Mechanisms of Diabetic Embryopathy and Molecular Pathways

说明（申请人提供）：妊娠期糖尿病是导致婴儿先天性缺陷的母体疾病之一。虽然妊娠期血糖水平的控制与婴儿畸形率的降低有关，然而，达到和维持满意的血糖控制是非常困难的。因此，预防或减少母体糖尿病相关的胎儿异常仍然是一个主要的临床挑战。这在一定程度上是因为脆弱期发生在器官发生期间，这是在怀孕早期，通常在母亲意识到自己怀孕之前。因此，在这一发育时期，血糖控制不足甚至暂时失去血糖控制可能导致胚胎异常和出生缺陷。为了改善公众健康和预防这些出生缺陷，我们需要创新和有效的治疗方法。为了开发这些治疗方法，了解由高血糖引起的胚胎畸形的机制至关重要。我们已发表的工作和正在进行的研究使我们假设高血糖启动了一个信号级联，通过该信号级联，蛋白激酶C （PKC）活性增加，激活胞质磷脂酶A2 （cPLA2）和花生四烯酸释放，导致膜脂过氧化和活性氧（ROS）产生增加。由此产生的氧化应激刺激异常的丝裂原活化蛋白激酶（MAPK）信号通路，如细胞外信号调节激酶（ERKs）和jun n-末端激酶（JNKs），导致过度凋亡和胚胎畸形发生。为了验证这些假设，我们将在Specific Aim 1中研究PKC作为高血糖依赖性胚胎畸形的中介的作用机制。我们将使用PKC基因敲除小鼠来确定PKC1、22和4在糖尿病胚胎病中的作用。我们将探讨每种PKC异构体调节cPLA2激活、磷脂过氧化和ROS产生的机制。在Specific Aim 2中，我们将研究ROS激活PKC和JNK的机制。我们将使用携带人类SOD1基因（将氧自由基转化为活性较低的分子）的转基因小鼠模型来确定PKC和JNK是否被氧化应激激活。我们将进一步研究JNK上游激酶凋亡信号调节激酶1 （apoptosis signal-regulating kinase 1, ASK1）和MAPK激酶4 （MAPK kinase 4, MKK4）是否在氧化应激对JNK活化的介导作用中发挥作用。在特异性目标3中，我们将研究JNK诱导糖尿病胚胎病细胞凋亡的机制。我们将使用缺乏jnk2基因的小鼠模型来确定JNK在糖尿病胚胎病中的作用。我们将进一步描述JNK调控的分子通路，涉及关键的凋亡调节因子，包括p66Shc，氧化应激信号的关键介质，以及在细胞凋亡中起重要作用的Bcl-2家族成员。通过仔细分析母体高血糖在胚胎发育过程中改变的信号机制，我们提出的研究将对了解糖尿病胚胎病的分子机制产生巨大影响，并为开发新的生化靶点提供重要信息，以进行潜在的治疗干预。公共卫生相关性：妊娠早期患有糖尿病的孕妇生下出生缺陷婴儿的风险很高。本研究的目的是了解高血糖如何改变胚胎发育中的分子和生化事件，导致新生儿异常。这项研究的结果将提供信息，作为未来发展治疗干预措施的基础，以预防孕产妇糖尿病相关的出生缺陷。

DESCRIPTION (provided by applicant): Diabetes mellitus in pregnancy is one of the maternal diseases that cause congenital defects in infants. Although control of glycemic level during pregnancy has been associated with reduced rate of malformed infants, nevertheless, achieving and maintaining satisfactory glycemic control is very difficult. Hence, prevention or reduction of maternal diabetes-associated fetal anomalies remains a major clinical challenge. This is in part because the period of vulnerability occurs during organogenesis, which is early in pregnancy and often before the mother is aware that she is pregnant. Thus, insufficient glycemic control or even transient loss of glycemic control during this developmental period may result in embryonic abnormalities and birth defects. To improve public health and prevent these birth defects, we need innovative and effective therapeutic approaches. To develop these therapies, it is critical to understand the mechanisms of embryonic malformations induced by hyperglycemia. Our published work and on-going studies lead us to hypothesize that hyperglycemia initiates a signaling cascade whereby increased activity of protein kinase C (PKC), activates cytosolic phospholipase A2 (cPLA2) and arachidonic acid release, resulting in increased membrane lipid peroxidation and reactive oxygen species (ROS) production. The resultant oxidative stress stimulates aberrant mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular signal-regulated kinases (ERKs) and jun N-terminal kinases (JNKs), leading to excessive apoptosis and embryonic dysmorphogenesis. To test these hypotheses, we will, in Specific Aim 1, investigate the mechanism of action of PKC as a mediator of hyperglycemia-dependent embryonic malformation. We will use PKC gene knockout mice to determine the role of PKC1, 22, and 4 in diabetic embryopathy. We will address the mechanisms whereby each PKC isoform regulates cPLA2 activation, phospholipid peroxidation, and ROS production. In Specific Aim 2, we will investigate the mechanisms of PKC and JNK activation by ROS. We will determine whether PKC and JNK are activated by oxidative stress, using a transgenic mouse model that carries the human SOD1 gene (converts oxygen free radicals into less reactive molecules). We will further investigate whether JNK upstream kinases, apoptosis signal-regulating kinase 1 (ASK1) and MAPK kinase 4 (MKK4), play a role in mediating the effect of oxidative stress on JNK activation. In Specific Aim 3, we will investigate the mechanisms by which JNK induces apoptosis in diabetic embryopathy. We will use a mouse model that lacks the jnk2 gene to determine the role of JNK in diabetic embryopathy. We will further delineate the JNK- regulated molecular pathway involving key apoptotic regulators, including p66Shc, a critical mediator of oxidative stress signaling, and members of the Bcl-2 family, which play essential roles in apoptosis. By carefully analyzing the signaling mechanisms altered by maternal hyperglycemia in the developing embryo, our proposed studies will have an enormous impact on understanding the molecular mechanisms of diabetic embryopathy, and provide important information for developing novel biochemical targets for potential therapeutic interventions. PUBLIC HEALTH RELEVANCE: Pregnant women with diabetes during the early gestation period have a high risk of having babies with birth defects. The aims of this study are to understand how hyperglycemia changes the molecular and biochemical events in the developing embryo, causing abnormalities in newborn infants. The results of the study will provide information that will serve as a basis for future development of therapeutic interventions to prevent maternal diabetes-associated birth defects.