Damerill Ian, Biggar Kyle K, Abu Shehab Majida, Li Shawn Shun-Cheng, Jansson Thomas, Gupta Madhulika B
Department of Biochemistry (I.D., K.K.B., S.S.-C.L., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada; Department of Obstetrics and Gynecology (T.J.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Department of Pediatrics (M.B.G.), University of Western Ontario, London, N6C 2V5 Canada; and Children's Health Research Institute (M.A.S., M.B.G.), University of Western Ontario, London, Ontario, N6C 2V5 Canada.
Mol Endocrinol. 2016 Feb;30(2):201-16. doi: 10.1210/me.2015-1194. Epub 2015 Dec 29.
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% O2) or basal (20% O2) 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.
在胎儿生长受限（FGR）中，胎儿生长因营养和氧气供应减少而受到限制。胰岛素样生长因子I（IGF-I）是胎儿生长的关键调节因子，而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。磷酸化增强了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生物利用度。我们最近报道，在FGR的非人灵长类动物模型中，胎儿肝脏IGFBP-1过度磷酸化与雷帕霉素作用靶点（mTOR）的抑制有关。在此，我们检验以下假设：缺氧诱导的IGFBP-1过度磷酸化是由mTOR抑制介导的。我们在缺氧（1% O₂）或基础（20% O₂）条件下培养的HepG2细胞中，通过雷帕霉素或靶向 Raptor（mTOR复合物[mTORC]1）和/或Rictor（mTORC2）的小干扰RNA（siRNA）抑制mTOR。相反，我们通过沉默内源性mTOR抑制剂（结节性硬化复合物2/含DEP结构域且与mTOR相互作用的蛋白）来激活mTORC1或mTORC1 + mTORC2。免疫印迹分析表明，缺氧以及mTORC1和/或mTORC2的抑制均诱导了相似程度的Ser101/119/169位点的IGFBP-1磷酸化，并降低了IGF-I受体的自磷酸化。mTORC1 + mTORC2或单独的mTORC1激活可防止缺氧诱导的IGFBP-1过度磷酸化。多反应监测质谱分析表明，雷帕霉素和/或缺氧还增加了Ser98和一个新位点Ser174的磷酸化。计算机模拟结构分析表明，Ser174靠近IGF结合位点。我们共同证明，通过mTORC1或mTORC2途径的信号传导足以诱导缺氧时的IGFBP-1过度磷酸化。这项研究为缺氧时控制胎儿IGFBP-1磷酸化的细胞机制提供了新的认识，并且我们提出mTOR抑制构成了缺氧、IGF-I生物利用度降低和FGR之间的机制联系。
