Free Radical Biology and Medicine

Identification of NOD2 as a novel target of RNA-binding protein HuR:

Evidence from NADPH oxidase-mediated HuR signaling in diabetic nephropathy

Jin Shang^1*, Qiang Wan^2*, Xiaojie Wang¹, Yiqi Duan¹, Ziying Wang¹, Xinbing Wei¹, Yan Zhang¹, Hui Wang¹, Rong Wang^2#, and Fan Yi^1#

¹Department of Pharmacology, Shandong University School of Medicine, Jinan, China, 250012

²Department of Nephrology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China, 250021

^*Co-first authors, equally contributes to this work.

Running title: HuR regulates hyperglycemia-induced NOD2 mRNA stability

^#To whom correspondence should be addressed:

Fan Yi, Ph.D

Department of Pharmacology,

Shandong University School of Medicine,

44 West Wenhua Rd., Jinan, Shandong 250012, China;

Tel.: 86-0531-8838-2616; Fax: 86-0531-8838-2616;

E-mail: fanyi@sdu.edu.cn;

or

Rong Wang,

Department of Nephrology,

Shandong Provincial Hospital Affiliated to Shandong University,

Jingwu Road No. 324, Jinan, Shandong, 250021, China;

Email: wangrongsdu@163.com

Abstract

Although our recent studies have demonstrated that NOD2 is one of critical components of a signal transduction pathway that links renal injury to inflammation in diabetic nephropathy (DN), the regulatory mechanisms for NOD2 expression under hyperglycemia have not yet been elucidated. Considering that NOD2 mRNA from different species bearing long 3′-UTR with various AU-rich elements (AREs), the present study was designed to investigate the potential contribution of the RNA binding protein human antigen R (HuR) on the posttranscriptional regulation of NOD2 expression. In this study, we firstly found that the up-regulation of HuR in the kidney from DN subjects, which was correlated with proteinuria, indicating the role of HuR in the pathogenesis of DN. In vitro, high glucose (HG) induced a distinct increase in cytoplasmic HuR in rat mesangial cells (RMCs). By RNA-EMSA analysis, we found that HuR binds to the 3'-UTR NOD2, while HuR silencing reduced HG-induced NOD2 expression and mRNA stability. Mechanistically, we further found that NADPH oxidase-mediated redox signaling contributed to the expression and translocation of HuR and NOD2 mRNA stability. Finally, we evaluated the role of HuR showing that in vivo gene silencing of HuR by intrarenal lentiviral gene delivery ameliorated renal injury as well as reduced NOD2 expression in diabetic rats. Collectively, our studies demonstrate that HuR acts as a key posttranscriptional regulator of NOD2 expression, suggesting that targeting of HuR-NOD2 signaling might be crucial for the treatment of DN.

Key words: NOD-like receptors; hyperglycemia; oxidative stress; mRNA stability; gene therapy