AMPK regulates angiogenesis by inhibition of the ALK1 signaling pathway

描述（由申请人提供）：本授权申请的目的是测试AMPK通过磷酸化和抑制ALK 1抑制血管生成的新假设。最近的研究发现，二甲双胍治疗2型糖尿病，一个众所周知的AMPK激活剂，降低癌症的发病率，促使其用于癌症治疗的临床试验。到目前为止，超过150项临床试验（www.clinicaltrial.gov）正在进行二甲双胍治疗不同癌症。虽然靶向AMPK治疗代谢综合征和癌症越来越受到关注，但其对血管生成的影响尚不清楚，血管生成是恶性细胞向相当大的肿瘤增殖的关键步骤，也是糖尿病并发症的罪魁祸首。这项赠款提案旨在促进这种理解。大量证据表明激活素受体样激酶1（activin receptor-like kinase 1，ALK 1）在血管系统的发育中起关键作用。此外，最近的研究表明，ALK 1参与了几种疾病（如癌症和糖尿病视网膜病变）的致病性血管生成，这为ALK 1可能成为抗血管生成治疗中有价值的靶标提供了一个有吸引力的理由。有趣的是，AMPK和ALK 1信号之间的功能相互作用从未被记录。在我们对人脐静脉内皮细胞的初步实验中，我们发现二甲双胍激活AMPK可有效抑制BMP 2诱导的Smad 1/5磷酸化和内皮细胞管形成。此外，序列比对指向ALK 1催化结构域的L45环中的潜在AMPK磷酸化位点（S269），其对于Smad结合是必需的。因此，我们推测AMPK对该位点的磷酸化可能导致ALK 1与Smad 1/5相互作用的破坏，并随后抑制血管生成。可能的是，该位点的磷酸化和去磷酸化的动力学在生理条件下维持，并且平衡的破坏可能导致致病性血管生成。作为验证这一假设的第一步，我们提出了以下两个具体目标：（1）确定AMPK是否抑制ALK 1介导的血管生成，（2）鉴定ALK 1上的AMPK磷酸化位点并阐明AMPK调节ALK 1的分子机制。这项研究将首次将AMPK和ALK 1信号通路与血管生成联系在一起。因此，该研究的完成将为血管生物学增加新的信息。此外，由于AMPK活性的抑制、ALK 1信号的上调和血管生成失调都已在2型糖尿病和癌症等疾病中发现，因此所提出的研究将有助于理解这些疾病的发病机制及其治疗。特别是，鉴于二甲双胍已经在临床上作为一种安全和廉价的药物用于治疗2型糖尿病，拟议研究的结果将有助于将其用于抗血管生成治疗。

DESCRIPTION (provided by applicant): The objective of this grant application is to test a novel hypothesis that AMPK inhibits angiogenesis by phosphorylation and inhibition of ALK1. Recent findings that treatment of type 2 diabetes with metformin, a well-known AMPK activator, reduces cancer incidence have prompted its use in clinical trials for cancer therapy. Thus far, more than 150 clinical trials (www.clinicaltrial.gov) are in progress with metformin for the treatment of different cancers. Although targeting AMPK for the therapy of metabolic syndrome and cancer is gaining more attention, its effects on angiogenesis, a crucial step for malignant cells to populate to a sizable tumor and a culprit for diabetic complications, are not clear. This grant proposal is aimed at advancing such understanding. Compelling evidence has demonstrated that activin receptor-like kinase 1 (ALK1) plays a key role in the development of vascular system. Additionally, recent studies have suggested that ALK1 is involved in pathogenic angiogenesis of several diseases (e.g. cancer and diabetic retinopathy), which provides an appealing rationale that ALK1 could be a valuable target in anti-angiogenesis therapy. Interestingly, the functional interaction between AMPK and ALK1 signaling has never been documented. In our preliminary experiments with human umbilical vein endothelial cells, we found that AMPK activation by metformin potently suppresses BMP2-induced phosphorylation of Smad1/5 and endothelial cell tube formation. Furthermore, sequence alignment points to a potential AMPK phosphorylation site (S269) in the L45 loop of the catalytic domain of ALK1, which is essential for Smad binding. Thus, we speculate that the phosphorylation of this site by AMPK may lead to disruption of ALK1 interaction with Smad1/5, and subsequent inhibition of angiogenesis. It is possible that dynamics of the phosphorylation and dephosphorylation of this site is maintained under physiological condition and disruption of the balance may lead to pathogenic angiogenesis. As a first step to test this hypothesis, we set out the following two specific aims: (1) To determine if AMPK inhibits ALK1- mediated angiogenesis and, (2) To identify AMPK phosphorylation site(s) on ALK1 and elucidate the molecular mechanism by which AMPK regulates ALK1. The proposed study will be the first to tether AMPK and ALK1 signaling pathways together to angiogenesis. Thus, completion of the study will add novel information to vascular biology. Moreover, since suppression of AMPK activity, upregulation of ALK1 signaling and dysregulated angiogenesis all have been found in such diseases as type 2 dabetes and cancer, the proposed study will be instrumental to understanding the pathogenesis of these diseases and to their treatment. Especially, given metformin has already been used clinically as a safe and inexpensive drug against type 2 diabetes, the outcome of the proposed study will help to expand its use to anti-angiogenesis therapies.