Abstract
Activation of Gs coupled receptors (e.g. β2-adrenoreceptor (β2AR)) expressed within the uterine muscle layer (myometrium), promotes intracellular cAMP generation, inducing muscle relaxation through short-term inhibition of contractile proteins, and longer-term modulation of cellular phenotype to promote quiescence. In the myometrium cAMP-driven modulation of cell phenotype is facilitated by CREB activity, however despite the importance of CREB signalling in the promotion of myometrial quiescence during pregnancy, little is currently known regarding the molecular mechanisms involved. Thus, we have characterised β-adrenoceptor-stimulated CREB signalling in the immortalised ULTR human myometrial cell line.The non-selective β-adrenoceptor agonist isoprenaline induced time- and concentration-dependent CREB phosphorylation, which was abolished by the β2AR selective antagonist ICI118,551. β2AR-stimulated CREB phosphorylation was mediated through a short-term PKA-dependent phase, and longer-term Src/p38 MAPK-dependent/PKA-independent phase. Since in model cells, arrestin2 can facilitate β2AR-mediated Src/p38 recruitment, we examined whether CREB signalling was activated through a similar process in myometrial cells. Depletion of arrestin2 attenuated p38 phosphorylation, whilst arrestin3 depletion enhanced and prolonged isoprenaline-stimulated p38 signals, which was reversed following inhibition of Src. Knockdown of arrestin2 led to enhanced short-term (up to 10min), and attenuated longer-term (>10min) isoprenaline-stimulated CREB phosphorylation. Contrastingly, removal of arrestin3 enhanced and prolonged isoprenaline-stimulated CREB phosphorylation, whilst depletion of both arrestins abolished CREB signals at time points >5min.In summary, we have delineated the molecular mechanisms coupling β2AR activity to CREB signalling in ULTR myometrial cells, revealing a biphasic activation process encompassing short-term PKA-dependent, and prolonged Src/arrestin2/p38-dependent components. Indeed, our data highlight a novel arrestin-mediated modulation of CREB signalling, suggesting a reciprocal relationship between arrestin2 and arrestin3, wherein recruitment of arrestin3 restricts the ability of β2AR to activate prolonged CREB phosphorylation by precluding recruitment of an arrestin2/Src/p38 complex.
Highlights
Throughout pregnancy the uterus undergoes dramatic remodelling in order to accommodate the growing foetus
In an attempt to bias cAMP response-element binding protein (CREB) signalling towards the PKA pathway, we examined the temporal-dependency of isoprenalinestimulated CREB signals to a low concentration (10 nM)
This low dose of isoprenaline induced a transient phosphorylation of CREB peaking at 5 min, which rapidly decayed to basal levels within 10 min, and was blocked by the PKA inhibitor (PKAI) peptide ( Fig 2G, H)
Summary
Throughout pregnancy the uterus undergoes dramatic remodelling in order to accommodate the growing foetus. During this time it is vital that the uterine muscle (myometrium) remains in a quiescent state to prevent unwanted or premature contractions that may lead to preterm delivery [1]. Myometrial quiescence is induced and maintained through many divergent changes in cellular physiology, many of which are mediated by the actions of sex hormones. Sex hormones promote the increased expression of proteins whose actions mediate myometrial relaxation. The net result is to increase the cellular concentrations of cAMP which activates a diverse array of signalling pathways within cells, and in the myometrium is known to mediate long and short term effects upon muscle contractile tone
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