QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02346

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shirakata, Y. Articles by Hashimoto, K. Search for Related Content PubMed PubMed Citation Articles by Shirakata, Y. Articles by Hashimoto, K.

Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing

Yuji Shirakata^1,*,, Rina Kimura^2,*, Daisuke Nanba³, Ryo Iwamoto², Sho Tokumaru¹, Chie Morimoto³, Koichi Yokota⁴, Masanori Nakamura⁴, Koji Sayama¹, Eisuke Mekada², Shigeki Higashiyama³ and Koji Hashimoto¹

¹ Department of Dermatology, Ehime University School of Medicine, Ehime 791-0295, Japan
² Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
³ Biochemistry and Molecular Genetics, Ehime University School of Medicine, Ehime 791-0295, Japan
⁴ Carna Biosciences Incorporated, Kobe, Hyogo 650-0047, Japan

View larger version (52K): [in a new window]   Fig. 1. Induction of expression of EGFR ligand mRNA, by scraping, in human and mouse keratinocytes. Confluent NHEK (A) and normal mouse epidermal keratinocytes (B) were scraped with a pipette tip; total RNA was harvested at several time points and mRNA expression was analyzed by RT-PCR. Right panels show densitometric analysis. In both cell types HB-EGF mRNA was rapidly and dramatically induced after scraping, whereas TGF-, AR, and EPR mRNA were slightly induced in NHEK and the normal keratinocytes, although in the latter cells EPR was also increased.

View larger version (47K): [in a new window]   Fig. 2. Genotype of the keratinocyte-specific HB-EGF-deficient mice. (A-C) Keratinocyte-specific HB-EGF-deficient mice were confirmed by PCR as lox homozygous (A), Cre-recombinase positive (B) and lox-out (C). (D) Keratinocyte-specific disruption of HB-EGF mRNA in HB–/– mice was confirmed by RT-PCR. KC, keratinocytes.

View larger version (47K): [in a new window]   Fig. 3. Impaired wound healing in HB–/– mice. Two 6-mm punch biopsies were made in the skin of the backs of HBlox/lox and HB–/– mice, and wound diameter was monitored. (A) Macroscopic view of wound healing assay in HBlox/lox and HB–/– mice at day 8. (B) Measurements of wound diameter during healing. *P<0.05.

View larger version (68K): [in a new window]   Fig. 4. BrdU-positive cell distribution at the leading edge and in the peripheral skin in HBlox/lox and HB–/– mice. (A) Skin sections were stained using anti-BrdU antibody, and cell numbers in the leading edge and in the peripheral skin were counted as indicated. (B) The total cell numbers in the peripheral skin (1.2 mm from the wound margin) and in the leading edge. (C) BrdU-positive cell number in the peripheral skin and in the leading edge. There were no differences in BrdU-positive cell numbers between HBlox/lox and HB–/– mice.

View larger version (48K): [in a new window]   Fig. 5. Impaired keratinocyte migration in HB–/– mice. (A) Serial sections were prepared, and the epidermis was stained with anti-keratin antibody. Computer-assisted morphometric analysis was performed and the ratio of the leading edge to initial wound length was calculated. (B) Immunohistochemical staining of wound healing assay at day 7. Scale bar: 500 µm. (C) Measurements of leading edge ratio in HBlox/lox and HB–/– mice. The leading edge ratio was significantly decreased in HB–/– mice (n=9) at day 7. *P<0.01.

View larger version (53K): [in a new window]   Fig. 6. HB-EGF expression in skin wound healing. The targeting vector contained the lacZ gene as a reporter for the expression of HB-EGF. When HB-EGF cDNA is deleted by Cre-recombinase, HB-EGF expression can be identified by X-gal staining in HB+/– mice. (A) Double staining for X-gal and BrdU at the wound healing stage. Scale bar: 0.2 mm. Arrow, wound margin. (B-E) Distribution of ß-gal-positive [lacZ(+)] cells (B) and BrdU-positive cells (C) from day 2 to 7 in HB+/– mice. HB-EGF is expressed predominantly at the tip of the leading edge, whereas BrdU-positive cells were distributed mainly at wound margin. Distribution of ß-gal-positive cells (D) and BrdU-positive cells (E) in HB–/– mice. There was no significant difference in the number of BrdU-positive cells between HB+/– and HB–/– mice at any stage of wound healing. (F) A proposed schematic illustration of skin wound healing. After injury, keratinocytes at the wound margin begin to migrate and express HB-EGF without proliferation. Next, focal release of HB-EGF may signal further migration and up-regulate HB-EGF expression in an autocrine manner. Values in B-E are number of cells per indicated area.