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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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Characterization of EGFR-ligand shedding in keratinocytes. (a) Gene expression of EGFR ligands in wounded keratinocytes. After incubation of keratinocytes for the indicated time after wounding, total RNA was extracted and analyzed by Northern blotting. Sequential hybridization using the same membrane is shown. HB-EGF (2.4 kb), AR (1.4 kb), and TGF-α (4.8 kb) messages were detected. (b) Effect of cycloheximide and HB-EGF neutralizing antibody No. 197 on wound-induced EGFR activation in cultured keratinocytes. Keratinocytes were stimulated by tip-scraping in the presence or absence of cycloheximide (50 μM) or HB-EGF neutralizing antibody No. 197 (10 μg/ml). After incubating for the indicated times, the cells were lysed and subjected to immunoprecipitation with EGFR antibodies, fractionation by SDS-PAGE, and Western blotting. Half of the immunocomplex was used for the detection of tyrosine phosphorylation with phosphotyrosine antibody PY-20, and the other half for detection of EGFR protein with EGFR antibody. Cycloheximide (50 μM) did not abrogate the wound-induced tyrosine phosphorylation of EGFR. On the other hand, antibody No. 197 (10 μg/ml) suppressed wound-induced tyrosine phosphorylation of EGFR by 70%. (c) Quantitative analyses of EGFR ligand shedding. Stable transfectants of HaCat cells expressing nearly equal amounts of AP-tag HB-EGF, AP-tag AR, or AP-Tag TGF-α were incubated with or without 1 μM OSU8-1 for 60 min after wounding. AP activities in the conditioned media were measured. Each bar is the average of triplicate values.
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Figure 4: Characterization of EGFR-ligand shedding in keratinocytes. (a) Gene expression of EGFR ligands in wounded keratinocytes. After incubation of keratinocytes for the indicated time after wounding, total RNA was extracted and analyzed by Northern blotting. Sequential hybridization using the same membrane is shown. HB-EGF (2.4 kb), AR (1.4 kb), and TGF-α (4.8 kb) messages were detected. (b) Effect of cycloheximide and HB-EGF neutralizing antibody No. 197 on wound-induced EGFR activation in cultured keratinocytes. Keratinocytes were stimulated by tip-scraping in the presence or absence of cycloheximide (50 μM) or HB-EGF neutralizing antibody No. 197 (10 μg/ml). After incubating for the indicated times, the cells were lysed and subjected to immunoprecipitation with EGFR antibodies, fractionation by SDS-PAGE, and Western blotting. Half of the immunocomplex was used for the detection of tyrosine phosphorylation with phosphotyrosine antibody PY-20, and the other half for detection of EGFR protein with EGFR antibody. Cycloheximide (50 μM) did not abrogate the wound-induced tyrosine phosphorylation of EGFR. On the other hand, antibody No. 197 (10 μg/ml) suppressed wound-induced tyrosine phosphorylation of EGFR by 70%. (c) Quantitative analyses of EGFR ligand shedding. Stable transfectants of HaCat cells expressing nearly equal amounts of AP-tag HB-EGF, AP-tag AR, or AP-Tag TGF-α were incubated with or without 1 μM OSU8-1 for 60 min after wounding. AP activities in the conditioned media were measured. Each bar is the average of triplicate values.

Mentions: To investigate the identity of the shed EGFR ligands and their origin at early times after wounding, Northern blot analyses, inhibition of de novo protein synthesis by cycloheximide, and neutralization experiments were carried out. In the early phase after wounding (0–6 h), little if any enhancement of HB-EGF, AR, or TGF-α gene expression was observed (Fig. 4 a). Cycloheximide failed to abrogate the EGFR activation induced by wounding (Fig. 4 b). These experiments indicated that the EGFR ligands were shed from transmembrane forms that were preexisting in the cells. HB-EGF neutralizing antibody No. 197 suppressed the wound-induced activation of EGFR by ∼70% (Fig. 4 b), suggesting that HB-EGF constitutes a major portion of the shed EGFR ligands in the keratinocyte conditioned medium.


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)

Characterization of EGFR-ligand shedding in keratinocytes. (a) Gene expression of EGFR ligands in wounded keratinocytes. After incubation of keratinocytes for the indicated time after wounding, total RNA was extracted and analyzed by Northern blotting. Sequential hybridization using the same membrane is shown. HB-EGF (2.4 kb), AR (1.4 kb), and TGF-α (4.8 kb) messages were detected. (b) Effect of cycloheximide and HB-EGF neutralizing antibody No. 197 on wound-induced EGFR activation in cultured keratinocytes. Keratinocytes were stimulated by tip-scraping in the presence or absence of cycloheximide (50 μM) or HB-EGF neutralizing antibody No. 197 (10 μg/ml). After incubating for the indicated times, the cells were lysed and subjected to immunoprecipitation with EGFR antibodies, fractionation by SDS-PAGE, and Western blotting. Half of the immunocomplex was used for the detection of tyrosine phosphorylation with phosphotyrosine antibody PY-20, and the other half for detection of EGFR protein with EGFR antibody. Cycloheximide (50 μM) did not abrogate the wound-induced tyrosine phosphorylation of EGFR. On the other hand, antibody No. 197 (10 μg/ml) suppressed wound-induced tyrosine phosphorylation of EGFR by 70%. (c) Quantitative analyses of EGFR ligand shedding. Stable transfectants of HaCat cells expressing nearly equal amounts of AP-tag HB-EGF, AP-tag AR, or AP-Tag TGF-α were incubated with or without 1 μM OSU8-1 for 60 min after wounding. AP activities in the conditioned media were measured. Each bar is the average of triplicate values.
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Related In: Results  -  Collection

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Figure 4: Characterization of EGFR-ligand shedding in keratinocytes. (a) Gene expression of EGFR ligands in wounded keratinocytes. After incubation of keratinocytes for the indicated time after wounding, total RNA was extracted and analyzed by Northern blotting. Sequential hybridization using the same membrane is shown. HB-EGF (2.4 kb), AR (1.4 kb), and TGF-α (4.8 kb) messages were detected. (b) Effect of cycloheximide and HB-EGF neutralizing antibody No. 197 on wound-induced EGFR activation in cultured keratinocytes. Keratinocytes were stimulated by tip-scraping in the presence or absence of cycloheximide (50 μM) or HB-EGF neutralizing antibody No. 197 (10 μg/ml). After incubating for the indicated times, the cells were lysed and subjected to immunoprecipitation with EGFR antibodies, fractionation by SDS-PAGE, and Western blotting. Half of the immunocomplex was used for the detection of tyrosine phosphorylation with phosphotyrosine antibody PY-20, and the other half for detection of EGFR protein with EGFR antibody. Cycloheximide (50 μM) did not abrogate the wound-induced tyrosine phosphorylation of EGFR. On the other hand, antibody No. 197 (10 μg/ml) suppressed wound-induced tyrosine phosphorylation of EGFR by 70%. (c) Quantitative analyses of EGFR ligand shedding. Stable transfectants of HaCat cells expressing nearly equal amounts of AP-tag HB-EGF, AP-tag AR, or AP-Tag TGF-α were incubated with or without 1 μM OSU8-1 for 60 min after wounding. AP activities in the conditioned media were measured. Each bar is the average of triplicate values.
Mentions: To investigate the identity of the shed EGFR ligands and their origin at early times after wounding, Northern blot analyses, inhibition of de novo protein synthesis by cycloheximide, and neutralization experiments were carried out. In the early phase after wounding (0–6 h), little if any enhancement of HB-EGF, AR, or TGF-α gene expression was observed (Fig. 4 a). Cycloheximide failed to abrogate the EGFR activation induced by wounding (Fig. 4 b). These experiments indicated that the EGFR ligands were shed from transmembrane forms that were preexisting in the cells. HB-EGF neutralizing antibody No. 197 suppressed the wound-induced activation of EGFR by ∼70% (Fig. 4 b), suggesting that HB-EGF constitutes a major portion of the shed EGFR ligands in the keratinocyte conditioned medium.

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