Oxytocin-stimulated NFAT transcriptional activation in human myometrial cells.
Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca(2+) stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1-C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency.
Bristol University, School of Clinical Sciences, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, United Kingdom. jp5620@bris.ac.uk
Jason N A
JN
Pont
Craig A
CA
McArdle
Andrés
A
López Bernal
D021581:Active Transport, Cell Nucleus; D019703:Calcineurin / Q000378:metabolism; D020013:Calcium Signaling; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D009215:Myometrium / Q000166:cytology*; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D010121:Oxytocin / Q000494:pharmacology / Q000502:physiology*; D010641:Phenotype; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; D020033:Protein Isoforms; C108612:RCAN1 protein, human; D020710:RGS Proteins; C087182:RGS2 protein, human; D010121:Oxytocin; D008156:Luciferases; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D019703:Calcineurin