Representative western blots to demonstrate transfection is shown above. After transfection, cells were incubated for 96 hours before being treated with progesterone (10μM), IL-1β (1ng/mL) and forskolin (100μM) in combination for 6 hours. The mRNA was extracted, and the levels of OTR (C, D), mRNA were measured using rt-PCR. Data are shown as the mean and SEM. A and B were compared using Wilcoxon matched pairs test for data that were not normally distributed and paired t test for data that were normally distributed. C and D were compared using a non-parametric paired test on the basis that we wanted to understand the impact of blocking PKA. *P<0.05, **P<0.01, ***P<0.001 (n = 8–9 myometrial samples from 8–9 different women in each experiment).

Fig 2. Forskolin represses IL-1β-induced total and nuclear and cytosolic p65 protein and c-jun levels.

Myometrial cells were isolated from myometrial biopsies obtained from women at the time of pre-labor term Caesarean section as described above in Materials and Methods, and treated with IL-1β (1ng/mL) and/or forskolin (100μM) either alone or in combination for 6 hours. Protein was extracted, and the levels of total p65 (A), and total c-jun (B) protein expression were measured using western blotting. Myometrial cells were isolated as described above in Materials and Methods, and treated with forskolin (100μM) or IL-1β (1ng/mL) either alone or in combination for 1 hour. Cells were lysed and samples purified for cytoplasmic or nuclear protein. Western blotting was performed using antibodies directed against phospho-p65 (Ser536) (C, D), and phospho c-jun (E, F). TATA-binding protein (TBP) and α-tubulin were used as the internal controls for nuclear and cytosolic fraction, respectively. MKP-1 (G, H) and IKBα (I, J) mRNA and protein levels were measured using rt-PCR and western blotting respectively. Data are shown as the mean and SEM, and were compared using (control vs. forskolin and IL-1β alone vs. IL-1β + forskolin) Wilcoxon matched pairs test for data that were not normally distributed and paired t test for data that were normally distributed. *P<0.05, **P<0.01, ***P<0.001 when compared to control. (n = 6–7 myometrial samples from 6–7 different women in each experiment).

Fig 3. The effect of the combination of forskolin and progesterone on the IL-1β-induced increase in OTR mRNA and protein levels.

Myometrial cells were isolated as described in Materials and Methods, and treated with progesterone (10μM), forskolin (100μM) or IL-1β (1ng/mL) either alone or in combination for 6 hours. mRNA and protein were extracted, and the levels of OTR (A) mRNA were measured using quantitative rt-PCR and the levels of OTR (B) protein were measured using Western blotting. A representative western blot is shown next to the graph displaying the densitometry of the protein levels. Data are expressed as mean SEM and were compared using Friedman’s Test, with a Dunn's Multiple Comparisons post hoc test for data that were not normally distributed, and using ANOVA, with Dunnett and Bonferroni’s post-test for data that were normally distributed, *P<0.05, **P<0.01 (n = 8–9 myometrial cells from 8–9 different women).

Fig 4. The combination of cAMP with progesterone represses IL-1β-induced nuclear transfer of phospho c-jun and phospho p65.

Myometrial cells were isolated from myometrial biopsies obtained from women at the time of pre-labor term Caesarean section as described above in Materials and Methods, and treated with progesterone (10μM), forskolin (100μM) or IL-1β (1ng/mL) either alone or in combination for for 0 minute, 30 minutes, 1 hour, 2 hours and 6 hours. Cells were lysed and samples purified for cytoplasmic or nuclear protein. Western blotting was performed using antibodies directed against phospho c-jun (A, B), and phospho-p65 (Ser536) (C, D). α-tubulin and TATA-bind protein (TBP) were used as the internal controls for cytosolic and nuclear fraction, respectively. Data were compared using Friedman’s Test, with a Dunn's Multiple Comparisons post hoc test for data that were not normally distributed, and using ANOVA, with Dunnett and Bonferroni’s post-test for data that were normally distributed, *P<0.05, **P<0.01, ***P<0.001. (n = 6 myometrial cells from 6 different women and only 30 minutes, 1 hour, and 2 hours time points were shown in the figure above).

Fig 5. The effect of PKA knockdown on the repression of the IL-1β-induced increase in OTR mRNA and protein levels by forskolin and progesterone.

Myometrial cells were isolated from myometrial biopsies obtained from women at the time of pre-labor term Caesarean section as described above in Materials and Methods. After the cells were about 80% confluent, PKAC-α were knocked down using siRNA (siPKAC-α, efficacy shown in inset figure) controlled with non-targeted siRNA [siNT]. Cells were knocked down using siNT. Representative western blots to demonstrate transfection is shown above. After transfection, cells were incubated for 96 hours before being treated with progesterone (10μM), IL-1β (1ng/mL) and forskolin (100μM) in combination for 6 hours. The mRNA was extracted, and the levels of OTR (A, B) mRNA were measured using rt-PCR. Data were compared using Friedman’s Test, with a Dunn's Multiple Comparisons post hoc test for data that were not normally distributed, and using ANOVA, with Dunnett and Bonferroni’s post-test for data that were normally distributed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (n = 6–7 myometrial cells from 6–7 different women).