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

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

Ann M Gregus et al. FASEB J. 2013 May.

Abstract

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.

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Figures

Figure 1.

Figure 1.

Putative activities of the rat 12/15-LOX enzyme family using arachidonic acid (AA) as substrate. AA can be metabolized by each of 6 rat 12-LOX family enzymes (see Table 1), which exhibit one or more of the following activities depicted above: 15-LOX, 12-LOX, or HXS.

Figure 2.

Figure 2.

Experimental validation of rat 12/15-LOX overexpression systems. mRNA expression of rat 12/15-LOX enzymes overexpressed in HEK-293T cells (HEK-12/15-LOX) as detected by qPCR. Transient transfection: U, untransfected control; 12, Alox12; 12b, Alox12b; 12e, Alox12e; e3, Aloxe3; 15, Alox15; 15b, Alox15b.

Figure 3.

Figure 3.

Experimental validation of rat 12/15-LOX antibodies. Antibodies detecting all six 12/15-LOX isozymes 12-LOX (pan; A) or those selectively targeting e3 (Aloxe3; B), 15 (Alox15; C), or 15b (Alox15b; D), as revealed by immunoblot of lysates from HEK-293T cells overexpressing each of the 6 rat 12/15-LOX enzymes. Samples were run with β-actin as a loading control in each lane as follows: U, untransfected control; 12, Alox12; 12b, Alox12b; 12e, Alox12e; e3, Aloxe3; 15, Alox15; 15b, Alox15b.

Figure 4.

Figure 4.

Activities of overexpressed rat 12/15-LOX enzymes. 15-LOX, 8-LOX, or 12-LOX activity with exposure of HEK-12/15-LOX cells to AA substrate as revealed by the release of 15-HETE (A), 8-HETE (B), or 12-HETE (C) from HEK-293T cells either untreated (ctrl) or treated with AA (70 μM), as follows: U, untransfected control; 12, Alox12; 12b, Alox12b; 12e, Alox12e; e3, Aloxe3; 15, Alox15; 15b, Alox15b. ***P < 0.001 vs. untransfected control; n = 4 wells/treatment.

Figure 5.

Figure 5.

Formation of hepoxilins observed from rat 12(S)- and (R)-type LOXs and Aloxe3/eLOX3 using AA or 12-HpETE as substrate. Hepoxilins (HXB3) are synthesized directly from AA (70 μM) by Alox12 or Alox15 (A); by Aloxe3 when it is coexpressed at a 1:1 ratio with Alox12b (B); from 12(S)-HpETE (5 μM) by Alox12, Aloxe3, or Alox15 (C); or from racemic 12(R,S)-HpETE (10 μM; D). U, untransfected control; 12, Alox12; 12b, Alox12b; 12e, Alox12e; e3, Aloxe3; 15, Alox15; 15b, Alox15b. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control or baseline; n = 4 wells/treatment.

Figure 6.

Figure 6.

Spinal expression of the hepoxilin synthase eLOX3 is increased following peripheral inflammation. Quantitative PCR and immunoblot reveal isozyme-specific expression of 12/15-LOX in rat spinal cord. Basal expression of Alox12, Alox12b, Aloxe3, and Alox15 mRNA (A) and induction of 12/15-LOX (as detected by pan antibody; B) and eLOX3 (Aloxe3; C) but not 12/15-LOX-l (Alox15) proteins (D) in spinal cord at 4 h after IPLT carrageenan. U, untreated/naive; C, 4 h postintraplantar carrageenan; +, transfected HEK293T cell lysate positive controls for Alox12 (B), Aloxe3 (C), or Alox15 (D). Total protein loaded: spinal cord, 30 μg; positive control, 10 μg. *P < 0.05 vs. control (untreated); n = 6 rats/group.

Figure 7.

Figure 7.

Spinal inhibition of eLOX3 or knockdown of Aloxe3 attenuates inflammatory hyperesthesia. IT pretreatment with either prevalidated antibody (5 μg) selectively targeting eLOX3 protein encoded by Aloxe3 (e3; A, B) or siRNA (2 μg; C, D) against Aloxe3 significantly reduced carrageenan (carra)-induced tactile allodynia. *P < 0.05, **P < 0.01 vs. controls (saline vehicle, 5 μg rabbit IgG, or 2 μg nontargeting siRNA); n = 6–10 rats/group.

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