Melatonin sensitizes human myometrial cells to oxytocin in a protein kinase C alpha/extracellular-signal regulated kinase-dependent manner

Abstract

Context: Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night.

Objective: The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells.

Design: To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP(3))/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation.

Results: I-MEL was found to activate PKC alpha via the phospholipase C/IP(3)/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1.

Conclusions: MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP(3)/DAG signaling pathway, resulting in specific activation of PKC alpha and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy.

Figure 1

MEL actions on the Rho kinase signaling pathway and MLCP activity. A, Gel contraction assay using human telomerase-immortalized hTERT myometrial smooth muscle cells. Results are presented as collagen disk surface area (square millimeters) normalized to disks without adherent cells (to control for shrinkage due to fixation). Cells were treated as noted at the following concentrations: I-MEL (1 nm), OT (1 nm), and the Rho kinase inhibitor Y27632 (10 μm). a, P < 0.05 vs. control; b, P < 0.05 vs. I-MEL-treated cells; c, P < 0.05 vs. OT-treated cells; d, P < 0.05 vs. I-MEL/OT-cotreated cells; e, P < 0.05 vs. Y27632-treated cells. B, Western blot for phospho-MLC20. hTert cells were treated with 1 nm I-MEL or 1 μm microcystin-LW (an MLCP inhibitor) or in combination. Microcystin was applied 30 min before treatment with I-MEL.

Figure 2

MEL actions on IP₃ and total IP turnover. A, IP₃ turnover after treatment with I-MEL (1 nm), OT (1 nm), the MT2R antagonist 4P-PDOT (10 nm), and the PLC inhibitor U73122 (1 μm). a, P < 0.05 vs. control; b, P < 0.05 vs. I-MEL-treated cells; c, P < 0.05 vs. OT-treated cells; d, P < 0.05 vs. I-MEL/OT-treated cells; e, P < 0.05 vs. I-MEL plus OT plus 4P-PDOT. B, Total IP turnover after treatments. a–e, Same as in A.

Figure 3

MEL activates PKCα. A, Treated samples were immunoprecipitated with anti-PKCα, and then Western blots were performed for phospho-PKCα/β. hTert cells were treated with 1 nm I-MEL with or without 10 nm 4P-PDOT. B, PKC activity assays were also performed on immunoprecipitated and immunodepleted samples for confirmation of the Western blot results. a, P < 0.05 vs. control and I-MEL plus 4P-PDOT samples.

Figure 4

MEL activates ERK1/2-dependent phosphorylation of caldesmon. Representative Western blots are shown for phospho- (P-)ERK1/2 and phospho- (P-)caldesmon for samples collected after 15-min treatments with I-MEL (1 nm) or OT (1 nm) or samples pretreated with the MEK inhibitor PD98059 (10 μm) or the PKC inhibitor C1 (10 μm) before I-MEL addition.

Figure 5

Proposed model for myometrial sensitization by MEL. MEL activates PLC, which generates DAG, resulting in PKCα activation. PKCα initiates the Raf/MEK/ERK1/2 signaling cascade leading to phosphorylation of caldesmon (hCaD), which increases actin availability for myosin binding and cross-bridge cycling, thereby enhancing contractions.