Xu Da, Wang Haoxun, Zhang Qiang, You Guofeng
Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey.
Department of Pharmaceutics, Rutgers University, Piscataway, New Jersey
Am J Physiol Renal Physiol. 2016 May 1;310(9):F821-31. doi: 10.1152/ajprenal.00522.2015. Epub 2016 Jan 28.
Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.
人有机阴离子转运体1（hOAT1）表达于肾近端小管细胞的膜上，介导多种临床上重要药物的体内处置，这些药物包括抗HIV治疗药物、抗肿瘤药物、抗生素、抗高血压药物和抗炎药物。因此，了解hOAT1的调节机制将为深入认识肾功能和功能障碍提供重要线索。我们之前已经确定，蛋白激酶C（PKC）的激活通过加速hOAT1从细胞表面内化到细胞内的内体并随后降解，从而抑制hOAT1的转运活性。我们进一步确定，PKC诱导的hOAT1泛素化是hOAT1内化之前的一个重要步骤。在当前的研究中，我们鉴定出两种密切相关的E3泛素连接酶，神经前体细胞表达、发育下调4-1和4-2（Nedd4-1和Nedd4-2），它们是hOAT1的重要调节因子：Nedd4-1或Nedd4-2的过表达增强了hOAT1的泛素化，减少了细胞表面hOAT1的量，并抑制了hOAT1的转运活性。在进一步探究PKC、Nedd4-1和Nedd4-2之间的关系时，我们发现，在用Nedd4-2的连接酶失活突变体（Nedd4-2/C821A）转染的细胞中，或者在用Nedd4-2特异性小干扰RNA敲低内源性Nedd4-2的细胞中，PKC依赖的hOAT1泛素化、表达和转运活性的变化被显著阻断，但在用Nedd4-1的连接酶失活突变体（Nedd4-1/C867S）转染的细胞中，或者在用Nedd4-1特异性小干扰RNA敲低内源性Nedd4-1的细胞中则未被阻断。总之，这是首次证明Nedd4-1和Nedd4-2都是hOAT1泛素化、表达和功能的重要调节因子。然而，它们发挥着不同的作用，因为Nedd4-2而非Nedd4-1是PKC调节的hOAT1泛素化、表达和转运活性的关键介导因子。
