Background & Aims
Following nerve injury, a strong local immune response is activated, resulting in the production of pro-inflammatory effectors believed to contribute to the development of chronic pain. A similar immune cell phenotype is triggered after soft tissue surgery, a cellular state that depends on the IRE1?-XBP1s arm of the endoplasmic reticulum (ER) stress response (1). We hypothesize that leukocytes infiltrating the injured injured nerve display an increase in XBP1 activation, which transactivates nuclear protein 1 (NUPR1), and that its blockade could prevent neuropathic pain behaviors in mice. Our aims were to establish the dependence of Nupr1 expression on XPB1 and to elucidate the role of XBP1/NUPR1 in a murine model of neuropathic pain.
Methods
First, we co-transfected Human 293T cells with luciferase-based transcriptional reporter vectors for the Nupr1 promoter and plasmids encoding Xbp1s alone or with ATF4 (for potential cooperation). Second, we measured Xbp1s and Nupr1 mRNA in wild type (WT), IRE1a knockout (KO), ATF6 KO, or PERK KO bone marrow-derived macrophages (BMDMs) stimulated with thapsigargin (1 uM). Third, we tested ZZW-115, an NUPR1 inhibitor, in mouse peritoneal macrophages stimulated with tunicamycin (10 uM) or thapsigargin (1 uM). Fourth, we measured Nupr1 mRNA levels in the injured sciatic nerve or related (L4-L6) DRGs before and after partial sciatic nerve ligation (PSNL) surgery in Ern1f/f-Vav1cre mice, which lack IRE1? exclusively in hematopoietic cells (conditional knockout, cKO) and Ern1f/f as controls. Fifth, we performed behavioral assessments in WT mice with PSNL surgery treated with ZZW-115 (5 mg/kg/day) during postop days 7-21.
Results
First, we observed that Xbp1s transactivates Nupr1 in cooperation with ATF4 in a concentration-dependent manner using luciferase activity assays in Human 293T cells. Second, thapsigargin induced Xbp1 activation (Xbp1s) and increased Nupr1 expression in WT and ATF6 KO BMDCs, an effect that was not observed in IRE1 and PERK KO BMDMs, confirming that Nupr1 induction depends on IRE1/ATF4 signaling. Third, ZZW-115 robustly blocked tunicamycin-induced Nupr1 and Xbp1s mRNA and partially blocked the effects of thapsigargin on these genes. Fourth, PSNL induced an increase in Nupr1 mRNA in the injured sciatic nerve (postop day 7) and L4-L6 DRGs (postop days 14-21) in Ern1f/f mice; however, this increase was not observed in Ern1f/f-Vav1cre mice tissues at those time points. Fifth, ZZW-115 significantly reduced cold allodynia, mechanical hypersensitivity, and partially diminished weight-bearing distribution between the injured and non-injured hind paws.
Conclusions
Our data demonstrate that XBP1 transactivates Nupr1 in cooperation with ATF4 in immune cells under ER stress conditions. Peripheral nerve injury induced an increase in Nupr1 along the peripheral sensorial neuroaxis, driven by XBP1. Pharmacological inhibition of NUPR1 reduced PSNL-induced pain-related behaviors in mice. Our studies uncover immune cell ER stress as a potential pathophysiological mechanism for neuropathic pain via NUPR1 and identify NUPR1 as a potential novel target to treat neuropathic pain.
References
1. IRE1?-XBP1 signaling in leukocytes controls prostaglandin biosynthesis and pain.
Chopra S, Giovanelli P, Alvarado-Vazquez PA, Alonso S, Song M, Sandoval TA, Chae CS, Tan C, Fonseca MM, Gutierrez S, Jimenez L, Subbaramaiah K, Iwawaki T, Kingsley PJ, Marnett LJ, Kossenkov AV, Crespo MS, Dannenberg AJ, Glimcher LH, Romero-Sandoval EA, Cubillos-Ruiz JR.
Science. 2019 Jul 19;365(6450):eaau6499.
Presenting Author
Alfonso Romero-Sandoval
Poster Authors
Alfonso Romero-Sandoval
MD, PhD
Wake Forest University School of Medicine, Winston Salem, North Carolina, United States
Lead Author
Miriam das Dores Mendes Fonseca
Wake Forest University
Lead Author
Oriana Gelblung
Wake Forest University
Lead Author
Juan R. Cubillos-Ruiz
Weill Cornell Medicine
Lead Author
Sung-Min Hwang
Weill Cornell Medicine
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology