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首页> 外文学位 >Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta.
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Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta.

机译：松弛素信号传导途径的分子机制：cAMP，PI3K和PKCzeta的激活。

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摘要

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production.; Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1--2 minutes with a second peak at 10--20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin.; To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C zeta (PKCzeta) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCzeta translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCzeta translocation is P13K-dependent and independent of cAMP production. Antisense PKCzeta oligodeoxynucleotides (PKCzeta-ODNs) deplete both PKCzeta transcript and protein levels in THP-1 cells. PKCzeta-ODNs abolish relaxin-mediated PKCzeta translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCzeta-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCzeta in relaxin stimulation of cAMP and provide the first example of the PKCzeta regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types.
机译：松弛素是一种多肽激素，对生殖和非生殖组织具有多种作用。松弛素激活G蛋白偶联受体LGR7和LRG8。早期研究描述了松弛素治疗后cAMP和蛋白激酶A活性增加，但仅cAMP积累不能解释松弛素介导的所有作用。我们利用人类单核细胞THP-1来研究松弛素刺激CAMP产生的机制。在THP-1细胞中进行松弛素处理会在cAMP积累中产生一个双相时间过程，其中第一个峰最早出现在1--2分钟，第二个峰出现在10--20分钟。磷酸肌醇3-激酶（P13K）的选择性抑制剂，如渥曼青霉素和LY294002，表现出剂量依赖性抑制松弛素刺激的cAMP积累，对松弛素时间过程的第二个峰具有特异性。磷酸二酯酶的活性既不介导松弛素的作用也不由LY294002抑制松弛素。此外，LY294002阻止了松弛素对血管内皮生长因子转录水平的上调。为了进一步描述松弛素信号传导途径，我们搜索了可以激活腺苷酸环化酶（AC）的PI3K下游靶标。蛋白激酶C Zeta（PKCzeta）是主要的候选药物，因为它可以激活II型和V AC型。氯屈菜红碱（一种一般的PKC抑制剂）与LY294002（〜40％）一样抑制松弛素诱导的cAMP产生。松弛素刺激THP-1，MCF-7，PHM1-31和MMC细胞中PKCzeta易位至质膜，如免疫细胞化学所示。 PKCzeta易位是P13K依赖性的，并且与cAMP的产生无关。反义PKCzeta寡脱氧核苷酸（PKCzeta-ODN）耗尽了THP-1细胞中的PKCzeta转录本和蛋白质水平。与模拟和随机ODN对照相比，PKCzeta-ODN消除了松弛素介导的PKCzeta易位，并抑制了40％的cAMP松弛素刺激。在PKCzeta-ODNs存在下用LY294002处理几乎没有进一步的抑制作用。两者合计，我们提出了PI3K和PKCzeta在松弛素刺激cAMP中的新作用，并提供了内源系统中AC的PKCzeta调控的第一个例子。此外，我们已经鉴定出AC同工型和PKA锚定蛋白的高级复合物，试图解释松弛素与各种细胞类型中cAMP和PI3K信号通路的差异偶联。

著录项

  • 作者

    Nguyen, Bao The.;

  • 作者单位

    The University of Texas Graduate School of Biomedical Sciences at Houston.;

  • 授予单位 The University of Texas Graduate School of Biomedical Sciences at Houston.;
  • 学科 Health Sciences Pharmacology.; Biology Molecular.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 146 p.
  • 总页数 146
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 药理学;分子遗传学;
  • 关键词

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