Regulation of Na absorption by NHE3 in the intestine

描述（由申请人提供）：在世界范围内，腹泻每年夺去数百万人的生命，其中大多数是婴儿。贫困、拥挤和受污染的水供应都是原因之一。虽然腹泻在较富裕国家的发病率要低得多，但腹泻仍然是儿科急诊室最常见的两种就诊方式之一，在机构化的老年人中也很常见。腹泻也是糖尿病的一种棘手的胃肠道并发症。肠中主要的盐和水吸收由电中性NaCl吸收介导，其由Na +/H+交换器与Cl-/HCO3-交换器偶联组成。Na +/H+交换器亚型3（NHE3）通常在许多牙周病中被靶向。然而，调节NHE 3的分子基础和生物学因素尚未完全了解。这是一个重要的方面，因为更好地理解激活NHE 3的机制应该有助于开发改进的治疗方式来对抗由Na +/H+交换抑制引起的腹泻。在之前的资助周期中，我们已经证明了溶血磷脂酸（LPA）通过结肠上皮细胞中的LPA5R激活NHE3，并表明LPA是NHE3和小鼠肠道液体吸收的有效激活剂。此外，我们发现LPA的作用涉及顶端表皮生长因子受体（EGFR）。然而，LPA诱导的EGFR反式激活和NHE3激活的机制尚未完全研究。我们的目的是确定LPA调节NHE3的分子机制，并测试LPA5R在体内的重要性（特异性目的1）。糖尿病腹泻是1型糖尿病的一种棘手的胃肠道并发症。我们的初步数据表明，糖尿病小鼠链脲佐菌素（STZ）诱导腹泻和NHE 3活性显着减弱。我们的研究进一步表明，胰岛素刺激STZ治疗小鼠的NHE3活性，尽管其潜在机制尚不清楚。我们将确定胰岛素激活NHE3的分子机制，并在啮齿动物1型糖尿病模型中测试LPA作为NHE3激活剂和液体吸收的效用（具体目标2）。我们的初步数据显示，二甲双胍通常用于2型糖尿病的血糖控制，抑制NHE 3活性，进一步表明NHE 3在糖尿病相关腹泻中的因果作用。我们的数据表明，NHE 3是泛素化的反应抑制剂，如二甲双胍，佛波酯，和毛喉素。令人惊讶的是，我们观察到人类和非人类之间NHE3的泛素化存在显著差异。NHE 3泛素化的状态及其作用从未被研究过。在具体目标3中，我们将研究调节NHE3泛素化的机制以及泛素化在NHE3调节和EPEC诱导的腹泻中的重要性。拟议工作的成功完成将增加NHE3调节的分子机制的知识，并将为治疗和预防牙周病的改进策略提供见解。 公共卫生相关性：腹泻是发展中国家的常见死因，也是全球婴儿死亡的第二大常见原因。分泌性腹泻发生在分泌增加、吸收减少或两者兼而有之时，从而导致电解质和液体的净损失。Na +/H+交换器3型（NHE 3）存在于小肠和结肠上皮细胞的顶膜中，在Na+和液体吸收中起主要作用。NHE 3在许多神经系统疾病中经常被靶向，并且NHE 3的抑制机制已被广泛研究。然而，重要的是要认识到，更好地了解激活NHE 3的生物学因素和机制，应有助于开发更好的治疗方式来对抗乳腺癌疾病。本申请的主要目的是继续我们的研究，以了解NHE 3激活的分子机制。

DESCRIPTION (provided by applicant): Worldwide, diarrhea claims several million lives annually, mostly those of infants. Poverty, crowding, and contaminated water supplies all contribute. Although its incidence is much lower in the more affluent nations, diarrhea remains one of the two most common visits to pediatric emergency rooms and is also common among the institutionalized elderly. Diarrhea is also a troublesome gastrointestinal complication of diabetes. Major salt and water absorption in the intestine is mediated by electroneutral NaCl absorption, consisting of a Na+/H+ exchanger coupled with a Cl-/HCO3- exchanger. The Na+/H+ exchanger isoform 3 (NHE3) is often targeted in many diarrheal diseases. However, the molecular basis and biological factors regulating NHE3 are not fully understood. This is an important aspect as a better understanding of the mechanisms activating NHE3 should aid the development of improved therapeutic modalities to combat diarrhea caused by the inhibition of Na+/H+ exchange. During the previous funding cycle, we have demonstrated that lysophosphatidic acid (LPA) activates NHE3 via LPA5R in colon epithelial cells and showed that LPA is a potent activator of NHE3 and fluid absorption in mouse intestine. In addition, we showed that the effect of LPA involves apical epidermal growth factor receptor (EGFR). However, the mechanism of LPA-induced EGFR transactivation and the activation of NHE3 are incompletely studied. We aim to determine the molecular mechanism of NHE3 regulation by LPA and test the importance of LPA5R in vivo (Specific Aim 1). Diabetic diarrhea is a troublesome gastrointestinal complication of type 1 diabetes. Our preliminary data show that mice with diabetes induced by streptozotocin (STZ) develop diarrhea and NHE3 activity is significantly attenuated. Our study further shows that insulin stimulates NHE3 activity in STZ-treated mice, although the underlying mechanisms are unknown. We will determine the molecular mechanism of NHE3 activation by insulin and test the utility of LPA as an activator of NHE3 and fluid absorption in the type 1 diabetic model in rodent (Specific Aim 2). Our preliminary data show that metformin commonly used for glycemic control in type 2 diabetes inhibits NHE3 activity, further suggesting a causal role of NHE3 in diabetes-related diarrhea. Our data show that NHE3 is ubiquitinated in response inhibitory agents, such as metformin, phorbol ester, and forskolin. Surprisingly, we observed a significant difference in ubiquitination of NHE3 between human and non-human. The status of NHE3 ubiquitination and its role have never been investigated. In Specific Aim 3, we will investigate the mechanisms that regulate NHE3 ubiquitination and the importance of ubiquitination in NHE3 regulation and EPEC-induced diarrhea. Successful completion of the proposed work will increase the knowledge of molecular mechanism of NHE3 regulation and will provide insights into improved strategies for treatment and prevention of diarrheal diseases. PUBLIC HEALTH RELEVANCE: Diarrhea is a common cause of death in developing countries and the second most common cause of infant deaths worldwide. Secretory diarrhea occurs when there is increased secretion, decreased absorption, or both so that there is a net loss of electrolytes and fluid. Na+/H+ exchanger type 3 (NHE3) present in the apical membrane of the small intestinal and colonic epithelia plays a major role in Na+ and fluid absorption. NHE3 is often targeted in many diarrheal diseases and the mechanisms of the inhibition of NHE3 have widely been studied. However, it is important to realize that a better understanding of biological factors and mechanisms activating NHE3 should aid the development of improved therapeutic modalities to counteract diarrheal diseases. The main objective of this application is to continue our investigation to understand the molecular mechanisms of NHE3 activation.