Department of Molecular Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster, Road Leicester, LE1 9HN, United Kingdom.
Department of Molecular Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster, Road Leicester, LE1 9HN, United Kingdom.
Cell Signal. 2017 Oct;38:182-191. doi: 10.1016/j.cellsig.2017.07.011. Epub 2017 Jul 18.
Activation of Gs coupled receptors (e.g. β-adrenoreceptor (βAR)) 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 βAR selective antagonist ICI118,551. βAR-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 βAR-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 βAR 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 βAR to activate prolonged CREB phosphorylation by precluding recruitment of an arrestin2/Src/p38 complex.
Gs 偶联受体（例如 β-肾上腺素受体（βAR））在子宫肌层（子宫肌）内的激活促进细胞内 cAMP 的产生，通过短期抑制收缩蛋白诱导肌肉松弛，并通过长期调节细胞表型来促进静止。在子宫肌中，cAMP 驱动的细胞表型调节是由 CREB 活性介导的，然而，尽管 CREB 信号在妊娠期间促进子宫静止很重要，但目前对于涉及的分子机制知之甚少。因此，我们在永生化的 ULTR 人子宫平滑肌细胞系中描述了 β-肾上腺素受体刺激的 CREB 信号。非选择性 β-肾上腺素受体激动剂异丙肾上腺素诱导时间和浓度依赖性 CREB 磷酸化，该磷酸化被 βAR 选择性拮抗剂 ICI118,551 消除。βAR 刺激的 CREB 磷酸化是通过短期 PKA 依赖性相和长期 Src/p38 MAPK 依赖性/ PKA 非依赖性相介导的。由于在模型细胞中，arrestin2 可以促进 βAR 介导的Src/p38 募集，我们检查了 CREB 信号是否通过子宫平滑肌细胞中的类似过程激活。arrestin2 的耗竭减弱了 p38 的磷酸化，而 arrestin3 的耗竭增强和延长了异丙肾上腺素刺激的 p38 信号，该信号在 Src 抑制后逆转。arrestin2 的敲低导致短期（长达 10 分钟）增强和长期（>10 分钟）异丙肾上腺素刺激的 CREB 磷酸化减弱。相反，arrestin3 的去除增强并延长了异丙肾上腺素刺激的 CREB 磷酸化，而两种 arrestin 的耗竭消除了>5 分钟时的 CREB 信号。总之，我们描绘了将 βAR 活性偶联到 ULTR 子宫平滑肌细胞中的 CREB 信号的分子机制，揭示了一个双相激活过程，包括短期 PKA 依赖性和长期 Src/arrestin2/p38 依赖性成分。事实上，我们的数据突出了一种新型的 arrestin 介导的 CREB 信号调节，表明 arrestin2 和 arrestin3 之间存在一种相互关系，其中 arrestin3 的募集通过排除 arrestin2/Src/p38 复合物的募集来限制 βAR 激活长期 CREB 磷酸化的能力。
