Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37

Sho Tokumaru, Koji Sayama, Yuji Shirakata, Hitoshi Komatsuzawa, Kazuhisa Ouhara, Yasushi Hanakawa, Yoko Yahata, Xiuju Dai, Mikiko Tohyama, Hiroshi Nagai, Lujun Yang, Shigeki Higashiyama, Akihiko Yoshimura, Motoyuki Sugai, Koji Hashimoto

Research output: Contribution to journalArticle

228 Citations (Scopus)

Abstract

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinecyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.

Original languageEnglish
Pages (from-to)4662-4668
Number of pages7
JournalJournal of Immunology
Volume175
Issue number7
Publication statusPublished - 2005 Oct 1
Externally publishedYes

Fingerprint

Keratinocytes
Epidermal Growth Factor Receptor
Transcriptional Activation
Skin
Transfection
Wounds and Injuries
Cytokines
antimicrobial peptide LL-37
Epidermal Growth Factor
Wound Healing
Heparin
Carrier Proteins
Phosphorylation
Peptides

ASJC Scopus subject areas

  • Immunology

Cite this

Tokumaru, S., Sayama, K., Shirakata, Y., Komatsuzawa, H., Ouhara, K., Hanakawa, Y., ... Hashimoto, K. (2005). Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. Journal of Immunology, 175(7), 4662-4668.

Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. / Tokumaru, Sho; Sayama, Koji; Shirakata, Yuji; Komatsuzawa, Hitoshi; Ouhara, Kazuhisa; Hanakawa, Yasushi; Yahata, Yoko; Dai, Xiuju; Tohyama, Mikiko; Nagai, Hiroshi; Yang, Lujun; Higashiyama, Shigeki; Yoshimura, Akihiko; Sugai, Motoyuki; Hashimoto, Koji.

In: Journal of Immunology, Vol. 175, No. 7, 01.10.2005, p. 4662-4668.

Research output: Contribution to journalArticle

Tokumaru, S, Sayama, K, Shirakata, Y, Komatsuzawa, H, Ouhara, K, Hanakawa, Y, Yahata, Y, Dai, X, Tohyama, M, Nagai, H, Yang, L, Higashiyama, S, Yoshimura, A, Sugai, M & Hashimoto, K 2005, 'Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37', Journal of Immunology, vol. 175, no. 7, pp. 4662-4668.
Tokumaru S, Sayama K, Shirakata Y, Komatsuzawa H, Ouhara K, Hanakawa Y et al. Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. Journal of Immunology. 2005 Oct 1;175(7):4662-4668.
Tokumaru, Sho ; Sayama, Koji ; Shirakata, Yuji ; Komatsuzawa, Hitoshi ; Ouhara, Kazuhisa ; Hanakawa, Yasushi ; Yahata, Yoko ; Dai, Xiuju ; Tohyama, Mikiko ; Nagai, Hiroshi ; Yang, Lujun ; Higashiyama, Shigeki ; Yoshimura, Akihiko ; Sugai, Motoyuki ; Hashimoto, Koji. / Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. In: Journal of Immunology. 2005 ; Vol. 175, No. 7. pp. 4662-4668.
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abstract = "The closure of skin wounds is essential for resistance against microbial pathogens, and keratinecyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.",
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AU - Ouhara, Kazuhisa

AU - Hanakawa, Yasushi

AU - Yahata, Yoko

AU - Dai, Xiuju

AU - Tohyama, Mikiko

AU - Nagai, Hiroshi

AU - Yang, Lujun

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AU - Sugai, Motoyuki

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N2 - The closure of skin wounds is essential for resistance against microbial pathogens, and keratinecyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.

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