AMPK regulates angiogenesis by inhibition of the ALK1 signaling pathway

描述(由申请人提供)：这项拨款申请的目的是测试AMPK通过磷酸化和抑制ALK1抑制血管生成的新假设。最近的研究结果表明，二甲双胍治疗2型糖尿病，一种著名的AMPK激活剂，可以降低癌症发病率，这促使它被用于癌症治疗的临床试验。到目前为止，150多项临床试验(www.ClinicalTrial.gov)正在进行中，二甲双胍用于治疗不同的癌症。尽管以AMPK为靶点治疗代谢综合征和癌症正受到越来越多的关注，但其对血管生成的影响尚不清楚。血管生成是恶性肿瘤细胞聚集成相当大的肿瘤的关键步骤，也是糖尿病并发症的罪魁祸首。这项赠款提案的目的是促进这种理解。越来越多的证据表明，激活素受体样激酶1(ALK1)在血管系统的发育中起着关键作用。此外，最近的研究表明，ALK1参与了几种疾病(如癌症和糖尿病视网膜病变)的病理性血管生成，这为ALK1可能成为抗血管生成治疗的一个有价值的靶点提供了一个有吸引力的理论基础。有趣的是，AMPK和ALK1信号之间的功能相互作用从未被记录在案。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可以有效地抑制BMP2诱导的Smad1/5的磷酸化和内皮细胞管的形成。此外，序列比对指向ALK1催化域L45环上潜在的AMPK磷酸化位点(S269)，这是Smad结合所必需的。因此，我们推测AMPK对该位点的磷酸化可能导致ALK1与Smad1/5的相互作用中断，从而抑制血管生成。在生理条件下，可能维持该位点的磷酸化和去磷酸化的动态，这种平衡的破坏可能导致致病的血管生成。作为验证这一假说的第一步，我们设定了以下两个具体目标：(1)确定AMPK是否抑制ALK1介导的血管生成；(2)鉴定ALK1上的AMPK磷酸化位点(S)，并阐明AMPK调节ALK1的分子机制。这项拟议的研究将首次将AMPK和ALK1信号通路捆绑在一起，以促进血管生成。因此，这项研究的完成将为血管生物学增加新的信息。此外，由于在2型糖尿病和癌症等疾病中都发现了AMPK活性抑制、ALK1信号上调和血管生成失调，因此本研究将有助于了解这些疾病的发病机制和治疗方法。特别是，鉴于二甲双胍已经作为一种安全和廉价的药物用于临床治疗2型糖尿病，拟议的研究结果将有助于将其扩大到抗血管生成治疗。

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.