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.

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.

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.

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.

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.

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.

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.

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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References

1. Buczynski M. W., Dumlao D. S., Dennis E. A. (2009) Thematic Review Series: Proteomics. An integrated omics analysis of eicosanoid biology. J. Lipid Res. 50, 1015–1038 - PMC - PubMed
1. Kuhn H., O'Donnell V. B. (2006) Inflammation and immune regulation by 12/15-lipoxygenases. Prog. Lipid Res. 45, 334–356 - PubMed
1. Ivanov I., Heydeck D., Hofheinz K., Roffeis J., O'Donnell V. B., Kuhn H., Walther M. (2010) Molecular enzymology of lipoxygenases. Arch. Biochem. Biophys. 503, 161–174 - PubMed
1. Krieg P., Marks F., Furstenberger G. (2001) A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression. Genomics 73, 323–330 - PubMed
1. Bendani M. K., Palluy O., Cook-Moreau J., Beneytout J. L., Rigaud M., Vallat J. M. (1995) Localization of 12-lipoxygenase mRNA in cultured oligodendrocytes and astrocytes by in situ reverse transcriptase and polymerase chain reaction. Neurosci. Lett. 189, 159–162 - PubMed

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