Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of OSU8-1 on TPA-inducible shedding of EGFR ligands. (a) AP activities in the conditioned media of CHO transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α treated with or without 60 nM of TPA in the presence or absence of OSU8-1. Cells were preincubated with the indicated concentration of OSU8-1 for 10 min at 37°C. The cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values. (b) CHO transfectants expressing wild-type (non–AP-tag fused) HB-EGF, AR, and TGF-α were biotinylated and preincubated with or without 10 μM OSU8-1 for 10 min at 37°C. Cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C, followed by cell lysis, immunoprecipitation with antibodies against the shed ligands, SDS-PAGE, Western blotting, and avidin-HRP/ECL detection. The wild-type HB-EGF shows different processing forms (Goishi et al. 1995).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192647&req=5

Figure 2: Effect of OSU8-1 on TPA-inducible shedding of EGFR ligands. (a) AP activities in the conditioned media of CHO transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α treated with or without 60 nM of TPA in the presence or absence of OSU8-1. Cells were preincubated with the indicated concentration of OSU8-1 for 10 min at 37°C. The cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values. (b) CHO transfectants expressing wild-type (non–AP-tag fused) HB-EGF, AR, and TGF-α were biotinylated and preincubated with or without 10 μM OSU8-1 for 10 min at 37°C. Cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C, followed by cell lysis, immunoprecipitation with antibodies against the shed ligands, SDS-PAGE, Western blotting, and avidin-HRP/ECL detection. The wild-type HB-EGF shows different processing forms (Goishi et al. 1995).

Mentions: Using the AP-tag EGFR ligand transfectants, >500 synthetic compounds were tested for the ability to inhibit shedding of the ligands. Since it has been reported that a matrix metalloprotease (MMP) is involved in the shedding of HB-EGF and TGF-α (Suzuki et al. 1997; Izumi et al. 1998; Peschon et al. 1998), these compounds were designed as potential MMP inhibitors. The most effective compound was OSU8-1. 1 μM OSU8-1 markedly blocked the shedding of AP-tag HB-EGF and AP-tag AR, and partially blocked the shedding of AP-tag TGF-α (Fig. 2 a). A 10-fold higher concentration of OSU8-1 (10 μM) significantly blocked the TPA-inducible shedding of all three AP-tagged EGFR ligands (Fig. 2 a). Cell-surface biotinylation and immunoprecipitation of wild-type HB-EGF, TGF-α, and AR also revealed the abrogation of their TPA-inducible shedding by 10 μM of OSU8-1 (Fig. 2 b). We examined the inhibitory spectrum of OSU8-1 with regard to the following three representative MMPs: MMP-1, MMP-3, and MMP-9. As shown in Table , OSU8-1 effectively inhibited the activities of all three MMPs with IC50s of 0.3–2.9 nM. We also selected two more compounds, OSU9-6 and OSU7-6 that showed similar inhibitory activities for the shedding of EGFR ligands, but which displayed distinguishable inhibitory activities for the three tested MMPs (Table ). These three inhibitors were used for characterization of EGFR ligand shedding in the wound experiments described below.


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)

Effect of OSU8-1 on TPA-inducible shedding of EGFR ligands. (a) AP activities in the conditioned media of CHO transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α treated with or without 60 nM of TPA in the presence or absence of OSU8-1. Cells were preincubated with the indicated concentration of OSU8-1 for 10 min at 37°C. The cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values. (b) CHO transfectants expressing wild-type (non–AP-tag fused) HB-EGF, AR, and TGF-α were biotinylated and preincubated with or without 10 μM OSU8-1 for 10 min at 37°C. Cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C, followed by cell lysis, immunoprecipitation with antibodies against the shed ligands, SDS-PAGE, Western blotting, and avidin-HRP/ECL detection. The wild-type HB-EGF shows different processing forms (Goishi et al. 1995).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Effect of OSU8-1 on TPA-inducible shedding of EGFR ligands. (a) AP activities in the conditioned media of CHO transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α treated with or without 60 nM of TPA in the presence or absence of OSU8-1. Cells were preincubated with the indicated concentration of OSU8-1 for 10 min at 37°C. The cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values. (b) CHO transfectants expressing wild-type (non–AP-tag fused) HB-EGF, AR, and TGF-α were biotinylated and preincubated with or without 10 μM OSU8-1 for 10 min at 37°C. Cells were incubated with fresh media containing the indicated concentrations of TPA and OSU8-1 for 30 min at 37°C, followed by cell lysis, immunoprecipitation with antibodies against the shed ligands, SDS-PAGE, Western blotting, and avidin-HRP/ECL detection. The wild-type HB-EGF shows different processing forms (Goishi et al. 1995).
Mentions: Using the AP-tag EGFR ligand transfectants, >500 synthetic compounds were tested for the ability to inhibit shedding of the ligands. Since it has been reported that a matrix metalloprotease (MMP) is involved in the shedding of HB-EGF and TGF-α (Suzuki et al. 1997; Izumi et al. 1998; Peschon et al. 1998), these compounds were designed as potential MMP inhibitors. The most effective compound was OSU8-1. 1 μM OSU8-1 markedly blocked the shedding of AP-tag HB-EGF and AP-tag AR, and partially blocked the shedding of AP-tag TGF-α (Fig. 2 a). A 10-fold higher concentration of OSU8-1 (10 μM) significantly blocked the TPA-inducible shedding of all three AP-tagged EGFR ligands (Fig. 2 a). Cell-surface biotinylation and immunoprecipitation of wild-type HB-EGF, TGF-α, and AR also revealed the abrogation of their TPA-inducible shedding by 10 μM of OSU8-1 (Fig. 2 b). We examined the inhibitory spectrum of OSU8-1 with regard to the following three representative MMPs: MMP-1, MMP-3, and MMP-9. As shown in Table , OSU8-1 effectively inhibited the activities of all three MMPs with IC50s of 0.3–2.9 nM. We also selected two more compounds, OSU9-6 and OSU7-6 that showed similar inhibitory activities for the shedding of EGFR ligands, but which displayed distinguishable inhibitory activities for the three tested MMPs (Table ). These three inhibitors were used for characterization of EGFR ligand shedding in the wound experiments described below.

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