Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia

FASEB J. 2013 May;27(5):1939-49. doi: 10.1096/fj.12-217414. Epub 2013 Feb 4.


Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Arachidonate 12-Lipoxygenase / drug effects
  • Arachidonate 12-Lipoxygenase / metabolism*
  • Arachidonate 15-Lipoxygenase / drug effects
  • Arachidonate 15-Lipoxygenase / metabolism*
  • HEK293 Cells
  • Humans
  • Hyperalgesia / etiology*
  • Intramolecular Oxidoreductases / metabolism*
  • Lipoxygenase Inhibitors / pharmacology
  • Male
  • Rats


  • 12-15-lipoxygenase
  • Lipoxygenase Inhibitors
  • Arachidonate 12-Lipoxygenase
  • Arachidonate 15-Lipoxygenase
  • Intramolecular Oxidoreductases
  • hepoxilin synthase