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12-Hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor.

Liu M, Saeki K, Matsunobu T, Okuno T, Koga T, Sugimoto Y, Yokoyama C, Nakamizo S, Kabashima K, Narumiya S, Shimizu T, Yokomizo T - J. Exp. Med. (2014)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan Department of Medical Biochemistry, Kyushu University, Fukuoka 812-8582, Japan.

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TNF and MMP9 participate in 12-HHT/BLT2–dependent mouse primary keratinocyte migration. (A) Relative mRNA levels of TNF were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were stimulated with 100 nM 12-HHT for the indicated time (n = 4 experimental replicates). The relative mRNA level in unstimulated BLT2 WT keratinocytes was set as 1. (B) Relative levels of MMP9 mRNA were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes (n = 4 experimental replicates). The relative mRNA level in BLT2 WT keratinocytes was set as 1. (C) Mouse MMP9 protein levels in the culture medium of mouse primary keratinocytes were measured by ELISA after 12 and 24 h of culture (n = 4 experimental replicates). (D) The effect of mouse TNF neutralizing antibody D2H4 on the migration of BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were cultured in medium containing 1 nM 12-HHT and 10 ng/ml control IgG or 10 ng/ml D2H4 for 39 h (n = 5–6 experimental replicates). Data represent the mean ± SEM. **, P < 0.01; *, P < 0.05 (A and D, two-way ANOVA with Bonferroni post hoc tests; B and C, unpaired Student’s t test). All the results are representative of at least two independent experiments.
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fig8: TNF and MMP9 participate in 12-HHT/BLT2–dependent mouse primary keratinocyte migration. (A) Relative mRNA levels of TNF were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were stimulated with 100 nM 12-HHT for the indicated time (n = 4 experimental replicates). The relative mRNA level in unstimulated BLT2 WT keratinocytes was set as 1. (B) Relative levels of MMP9 mRNA were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes (n = 4 experimental replicates). The relative mRNA level in BLT2 WT keratinocytes was set as 1. (C) Mouse MMP9 protein levels in the culture medium of mouse primary keratinocytes were measured by ELISA after 12 and 24 h of culture (n = 4 experimental replicates). (D) The effect of mouse TNF neutralizing antibody D2H4 on the migration of BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were cultured in medium containing 1 nM 12-HHT and 10 ng/ml control IgG or 10 ng/ml D2H4 for 39 h (n = 5–6 experimental replicates). Data represent the mean ± SEM. **, P < 0.01; *, P < 0.05 (A and D, two-way ANOVA with Bonferroni post hoc tests; B and C, unpaired Student’s t test). All the results are representative of at least two independent experiments.

Mentions: The involvement of TNF and MMP9 in cell migration was also investigated in mouse primary keratinocytes isolated from BLT2 WT and BLT2 KO mice. 12-HHT significantly up-regulated the transcription of TNF only in BLT2 WT keratinocytes but not in BLT2 KO keratinocytes (Fig. 8 A). Both MMP9 mRNA (Fig. 8 B) and MMP9 protein levels (Fig. 8 C) were significantly higher in BLT2 WT keratinocytes. The mouse TNF-neutralizing antibody D2H4 only reduced the migration of BLT2 WT keratinocytes (Fig. 8 D). Collectively, these results clearly indicate that the 12-HHT/BLT2 axis accelerates keratinocyte migration by stimulating NF-κB signaling, which then induces the expression of TNF and MMP9 to promote wound healing.


12-Hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor.

Liu M, Saeki K, Matsunobu T, Okuno T, Koga T, Sugimoto Y, Yokoyama C, Nakamizo S, Kabashima K, Narumiya S, Shimizu T, Yokomizo T - J. Exp. Med. (2014)

TNF and MMP9 participate in 12-HHT/BLT2–dependent mouse primary keratinocyte migration. (A) Relative mRNA levels of TNF were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were stimulated with 100 nM 12-HHT for the indicated time (n = 4 experimental replicates). The relative mRNA level in unstimulated BLT2 WT keratinocytes was set as 1. (B) Relative levels of MMP9 mRNA were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes (n = 4 experimental replicates). The relative mRNA level in BLT2 WT keratinocytes was set as 1. (C) Mouse MMP9 protein levels in the culture medium of mouse primary keratinocytes were measured by ELISA after 12 and 24 h of culture (n = 4 experimental replicates). (D) The effect of mouse TNF neutralizing antibody D2H4 on the migration of BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were cultured in medium containing 1 nM 12-HHT and 10 ng/ml control IgG or 10 ng/ml D2H4 for 39 h (n = 5–6 experimental replicates). Data represent the mean ± SEM. **, P < 0.01; *, P < 0.05 (A and D, two-way ANOVA with Bonferroni post hoc tests; B and C, unpaired Student’s t test). All the results are representative of at least two independent experiments.
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Related In: Results  -  Collection

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fig8: TNF and MMP9 participate in 12-HHT/BLT2–dependent mouse primary keratinocyte migration. (A) Relative mRNA levels of TNF were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were stimulated with 100 nM 12-HHT for the indicated time (n = 4 experimental replicates). The relative mRNA level in unstimulated BLT2 WT keratinocytes was set as 1. (B) Relative levels of MMP9 mRNA were measured by Q-PCR in BLT2 WT and BLT2 KO mouse primary keratinocytes (n = 4 experimental replicates). The relative mRNA level in BLT2 WT keratinocytes was set as 1. (C) Mouse MMP9 protein levels in the culture medium of mouse primary keratinocytes were measured by ELISA after 12 and 24 h of culture (n = 4 experimental replicates). (D) The effect of mouse TNF neutralizing antibody D2H4 on the migration of BLT2 WT and BLT2 KO mouse primary keratinocytes. Cells were cultured in medium containing 1 nM 12-HHT and 10 ng/ml control IgG or 10 ng/ml D2H4 for 39 h (n = 5–6 experimental replicates). Data represent the mean ± SEM. **, P < 0.01; *, P < 0.05 (A and D, two-way ANOVA with Bonferroni post hoc tests; B and C, unpaired Student’s t test). All the results are representative of at least two independent experiments.
Mentions: The involvement of TNF and MMP9 in cell migration was also investigated in mouse primary keratinocytes isolated from BLT2 WT and BLT2 KO mice. 12-HHT significantly up-regulated the transcription of TNF only in BLT2 WT keratinocytes but not in BLT2 KO keratinocytes (Fig. 8 A). Both MMP9 mRNA (Fig. 8 B) and MMP9 protein levels (Fig. 8 C) were significantly higher in BLT2 WT keratinocytes. The mouse TNF-neutralizing antibody D2H4 only reduced the migration of BLT2 WT keratinocytes (Fig. 8 D). Collectively, these results clearly indicate that the 12-HHT/BLT2 axis accelerates keratinocyte migration by stimulating NF-κB signaling, which then induces the expression of TNF and MMP9 to promote wound healing.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan Department of Medical Biochemistry, Kyushu University, Fukuoka 812-8582, Japan.

Show MeSH
Related in: MedlinePlus