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

Expression of AP-tagged EGFR ligands. (a) Structure of the AP-tag parental expression plasmid, pSS-AlPh. (b) A schematic diagram of the region within the expression plasmids derived from pss-AlPh that encode the fusions of AP-tag and EGFR ligands. The coding region for the AP-tag HB-EGF fusion was constructed such that, in the encoded fusion, the AP sequence (488 amino acids) was inserted at Ala84 of the HB-EGF precursor. In the AP-tag AR and AP-tag TGF-α fusion proteins, AR (residues 102–252) and TGF-α (residues 45–160), respectively, were substituted for HB-EGF (residues 85–208). (c) Expression of AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α on the surface of transfected CHO cells. Biotinylated AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α were immunoprecipitated with human placental AP antibody and analyzed by SDS-PAGE and Western blotting. Biotinylated proteins on the Western blot membranes were detected by HRP-conjugated avidin and ECL. (d) AP activity in the conditioned media of transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α. Cells were treated with or without 60 nM TPA for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2192647&req=5

Figure 1: Expression of AP-tagged EGFR ligands. (a) Structure of the AP-tag parental expression plasmid, pSS-AlPh. (b) A schematic diagram of the region within the expression plasmids derived from pss-AlPh that encode the fusions of AP-tag and EGFR ligands. The coding region for the AP-tag HB-EGF fusion was constructed such that, in the encoded fusion, the AP sequence (488 amino acids) was inserted at Ala84 of the HB-EGF precursor. In the AP-tag AR and AP-tag TGF-α fusion proteins, AR (residues 102–252) and TGF-α (residues 45–160), respectively, were substituted for HB-EGF (residues 85–208). (c) Expression of AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α on the surface of transfected CHO cells. Biotinylated AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α were immunoprecipitated with human placental AP antibody and analyzed by SDS-PAGE and Western blotting. Biotinylated proteins on the Western blot membranes were detected by HRP-conjugated avidin and ECL. (d) AP activity in the conditioned media of transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α. Cells were treated with or without 60 nM TPA for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values.

Mentions: The coding region for human placental alkaline phosphatase (AP) was obtained from the APtag-1 expression vector (a gift from Dr. J. Flanagan, Harvard Medical School, Boston, MA) and inserted into the multiple cloning site of pRc/CMV (Invitrogen). A BstBI-MscI fragment of the human HB-EGF cDNA, which encodes the signal sequence and prosequence, was fused to the 5′ end of the AP sequence to create the plasmid pSS-AlPh (see Fig. 1 a). As pSS-AlPh has a unique HpaI site overlapping the termination codon of the AP coding region, one of the following DNA fragments encoding human EGF–related ligands were fused in-frame at this site: MscI-PstI fragment of the HB-EGF cDNA (0.45 kb); HinfI fragment of the AR cDNA (0.5 kb); or MseI-SauIII fragment of the TGF-α cDNA (0.38 kb). The plasmids resulting from these ligations were introduced into CHO cells using the calcium phosphate method (Chen and Okayama 1988), and stably transfected clones were isolated.


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)

Expression of AP-tagged EGFR ligands. (a) Structure of the AP-tag parental expression plasmid, pSS-AlPh. (b) A schematic diagram of the region within the expression plasmids derived from pss-AlPh that encode the fusions of AP-tag and EGFR ligands. The coding region for the AP-tag HB-EGF fusion was constructed such that, in the encoded fusion, the AP sequence (488 amino acids) was inserted at Ala84 of the HB-EGF precursor. In the AP-tag AR and AP-tag TGF-α fusion proteins, AR (residues 102–252) and TGF-α (residues 45–160), respectively, were substituted for HB-EGF (residues 85–208). (c) Expression of AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α on the surface of transfected CHO cells. Biotinylated AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α were immunoprecipitated with human placental AP antibody and analyzed by SDS-PAGE and Western blotting. Biotinylated proteins on the Western blot membranes were detected by HRP-conjugated avidin and ECL. (d) AP activity in the conditioned media of transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α. Cells were treated with or without 60 nM TPA for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Expression of AP-tagged EGFR ligands. (a) Structure of the AP-tag parental expression plasmid, pSS-AlPh. (b) A schematic diagram of the region within the expression plasmids derived from pss-AlPh that encode the fusions of AP-tag and EGFR ligands. The coding region for the AP-tag HB-EGF fusion was constructed such that, in the encoded fusion, the AP sequence (488 amino acids) was inserted at Ala84 of the HB-EGF precursor. In the AP-tag AR and AP-tag TGF-α fusion proteins, AR (residues 102–252) and TGF-α (residues 45–160), respectively, were substituted for HB-EGF (residues 85–208). (c) Expression of AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α on the surface of transfected CHO cells. Biotinylated AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α were immunoprecipitated with human placental AP antibody and analyzed by SDS-PAGE and Western blotting. Biotinylated proteins on the Western blot membranes were detected by HRP-conjugated avidin and ECL. (d) AP activity in the conditioned media of transfectants expressing AP-tag HB-EGF, AP-tag AR, and AP-tag TGF-α. Cells were treated with or without 60 nM TPA for 30 min at 37°C. AP activity was measured as described in Materials and Methods. Each bar is the average of triplicate values.
Mentions: The coding region for human placental alkaline phosphatase (AP) was obtained from the APtag-1 expression vector (a gift from Dr. J. Flanagan, Harvard Medical School, Boston, MA) and inserted into the multiple cloning site of pRc/CMV (Invitrogen). A BstBI-MscI fragment of the human HB-EGF cDNA, which encodes the signal sequence and prosequence, was fused to the 5′ end of the AP sequence to create the plasmid pSS-AlPh (see Fig. 1 a). As pSS-AlPh has a unique HpaI site overlapping the termination codon of the AP coding region, one of the following DNA fragments encoding human EGF–related ligands were fused in-frame at this site: MscI-PstI fragment of the HB-EGF cDNA (0.45 kb); HinfI fragment of the AR cDNA (0.5 kb); or MseI-SauIII fragment of the TGF-α cDNA (0.38 kb). The plasmids resulting from these ligations were introduced into CHO cells using the calcium phosphate method (Chen and Okayama 1988), and stably transfected clones were isolated.

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