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Purpose: To investigate the effects of epiregulin, a newly identified member of the epidermal growth factor (EGF) family, on the proliferation of human corneal epithelial cells (HCECs)., Methods: The proliferation of HCECs was determined by cell counting and BrdU incorporation assays at specific times after exposure to different concentrations of human recombinant epiregulin (0 to 20 ng/ml). Immunohistochemical staining was used to localize epiregulin in cadaveric corneas. RT-PCR and real-time PCR were used to determine the expression levels of epiregulin in cultured and cadaveric HCECs. To examine the interaction between epiregulin and epidermal growth factor receptors (EGFRs), the phosphorylation of ErbB1 and ERK1/ERK2 (ERK1/2) was estimated by western blot analysis in the presence or absence of AG1478, a specific inhibitor of EGFR kinase activity. To search for cross-induction of epiregulin by other EGF family members, the expressions of EGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR), and transforming growth factor-alpha (TGF-alpha) mRNA were determined by real-time PCR in the presence of 10 ng/ml of epiregulin. Conversely, the expression of epiregulin was also determined following the incubation of HCECs with 10 nM of either of EGF, HB-EGF, TGF-alpha, or AR., Results: The mRNA of epiregulin was expressed in cultured HCECs and HCECs obtained from cadaveric eyes. Epiregulin was strongly detected in the limbal epithelium and basal epithelium of the peripheral cornea, but it was weakly detected in the central corneal epithelium. HCECs proliferated in the presence of epiregulin in a dose-dependent manner as detected by an increase in cell numbers or in BrdU incorporation. When HCECs were incubated with exogenous epiregulin, the expression of the mRNA of epiregulin was upregulated as detected by real-time PCR, and the phosphorylation of ErbB1 and ERK1/2 was upregulated in a dose-dependent manner as shown by western blot analysis. These upregulations were inhibited by AG1478, a specific inhibitor of EGFR kinase activity. Epiregulin increased the expression of HB-EGF and AR, while TGF-alpha, HB-EGF, AR, and EGF increased the expression of epiregulin in HCECs., Conclusions: These findings indicate that epiregulin played an autocrine role in the proliferation of HCECs presumably through cross-induction with other EGF family members.

The role of angiotensin II (Ang II) in the control of systemic blood pressure and volume homeostasis is well known and has been extensively studied. Recently, Ang II was suggested to also have a function in skin wound healing. In the present study, the in vivo function of Ang II in skin wound healing was investigated using Ang II type 1 receptor (AT1R) knock-out mice. Wound healing in these mice was found to be markedly delayed. Keratinocytes and fibroblasts play important roles in wound healing, and thus the effect of Ang II on the migration of these cells was examined. Ang II stimulated keratinocyte and fibroblast migration in a dose-dependent manner. It has been reported that G protein-coupled receptor (GPCR) activation induces epidermal growth factor (EGF) receptor (EGFR) transactivation through the shedding of heparin-binding EGF-like growth factor (HB-EGF). As AT1R is a GPCR, it was hypothesized that Ang II-induced keratinocyte and fibroblast migration is mediated by EGFR transactivation. Ang II induced EGFR phosphorylation, which was inhibited by an AT1R antagonist, HB-EGF neutralizing antibody, and an HB-EGF antagonist in both keratinocytes and in fibroblasts. Moreover, Ang II-induced migration of keratinocytes and fibroblasts was also prevented by these inhibitors. Taken together, these findings clearly demonstrate, for the first time, that Ang II plays an important role in skin wound healing and that it functions by accelerating keratinocyte and fibroblast migration in a process mediated by HB-EGF shedding.

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.

Background: Cultured epidermal sheet (CES) grafts accelerate wound healing as a result of so-called biological dressing effect, which is thought to be mediated by various growth factors. However, the profile of growth factor expression in CESs is unclear., Objective: To investigate whether CESs produce growth factors along with stratification we investigated the production of growth factors and their regulation in CESs., Methods: CESs conditioned medium was harvested and the concentration of TGF-alpha, TGF-beta1, TGF-beta2, and VEGF was measured using ELISA. The mRNA of EGF family, TGF-beta family and VEGF was detected by Northern blot or RNase protection assay., Results: The concentration of TGF-alpha was 100 pg/ml in the monolayer culture, but dramatically increased to 600 pg/ml 2 days after stratification. It decreased to baseline, and then gradually increased to 300 pg/ml in the presence of EGF and remained at that level until day 20. TGF-beta1 increased from 50 to 400 pg/ml after stratification, and remained at that level day 20. TGF-beta2 was undetectable in the monolayer culture, but dramatically increased to 200 pg/ml 2 days after stratification. Unlike TGF-beta1, TGF-beta2 gradually increased over time after stratification. VEGF increased with stratification from 500 to 1500 pg/ml. The addition of EGF upregulated EGF family, TGF-beta, and VEGF production in CESs, as confirmed by ELISA, Northern blot, and RNase protection assay., Conclusion: These results indicate that so-called biological dressing effect of CESs is mediated by production of the EGF family, TGF-beta, and VEGF. Our results also demonstrate the ability of EGF to enhance growth factor production in CESs.

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

Abstract: Epidermal keratinocytes are the main component cells of the epidermis and their function is regulated by various kinds of growth factors, cytokines, and chemokines. Of these, members of the epidermal growth factor and fibroblast growth factor families, as wells as hepatocyte growth factor and insulin-like growth factor, play central roles in keratinocyte proliferation, while transforming growth factor-β, vitamin D3, and interferon-γ are important inhibitors of keratinocyte growth. Keratinocytes are known to produce many of the currently identified growth factors, cytokines and chemokines. Keratinocyte-derived growth factors and cytokines regulate immune and inflammatory responses, and play important roles in pathological skin conditions. This review focuses on the regulation of keratinocytes by growth factors, cytokines, and chemokines. [Copyright &y& Elsevier]