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Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.

Tokumaru S, Higashiyama S, Endo T, Nakagawa T, Miyagawa JI, Yamamori K, Hanakawa Y, Ohmoto H, Yoshino K, Shirakata Y, Matsuzawa Y, Hashimoto K, Taniguchi N - J. Cell Biol. (2000)

Bottom Line: The resulting soluble ligands stimulated transient activation of EGFR.The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1.These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565-0871, Japan.

ABSTRACT
Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

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Dose effects of EGFR-Fc (a) and OSU8-1, OSU7-6, and OSU9-6 (b) on keratinocyte migration after wounding. Each point is the average of quadruplicate measurements. Migration was evaluated by the distance between the furthest migrated cell and the scraped edge.
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Figure 6: Dose effects of EGFR-Fc (a) and OSU8-1, OSU7-6, and OSU9-6 (b) on keratinocyte migration after wounding. Each point is the average of quadruplicate measurements. Migration was evaluated by the distance between the furthest migrated cell and the scraped edge.

Mentions: Cultured keratinocytes grew and spread into a wound area during a 3-d incubation as shown in Fig. 5. To confirm the involvement of EGFR signaling in the wound-induced growth and migration of these keratinocytes, EGFR neutralizing antibody No. 425 or a tyrosine kinase inhibitor specific for EGFR (tyrphostin AG1478) were introduced into the wounded keratinocyte cultures. Cells treated with antibody No. 425 or AG1478 were both suppressed in terms of proliferation (to approximately 10–15% of the control) and migration (Fig. 5; see also Fig. 7 b). We also employed an EGFR-Fc fusion protein to neutralize all EGFR ligands released by the keratinocytes, and achieved a 70% inhibition of migration (Fig. 6 a). These data indicate that EGFR activation and the subsequent molecular signaling play an essential role in the wound-induced growth and migration of keratinocytes. The addition of 1 μM OSU8-1 also significantly suppressed cell migration to a level comparable to that seen with antibody No. 425 and AG1478 (Fig. 5). In contrast, growth of the cells was not suppressed to the extent observed for No. 425 and AG1478 (only ∼50% inhibition as shown in Fig. 7 b). Colony growth in the nonscraped area of the cultured keratinocytes was observed in the presence of OSU8-1, again suggesting that this compound is more effective in inhibiting cell migration than cell growth. Cells treated with both 20 ng/ml of human recombinant HB-EGF and 1 μM of OSU8-1 showed superior growth and migration into the scraped area, compared with OSU8–treated cells and control cells (Fig. 5). On the other hand, the addition of HB-EGF to wounded keratinocytes that were also treated with No. 425 or AG1478 had no effect on cell growth or migration (data not shown). These data suggest that OSU8-1 does not interfere directly with EGFR activation and its downstream signaling pathway.


Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.

Tokumaru S, Higashiyama S, Endo T, Nakagawa T, Miyagawa JI, Yamamori K, Hanakawa Y, Ohmoto H, Yoshino K, Shirakata Y, Matsuzawa Y, Hashimoto K, Taniguchi N - J. Cell Biol. (2000)

Dose effects of EGFR-Fc (a) and OSU8-1, OSU7-6, and OSU9-6 (b) on keratinocyte migration after wounding. Each point is the average of quadruplicate measurements. Migration was evaluated by the distance between the furthest migrated cell and the scraped edge.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192647&req=5

Figure 6: Dose effects of EGFR-Fc (a) and OSU8-1, OSU7-6, and OSU9-6 (b) on keratinocyte migration after wounding. Each point is the average of quadruplicate measurements. Migration was evaluated by the distance between the furthest migrated cell and the scraped edge.
Mentions: Cultured keratinocytes grew and spread into a wound area during a 3-d incubation as shown in Fig. 5. To confirm the involvement of EGFR signaling in the wound-induced growth and migration of these keratinocytes, EGFR neutralizing antibody No. 425 or a tyrosine kinase inhibitor specific for EGFR (tyrphostin AG1478) were introduced into the wounded keratinocyte cultures. Cells treated with antibody No. 425 or AG1478 were both suppressed in terms of proliferation (to approximately 10–15% of the control) and migration (Fig. 5; see also Fig. 7 b). We also employed an EGFR-Fc fusion protein to neutralize all EGFR ligands released by the keratinocytes, and achieved a 70% inhibition of migration (Fig. 6 a). These data indicate that EGFR activation and the subsequent molecular signaling play an essential role in the wound-induced growth and migration of keratinocytes. The addition of 1 μM OSU8-1 also significantly suppressed cell migration to a level comparable to that seen with antibody No. 425 and AG1478 (Fig. 5). In contrast, growth of the cells was not suppressed to the extent observed for No. 425 and AG1478 (only ∼50% inhibition as shown in Fig. 7 b). Colony growth in the nonscraped area of the cultured keratinocytes was observed in the presence of OSU8-1, again suggesting that this compound is more effective in inhibiting cell migration than cell growth. Cells treated with both 20 ng/ml of human recombinant HB-EGF and 1 μM of OSU8-1 showed superior growth and migration into the scraped area, compared with OSU8–treated cells and control cells (Fig. 5). On the other hand, the addition of HB-EGF to wounded keratinocytes that were also treated with No. 425 or AG1478 had no effect on cell growth or migration (data not shown). These data suggest that OSU8-1 does not interfere directly with EGFR activation and its downstream signaling pathway.

Bottom Line: The resulting soluble ligands stimulated transient activation of EGFR.The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1.These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565-0871, Japan.

ABSTRACT
Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

Show MeSH
Related in: MedlinePlus