Hypoxia Increases IGFBP-1 Phosphorylation Mediated by mTOR Inhibition
Hypoxia Increases IGFBP-1 Phosphorylation Mediated by mTOR Inhibition
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DOI：
10.1210/me.2015-1194
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发表时间：
2016-02-01
影响因子：
--
通讯作者：
Gupta, Madhulika B.
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文献类型：
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作者：
Damerill, Ian;Biggar, Kyle K.;Gupta, Madhulika B.
在胎儿生长受限（FGR）中，胎儿生长受到营养和氧气供应减少的限制。胰岛素样生长因子I（IGF-I）是胎儿生长的关键调节因子，IGF结合蛋白-1（IGFBP-1）是胎儿IGF-I生物利用度的主要调节因子。磷酸化增强IGFBP-1对IGF-I的亲和力。缺氧诱导IGFBP-1过度磷酸化，显著降低IGF-I的生物利用度。我们最近报道了胎肝IGFBP-1过度磷酸化与非人灵长类FGR模型中雷帕霉素（mTOR）机制靶点的抑制有关。在这里，我们测试的假设，IGFBP-1的过度磷酸化反应缺氧是由mTOR抑制介导的。我们在低氧（1%O-2）或基础（20%O-2）条件下培养的HepG 2细胞中通过雷帕霉素或靶向raptor（mTOR复合物[mTORC] 1）和/或rictor（mTORC 2）的小干扰RNA（siRNA）抑制mTOR。相反，我们通过沉默内源性mTOR抑制剂（结节性硬化症复合物2/含DEP结构域和mTOR相互作用蛋白）来激活mTORC 1或mTORC 1 + mTORC 2。免疫印迹分析表明，缺氧和抑制mTORC 1和/或mTORC 2诱导相似程度的IGFBP-1磷酸化Ser 101/119/169和减少IGF-I受体自身磷酸化。mTORC 1 + mTORC 2或mTORC 1单独激活可防止IGFBP-1对缺氧的过度磷酸化反应。多反应监测质谱表明，雷帕霉素和/或缺氧增加磷酸化也在Ser 98和一个新的网站Ser 174。计算机结构分析表明Ser 174与IGF结合位点非常接近。总之，我们证明了通过mTORC 1或mTORC 2通路的信号传导足以诱导IGFBP-1在缺氧反应中的过度磷酸化。这项研究提供了新的理解的细胞机制，控制胎儿IGFBP-1磷酸化缺氧，我们提出，mTOR抑制构成缺氧，降低IGF-I生物利用度和FGR之间的机制联系。
In fetal growth restriction (FGR), fetal growth is limited by reduced nutrient and oxygen supply. Insulin-like growth factor I (IGF-I) is a key regulator of fetal growth and IGF binding protein -1(IGFBP-1) is the principal regulator of fetal IGF-I bioavailability. Phosphorylation enhances IGFBP-1's affinity for IGF-I. Hypoxia induces IGFBP-1 hyperphosphorylation, markedly decreasing IGF-I bioavailability. We recently reported that fetal liver IGFBP-1 hyperphosphorylation is associated with inhibition of the mechanistic target of rapamycin (mTOR) in a nonhuman primate model of FGR. Here, we test the hypothesis that IGFBP-1 hyperphosphorylation in response to hypoxia is mediated by mTOR inhibition. We inhibited mTOR either by rapamycin or small interfering RNA (siRNA) targeting raptor (mTOR complex [mTORC] 1) and/or rictor (mTORC2) in HepG2 cells cultured under hypoxia (1% O-2) or basal (20% O-2) conditions. Conversely, we activated mTORC1 or mTORC1 + mTORC2 by silencing endogenous mTOR inhibitors (tuberous sclerosis complex 2/DEP-domain-containing and mTOR-interacting protein). Immunoblot analysis demonstrated that both hypoxia and inhibition of mTORC1 and/or mTORC2 induced similar degrees of IGFBP-1 phosphorylation at Ser101/119/169 and reduced IGF-I receptor autophosphorylation. Activation of mTORC1 + mTORC2 or mTORC1 alone prevented IGFBP-1 hyperphosphorylation in response to hypoxia. Multiple reaction monitoring-mass spectrometry showed that rapamycin and/or hypoxia increased phosphorylation also at Ser98 and at a novel site Ser174. In silico structural analysis indicated that Ser174 was in close proximity to the IGF-binding site. Together, we demonstrate that signaling through the mTORC1 or mTORC2 pathway is sufficient to induce IGFBP-1 hyperphosphorylation in response to hypoxia. This study provides novel understanding of the cellular mechanism that controls fetal IGFBP-1 phosphorylation in hypoxia, and we propose that mTOR inhibition constitutes a mechanistic link between hypoxia, reduced IGF-I bioavailability and FGR.