12-Hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor.
Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4.Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive.These results identify a novel mechanism underlying the action of the 12-HHT/BLT2 axis in epidermal keratinocytes and accordingly suggest the use of BLT2 agonists as therapeutic agents to accelerate wound healing, particularly for intractable wounds, such as diabetic ulcers.
Affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan Department of Medical Biochemistry, Kyushu University, Fukuoka 812-8582, Japan.
- Cell Movement*
- Fatty Acids, Unsaturated/metabolism*
- Receptors, Leukotriene B4/agonists/genetics/metabolism*
- Wound Healing/drug effects/physiology*
- Cell Line
- Cells, Cultured
- Diabetes Mellitus, Type 2/genetics/metabolism/physiopathology
- Enzyme-Linked Immunosorbent Assay
- Flow Cytometry
- Gene Expression Profiling
- Matrix Metalloproteinase 9/genetics
- Mice, Inbred C57BL
- Mice, Knockout
- Oligonucleotide Array Sequence Analysis
- Reverse Transcriptase Polymerase Chain Reaction
- Tumor Necrosis Factor-alpha/genetics
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fig7: TNF and MMP9 participate in 12-HHT/BLT2–dependent HaCaT cell migration. (A) Relative levels of TNF, IL-1β, and MMP9 mRNA were measured by Q-PCR in BLT2 WT and BLT2 KO mouse skin (n = 3–7 mice per group). Punch biopsies (5 mm in diameter) were obtained at 2 d after skin punching and used for the assay. The relative mRNA level in uninjured BLT2 WT mice was set as 1. (B) HaCaT-mock and HaCaT-BLT2 cells were stimulated with 1 µM 12-HHT, and the relative levels of TNF, IL-1β, and MMP9 mRNA were measured by Q-PCR (n = 3 experimental replicates). (C) HaCaT-mock and HaCaT-BLT2 cells were stimulated with EtOH vehicle or 1 µM 12-HHT for 6 h, and TNF protein levels were assessed in the culture medium (n = 3 experimental replicates). (D and E) MMP9 activity in the culture medium was measured by zymography. HaCaT-mock and HaCaT-BLT2 cells were serum-starved for 3 h and then stimulated for 24 h with EtOH vehicle, 1 µM 12-HHT, or 10 ng/ml TNF (D, n = 3 experimental replicates), or with 1 µM 12-HHT and 100 µg/ml control IgG or 100 µg/ml Infliximab (E, n = 3 experimental replicates). (F) Determination of TNF and IL-1β mRNA stability in HaCaT-mock and HaCaT-BLT2 cells. The cells were cultured in medium containing 5 µg/ml actinomycin d and EtOH vehicle or 1 µM 12-HHT and then harvested at the indicated times. TNF and IL-1β mRNA levels were quantified by Q-PCR (n = 3 experimental replicates). (G) Determination of NF-κB activity in HaCaT-mock and HaCaT-BLT2 cells. Cells were stimulated with EtOH vehicle or 1 µM 12-HHT, and subjected to the dual luciferase assay (n = 3 experimental replicates). (H and I) Quantification of cell migration of HaCaT-mock and HaCaT-BLT2 cells at 15 h after scratching. Cells were cultured in serum-free medium without 12-HHT or with 100 nM 12-HHT, and control IgG or 100 µg/ml Infliximab (H, n = 5–8 experimental replicates); or DMSO and 10 µM MMP9 inhibitor I (I, left, n = 6–8 experimental replicates) or 10 µM MMP inhibitor II (I, right, n = 6–10 experimental replicates). Data represent the mean ± SEM. **, P < 0.01; *, P < 0.05 (A and B, unpaired Student’s t test; C–E and G–I, two-way ANOVA with Bonferroni post hoc tests). All the results are representative of at least two independent experiments.
To gain insight into the molecular mechanism by which the 12-HHT/BLT2 axis stimulates keratinocyte migration, we investigated global transcription in mouse skin by DNA microarray analysis. Total RNA was isolated from the skin of BLT2 WT and BLT2 KO mice at 2 d after skin punching or without skin punching. The RNA was then evaluated for its content of injury-related transcripts. The analysis revealed that injury-related cytokines, chemokines, and MMPs, which are reported to enhance keratinocyte migration (Gillitzer and Goebeler, 2001; Kyriakides et al., 2009), were down-regulated in uninjured BLT2 KO mouse skin (data deposited to GEO repository under accession no. GSE53400). Q-PCR confirmed that the mRNA levels of TNF, IL-1β, and MMP9 were significantly lower in the uninjured skin of BLT2 KO mice than those in BLT2 WT mice, and these transcripts were all up-regulated after skin punching in both groups (Fig. 7 A). Consistent with these observations, the transcription levels of TNF, IL-1β, and MMP9 were up-regulated in 12-HHT–treated HaCaT-BLT2 cells (Fig. 7 B). Interestingly, 12-HHT initially induced expression of TNF and IL-1β mRNA, followed somewhat later by MMP9 mRNA, suggesting that MMP9 transcription occurs downstream of TNF and IL-1β transcription.