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
Author for correspondence (e-mail: shirakat{at}m.ehime-u.ac.jp)
Accepted 14 February 2005
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Summary |
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Key words: Conditional knockout, HB-EGF, Keratinocytes, Migration, Wound healing
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Introduction |
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The EGF family consists of EGF, transforming growth factor (TGF)-, heparin binding EGF-like growth factor (HB-EGF), amphiregulin (AR), epiregulin (EPR), betacellulin (BTC), epigen and neuregulin (NRG)-1, NRG-2, NRG-3 and NRG-4 (Falls, 2003
; Harari et al., 1999
). The EGF receptor (EGFR) family consists of EGFR (also called ErbB1), ErbB2, ErbB3 and ErbB4 (Jorissen et al., 2003
). The mammalian ligands that bind EGFR include EGF, HB-EGF, TGF-
, AR, BTC, EPR and epigen. Recent studies using gene targeting or transgenic models have revealed that EGFR is essential for epithelial development in the skin, lung and gastrointestinal tract, whereas ErbB2, ErbB3, ErbB4 and neuregulins are essential for the development of cardiac muscle and the central nervous system (Erickson et al., 1997
; Gassmann et al., 1995
; Lee et al., 1995
; Meyer and Birchmeier, 1995
; Miettinen et al., 1995
; Murillas et al., 1995
; Riethmacher et al., 1997
; Sibilia and Wagner, 1995
).
Previous reports have shown that TGF-, AR, HB-EGF and EPR are autocrine growth factors in normal human epidermal keratinocytes (NHEK) (Coffey et al., 1987
; Cook et al., 1991
; Hashimoto et al., 1994
; Shirakata et al., 2000
). It has been reported that keratinocyte migration and proliferation are predominantly mediated by autocrine EGFR activation (Stoll et al., 1997
). However, the importance of the role that the EGF family plays in skin wound healing has not been confirmed in vivo using knockout mice. Previously, Marikovsky et al. (Marikovsky et al., 1993
) reported that HB-EGF is a major component of the mix of growth factors found in wound fluid. Therefore, we speculated that HB-EGF was an important member of the EGF family in cutaneous wound healing. To test this hypothesis, we generated keratinocyte-specific HB-EGF knockout mice, and clearly demonstrated that HB-EGF is an important growth factor for epithelialization in skin wound healing in vivo.
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Materials and Methods |
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Generation of HB-EGF knockout mice using a gene targeting Cre-loxP strategy and PCR
The targeting construct has been described previously (Iwamoto et al., 2003). Homozygous HBlox/lox mice were bred with K5 promoter-driven Cre-recombinase transgenic mice to generate K5-Cre-HBlox/+ mice (Takeda et al., 2000
). Subsequently, K5-Cre-HBlox/+ mice were bred with HBlox/lox mice to generate HBlox/lox: K5-Cre (HB/) mice. The genotype of each mouse was confirmed by PCR. Primers are shown in Table 1.
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RT-PCR analysis
Keratinocytes were cultured in MCDB153 complete medium on type I collagen-coated dishes until they reached confluency. Keratinocytes were treated by tip scraping and total RNA was harvested at several time points. mRNA expression of HB-EGF, TGF-, AR, EPR and GAPDH was analyzed by RT-PCR. The absence of HB-EGF mRNA in keratinocytes from HB/ mice was confirmed by RT-PCR. Primers are shown in Table 2. The RT-PCR was performed using RT-PCR High Plus (Toyobo Co. Ltd, Osaka, Japan) according to the manufacturer's instructions. cDNA was reverse-transcribed from total RNA for 30 minutes at 60°C and heated to 94°C for 2 minutes. Amplification was performed using a DNA thermal cycler (Astec, Fukuoka, Japan) for 25 cycles. A cycle profile consisted of 1 minute at 94°C for denaturation, 1.5 minutes at 60°C for annealing and primer extension.
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Wound healing studies
Wound healing experiments were performed in HB/ and HBlox/lox mice. Under sodium pentobarbital anesthesia, two full-thickness wounds were created on the skin of the backs of each of nine 9- to 10-week-old female mice using 6-mm skin biopsy punches. Each wound diameter was determined as the average of longitudinal and lateral diameter. Wound closure was monitored, and skin sections were harvested at 3, 5, 7, 9 and 11 days after wounding. For BrdU labeling, mice received intraperitoneal injections of BrdU (250 µg/g; Sigma, Tokyo, Japan) 2 hours prior to sacrifice.
Histological analysis
Mouse tissues were fixed in 4% paraformaldehyde or formaldehyde, dehydrated and embedded in paraffin. Four-µm sections were stained with Hematoxylin and Eosin. For ß-gal staining, after fixation with 0.2% glutaraldehyde and 1% formalin, the tissues were stained with 5-bromo-4-chloro-3-indol ß-D-galactoside (X-gal). Skin sections were stained with rabbit anti-keratin IgG or anti-BrdU IgG, and immunopositive reactions were visualized using a streptavidin-biotin-peroxidase staining kit (Nichirei Co. Inc., Tokyo, Japan) according to the manufacturer's instructions. Morphometric analysis was performed using MacSCOPE Ver2.61 software. Statistical analysis was performed using Student's t-test.
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Results |
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Impaired wound healing in keratinocyte-specific HB-EGF-deficient mice
To examine the role of HB-EGF in skin wound healing in vivo, we performed a wound-healing assay using HBlox/lox and HB/ mice. Two 6-mm punch skin biopsies were made in the back of each mouse and the wound diameter was measured at various times after wounding as a measure of healing. There was no difference in wound diameter up to day 3 post-wounding; however, wound healing was noticeably retarded from day 5 to 11 in the HB/ mice. Wound closure was delayed significantly in HB/ mice compared with HBlox/lox mice on day 8 (Fig. 3A). The wound diameter was reduced to 34% in HBlox/lox mice on day 8, whereas it was still 58% in the HB/ mice (Fig. 3B). These results indicate that HB-EGF expression by keratinocytes is important for skin wound healing in vivo.
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Expression of HB-EGF at wound sites
It has been reported that HB-EGF was upregulated in burn wound healing and that topical application of HB-EGF accelerated re-epithelialization of partial-thickness burns (Cribbs et al., 2002; Cribbs et al., 1998
; McCarthy et al., 1996
). It has been also reported that addition of HB-EGF into c-jun null keratinocyte growth medium can rescue the migration defect and induce phosphorylation of EGF receptor (Li et al., 2003
). Since HB-EGF may play an important role in skin wound healing, we investigated the HB-EGF expression and keratinocyte proliferation pattern in skin wound healing using HBlox/+:K5-Cre (HB+/) mice. With the targeting vector containing the lacZ gene as a reporter for the expression of HB-EGF, it is possible to ascertain the expression of HB-EGF by staining for ß-gal in HB+/ mice. HB-EGF was expressed at the leading edge of the epithelium at day 2 post-wounding, and was predominantly expressed at the tip of the leading edge until day 7 (Fig. 6A). Unlike the HB-EGF expression pattern, BrdU-positive (BrdU+) cells were detected mainly within the peripheral skin on days 2 and 3. On days 5 and 7, BrdU+ cells were found toward the leading edge, although they were preferentially located near the wound margin. To quantify the distribution of HB-EGF-expressing cells and proliferating cells, we counted the ß-gal-positive (ß-gal+) cells and BrdU+ cells in 0.2 mm ranges in the leading edge and in the peripheral skin in HB+/ mice. On day 2 the peak of the ß-gal+ cells was between 0 +0.2 mm into the leading edge, whereas the peak of the BrdU+ cells was 0.2 to 0.4 mm into the peripheral skin. On day 3, the peak of the ß-gal+ cells was between +0.4 and +0.6 mm, whereas the peak of the BrdU+ cells was between 0 and +0.2 mm. From day 5 to day 7, BrdU+ cells were located at the leading edge, although they always appeared just behind the ß-gal+ cells (Fig. 6B,C).
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We also examined ß-gal+ and BrdU+ cells in the wound-healing assay using HB/ mice. As in HB+/ mice, in HB/ mice, on day 2 post-wounding, ß-gal+ cells were localized mostly 0 to +0.2 mm into the leading edge, whereas BrdU+ cells were detected mostly between 0.2 and 0.4 mm into the peripheral skin. On day 3, the peak of the ß-gal+ cells were localized at 0 to +0.2 mm, whereas the peak of the BrdU+ cells were between 0 and 0.2 mm. On days 5 and 7, the peak aggregation of BrdU+ cells in HB/ mice was at almost the same location as the ß-gal+ cells, at the leading edge (Fig. 6D,E). The overlapping distribution patterns of BrdU+ and ß-gal+ cells in these mice may be due to the impaired cell migration (Fig. 5B,C). However, the total counts of BrdU+ cells were similar in HBlox/lox and HB/ mice (Fig. 4B,C).
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Discussion |
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HB-EGF is produced and secreted by human keratinocytes and acts as an autocrine growth factor (Hashimoto et al., 1994). HB-EGF mRNA was rapidly and dramatically induced after scrape-wounding, although slight increases in TGF-
, amphiregulin and epiregulin mRNAs were observed. Furthermore, blocking HB-EGF by addition of neutralizing antibody to the medium inhibited keratinocyte migration (Tokumaru et al., 2000
). In contrast, the addition of recombinant HB-EGF to the medium accelerates keratinocyte migration (Tokumaru et al., 2000
). These results indicate that HB-EGF plays an important role in skin wound healing, and led us to investigate the in vivo function of HB-EGF. Since germline targeting of the HB-EGF gene resulted in embryonic lethality (Iwamoto et al., 2003
), we generated keratinocyte-specific HB-EGF-deficient mice (HB/) using Cre/loxP technology in combination with the keratin 5 promoter (Takeda et al., 2000
). There was no difference in wound closure between HB/ and HBlox/lox mice on day 3. However, wound closure was markedly retarded in HB/ mice compared to HBlox/lox mice. We clearly demonstrated for the first time that endogenous HB-EGF is the most important growth factor in the epithelialization of skin wound healing in vivo, using keratinocyte-specific HB-EGF-deficient mice.
EGF family members are well known to promote keratinocyte growth in vitro (Hashimoto, 2000). It has been reported that TGF-
, amphiregulin, HB-EGF and epiregulin are autocrine growth factors in normal human keratinocytes (Coffey et al., 1987
; Cook et al., 1991
; Hashimoto et al., 1994
; Shirakata et al., 2000
). In vitro observation suggests that these EGF family members play important roles in development, epidermal morphogenesis, skin homeostasis and wound healing. In this study, we investigated HB-EGF function in cell migration and proliferation. HB-EGF stimulates keratinocyte migration in vitro and in vivo. However, there was little difference in proliferation between HBlox/lox and HB/ mice. HB-EGF promoter activity was up-regulated at the migrating epidermal edge, whereas the distribution of proliferating cells (BrdU-positive) was not identical to that of HB-EGF mRNA-positive cells. Interestingly, the wound margin of normal epidermis expressed HB-EGF mRNA and was positive for BrdU, although HB-EGF promoter activity could not be detected in normal skin far from the wound margin. Therefore, normal skin does not require much HB-EGF, but after injury HB-EGF is induced and plays a crucial role in wound healing by up-regulating keratinocyte migration but not proliferation.
Combined, the evidence suggests that the synthesis of HB-EGF at the leading epithelial edge stimulates cells, via an autocrine loop, to migrate towards the center of the wound rather than to proliferate. Interestingly, there were few ß-gal-positive cells and little HB-EGF expression in normal skin far from the wound margin. Therefore, HB-EGF expression induced by wounding might itself stimulate further expression of HB-EGF at the leading edge via an autocrine loop. Fig. 6F shows a schematic illustration of our proposed skin wound healing mechanism. After injury, keratinocytes at the wound margin begin to express HB-EGF and migrate toward the wound site without proliferating. Next, the focal release of HB-EGF may trigger the migration of additional cells, rather than cell proliferation at the leading edge. Therefore, we conclude that HB-EGF is rapidly induced after injury and plays an important role in wound healing by up-regulating keratinocyte migration.
Nuclear transcription factors play important roles in almost all biological events resulting from growth factor signaling, and several nuclear transcription factors are thought to be involved in skin wound healing. Several mouse models with gene-targeted disruption of nuclear transcriptional factors have been analyzed for skin wound healing. Sano et al. (Sano et al., 1999) reported severe retardation of wound healing in keratinocyte-specific STAT3 knockout mice. D'Souza et al. (D'Souza et al., 2002
) reported impaired skin wound healing in E2F-1 knockout mice. Recently, the development of keratinocyte-specific c-jun knockout mice was reported (Li et al., 2003
; Zenz et al., 2003
). These mice showed retarded wound healing, and the activation of EGFR was greatly decreased (Li et al., 2003
). Since EGF itself is not produced by keratinocytes, the autocrine loop consisting of HB-EGF, EGFR and c-jun might be one of the major regulatory signal transduction mechanisms in skin wound healing.
In conclusion, HB-EGF is an important growth factor in epithelialization during skin wound healing in vivo, and acts mainly by stimulating migration, rather than proliferation, of keratinocytes.
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Acknowledgments |
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Footnotes |
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References |
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