Yu Zhou, Zhang Jinghui, Feng Jiaxu, You Guofeng
Department of Pharmaceutics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Pharmaceutics. 2025 Jun 25;17(7):825. doi: 10.3390/pharmaceutics17070825.
Organic anion transporter 3 (OAT3) in the kidney proximal tubule cells plays a critical role in renal clearance of numerous endogenous metabolites and exogenous drugs and toxins. In this study, we discovered that epidermal growth factor (EGF) regulates the expression and activity of OAT3 through palmitoylation, a novel mechanism that has never been described in the OAT field. Our results showed that treatment of OAT3-expressing cells with EGF led to a ~40% increase in OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate for OAT3. EGF-stimulated OAT3 transport activity was abrogated by H-89, a protein kinase A (PKA) inhibitor, indicating that an EGF-PKA signaling pathway is involved in the regulation of OAT3. We also showed that treatment of OAT3-expressing cells with EGF resulted in an enhancement of OAT3 palmitoylation, a novel type of post-translational modification for OATs, and such an enhancement was blocked by H-89, suggesting that the EGF-PKA signaling pathway participated in the modulation of OAT3 palmitoylation. Palmitoylation was catalyzed by a group of palmitoyltransfereases, and we showed that OAT3 palmitoylation and expression were inhibited by 2-BP, a general inhibitor for palmitoyltransfereases. We also explored the relationship among EGF/PKA signaling, OAT palmitoylation, and OAT transport activity. We treated OAT3-expressing cells with EGF or Bt2-cAMP, a PKA activator, in the presence and absence of 2-BP, followed by the measurement of OAT3-mediated transport of estrone sulfate. We showed that both EGF- and Bt2-cAMP-stimulated OAT3 transport activity were abolished by 2-BP, suggesting that palmitoylation mediates the regulation of EGF/PKA on OAT3. Finally, we showed that osimertinib, an anti-cancer drug/EGFR inhibitor, blocked EGF-stimulated OAT3 transport activity. In summary, we provided the first evidence that palmitoylation transduces the EGF/PKA signaling pathway to the modulation of OAT3 expression and function. Our study also provided an important implication that during comorbidity therapies, EGFR inhibitor drugs could potentially decrease the transport activity of renal OAT3, which would subsequently alter the therapeutic efficacy and toxicity of many co-medications that are OAT3 substrates.
肾脏近端小管细胞中的有机阴离子转运体3（OAT3）在众多内源性代谢产物、外源性药物及毒素的肾脏清除过程中发挥着关键作用。在本研究中，我们发现表皮生长因子（EGF）通过棕榈酰化作用调节OAT3的表达及活性，这是一种在OAT领域从未被描述过的新机制。我们的结果表明，用EGF处理表达OAT3的细胞会导致OAT3表达增加约40%，且OAT3介导的硫酸雌酮转运增加，硫酸雌酮是OAT3的典型底物。蛋白激酶A（PKA）抑制剂H - 89消除了EGF刺激的OAT3转运活性，表明EGF - PKA信号通路参与了OAT3的调节。我们还表明，用EGF处理表达OAT3的细胞会导致OAT3棕榈酰化增强，这是OATs一种新型的翻译后修饰，且这种增强被H - 89阻断，提示EGF - PKA信号通路参与了OAT3棕榈酰化的调节。棕榈酰化由一组棕榈酰转移酶催化，我们发现棕榈酰转移酶的通用抑制剂2 - BP可抑制OAT3棕榈酰化和表达。我们还探究了EGF/PKA信号、OAT棕榈酰化和OAT转运活性之间的关系。我们在有或没有2 - BP存在的情况下，用EGF或PKA激活剂Bt2 - cAMP处理表达OAT3的细胞，随后测定OAT3介导的硫酸雌酮转运。我们发现2 - BP消除了EGF和Bt2 - cAMP刺激的OAT3转运活性，提示棕榈酰化介导了EGF/PKA对OAT3的调节。最后，我们发现抗癌药物/EGFR抑制剂奥希替尼可阻断EGF刺激的OAT3转运活性。总之，我们首次提供证据表明棕榈酰化将EGF/PKA信号通路传导至对OAT3表达和功能的调节。我们的研究还提供了一个重要启示，即在合并症治疗期间，EGFR抑制剂药物可能会降低肾脏OAT3的转运活性，这随后会改变许多作为OAT3底物的联合用药的治疗效果和毒性。
