|Molecular Vision 2007;
Received 26 January 2007 | Accepted 12 November 2007 | Published 15 November 2007
Human corneal epithelial cell proliferation by epiregulin and its cross-induction by other EGF family members
1Department of Ophthalmology, 2Dermatology, 3Center for Regenerative Medicine, and 4Biochemistry and Molecular Genetics, Ehime University School of Medicine, Ehime, Japan
Correspondence to: Shin-ichi Morita, Department of Ophthalmology, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan; Phone: 81-89-960-5361; FAX: 81-89-960-5364; email: firstname.lastname@example.org
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-α) 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-α, 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 up-regulated as detected by real-time PCR, and the phosphorylation of ErbB1 and ERK1/2 was up-regulated in a dose-dependent manner as shown by Western blot analysis. These up-regulations were inhibited by AG1478, a specific inhibitor of EGFR kinase activity. Epiregulin increased the expression of HB-EGF and AR, while TGF-α, 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 corneal epithelium is a multi-layered stratified epithelium that covers the surface of the cornea and acts as a physical barrier to noxious agents. To maintain an effective barrier, a constant renewal of corneal epithelial cells is necessary, and this was accomplished with the aid of different growth factors [1,2]. Of these growth factors, the members of the epidermal growth factor (EGF) family have been most extensively studied. The EGF family consists of EGF, transforming growth factor-alpha (TGF-α), heparin binding EGF-like growth factor (HB-EGF), amphiregulin (AR), epiregulin, betacellulin (BTC), neuregulin 1, neuregulin 2, and neuregulin 3 [3-12]. Among these, TGF-α and HB-EGF are known to stimulate not only the migration and proliferation of corneal epithelial cells, but also the synthesis of the basement membrane and extracellular components [6,11,13-16].
In general the growth factors of the EGF family consist of a transmembrane domain and one or more EGF domains. Soluble mature peptides are released from the extracellular domains by proteolytic cleavage as ligands for EGF receptors . There are four types of EGF receptors: ErbB1 (EGFR/Her1), ErbB2 (Neu/Her2), ErbB3 (Her3), and ErbB4 (Her4) [18-21]. The members of the EGF family can be divided into two groups according to their binding specificity to transmembrane receptors. The first group, which includes EGF, HB-EGF, TGF-α, AR, BTC, and epiregulin, generally binds to ErbB1 [4,5,12,22-24] and plays a crucial role in epithelial development and wound-healing for skin, lungs, and the gastrointestinal tract [25-27]. The members of the second group, the neuregulins, bind to ErbB3 and ErbB4 [3,7,8,28] and are crucial for the development of cardiac muscle and the central nervous system [29-32].
It has been shown that the first group of the EGF family members plays important roles during the healing of corneal injuries [1,16,33-35]. These binding EGFR ligands activate ErbB1, thus activating subsequent intracellular signals such as extracellular signaling-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), which lead to corneal epithelial cell proliferation and migration [36-39].
Epiregulin is a relatively new member of the EGF family and was originally purified from conditioned medium of the NIH3T3 cell line T7 . It is made up of 46 amino acids that form a single-chain polypeptide that exhibits 24-25% amino sequence homology with other EGFR ligands [12,40,41]. Epiregulin is unique due to its affinity to bind to all of the ErbB family members . It has been demonstrated that epiregulin plays a role in the proliferation of epithelial cells, e.g., epidermal and urothelial cells [43,44]. In the eye, Zhou et al.  reported that the mRNA of epiregulin was preferentially expressed in limbal basal cells of mice; however, the precise role of epiregulin in human corneal epithelial cells has not been determined. Like other members of the first group of the EGF family, epiregulin also binds to ErbB1, and it is thus reasonable to consider that epiregulin may play a role in corneal epithelial cell proliferation.
The purpose of this study was to determine whether the proliferation of HCECs is up-regulated in the presence of epiregulin, and whether epiregulin can act on the HCECs together with other members of the EGF family including EGF, TGF-α, HB-EGF, and AR.
All procedures involving human subjects were conducted in accordance with the tenets of the Declaration of Helsinki (JAMA 1997; 277:925-926). The experimental protocol for these experiments was approved by the Institutional Review Board of Ehime University.
All reagents used for cell cultures were purchased from Invitrogen (Carlsbad, CA) except for the growth factors. Recombinant human epiregulin, EGF, TGF-α, AR, and HB-EGF were purchased from R&D Systems (Minneapolis, MN).
Primary HCECs were isolated from human corneoscleral buttons dissected from eyes acquired from an American eye bank. The buttons were carefully denuded of the endothelial cells and adherent iris tissues. After digestion with 1.2 U/ml Dispase at 4 °C for 24 h, the loosened epithelial sheets were removed and dissociated into single cells by enzyme digestion with 0.25% trypsin. The isolated HCECs were cultured in serum-free, modified MCDB153 medium containing insulin (5 μg/ml), hydrocortisone (0.5 μM), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), and bovine hypothalamic extract (50 μg/ml). The medium was changed every 2 days, and cells from the third or fourth passage were used for all experiments.
Expression of epiregulin mRNA in cadaveric corneal epithelium
The corneal epithelium was collected from the cadaveric corneoscleral buttons using a surgical knife, and then total RNA was immediately extracted using an RNeasy kit (Qiagen, Valencia, CA) and measured by spectrophotometry (OD at 260 and 280 nm). Total RNA was reverse-transcribed to cDNA using Omniscript Reverse Transcription Reagents (Qiagen) according to the instructions of the manufacturer. The PCR conditions for the initial denaturation step were 95 °C for 15 min and 40 cycles at 95 °C for 10 s, followed by 64 °C for 20 s for annealing and 72 °C for 20 s for extension.
Proliferation of HCECs by epiregulin
HCECs were seeded in 6 well plates at a density of 8x104 cells/well in serum-free modified MCDB153 medium. On the following day, the cells were subcultured in fresh medium containing serial concentrations of recombinant epiregulin (0-20 ng/ml) in the absence of bovine hypothalamic extract because the cells were still 20% confluent. After 6 days, the cells were collected and counted using a hemocytometer.
To determine whether the HCECs had proliferated, the level of BrdU incorporated into the HCECs was determined. HCECs were seeded in 6 well plates at a density of 8x104 cells/well in serum-free modified MCDB153 medium. After reaching about 50% confluency, the cells were subcultured in a medium without bovine hypothalamic extract. On the following day, the cells were subcultured in the same medium containing serial concentrations of recombinant human epiregulin (0-20 ng/ml) and incubated for 48 h. The cells were then incubated with medium containing BrdU for 2 h. BrdU was detected immunohistochemically using the 5-Bromo-2'-deoxy-uridine Labeling and Detection kit 2 (Roche, Indianapolis, IN) according to the instructions of the manufacturer. The number of BrdU-labeled cells/5 mm2 were counted (n=4).
Phosphorylation of ErbB1 and ERK1/ERK2 by epiregulin
HCECs were seeded in 24 well plates at a density of 5x104 cells/well in serum-free, modified MCDB153 medium. After reaching about 80% confluency, the cells were subcultured in a medium without bovine hypothalamic extract. On the following day, the cells were subcultured in the same medium containing serial concentrations of recombinant human epiregulin (0-20 ng/ml) for 5 min. The cells were rinsed with cold Ca++- free and Mg++-free phosphate buffer saline (PBS-) and then harvested on ice with a cell scraper. The cells were lysed in 200 μl of lysis buffer containing 62.5 mM Tris-HCl, 2% SDS, 25% glycerol, 0.01% bromophenol blue, and proteinase inhibitors at pH 6.8. After centrifugation, the cell lysates were separated on 7.5% polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore, Billerica, MA). After blocking with 5% non-fat dry milk, the membrane was incubated with a 1:1,000 dilution of an anti-phospho-EGFR(ErbB1, Tyr1173) antibody (Upstate Biotechnology, Lake Placid, NY) or an anti-phospho-ERK1/ERK2 (ERK1/2) antibody (R&D systems) at 4 °C for 12 h. After washing with PBS, the membrane was incubated with a 1:2,500 dilution of fluorescein-labeled goat anti mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at RT. The signal was amplified with an anti-fluorescein alkaline phosphatase conjugate followed by the addition of a fluorescent substrate, Attophos (Amersham Pharmacia Biotech). The membrane was scanned using a FluorImager (Molecular Dynamics, Sunnyvale, CA), and the expression levels of ErbB1 and ERK1/2 were determined relative to that of beta-actin in the same sample using the NIH Image program. The phosphorylation of ErbB1 and ERK1/2 was also determined by Western blot analysis in HCECs exposed to serial concentrations of AG1478 (Calbiochem, La Jolla, CA), a specific inhibitor of EGFR kinase activity, with 100 ng/ml of epiregulin.
Quantitative real time PCR analysis
To determine whether exogenous epiregulin affected the expression of endogenous epiregulin, i.e., an auto-induction mechanism, or other members of the EGF family, i.e., a cross-induction mechanism, HCECs were stimulated by epiregulin, and the expression of the mRNA of the other growth factors was examined by real time PCR. Briefly, HCECs were seeded in 24 well plates at a density of 5x104 cells/well in serum-free, modified MCDB153 medium. After reaching about 80% confluency, the cells were subcultured in a medium without bovine hypothalamic extract. On the following day, the cells were again subcultured in the same medium containing 10 ng/ml of epiregulin and harvested at 0.5, 1, 2, 3, 6, and 12 h. To determine whether other EGF family members affected the expression of epiregulin (a cross-induction mechanism by other EGF family members), HCECs were exposed to 10 nM of HB-EGF, TGF-α, AR, or EGF. In addition, to determine whether AG1478 affected the expression of endogenous epiregulin by exogenous epiregulin stimulation, HCECs were exposed to 10 ng/ml of epiregulin for 2 h with or without 1 h of preincubation in AG1478 (1 μM). Then the total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA) and measured by spectrophotometry (OD at 260 and 280 nm). Equal amounts (1 μg) of total RNA were reverse-transcribed to cDNA using Omniscript Reverse Transcription Reagents (Qiagen) according to the instructions of the manufacturer. The PCR primers are listed in Table 1. These primers were designed from the full-length cDNA sequence in Genbank, and their specificities were confirmed by BLAST (National Library of Medicine, Bethesda, MD).
Real-time PCR was performed using a DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland). Amplifications were performed in a final volume of 20 μl containing 0.5 μM of primer mixture and 2 μl of cDNA. The PCR conditions for the initial denaturation step were 95 °C for 15 min and 40 cycles at 95 °C for 10 s, followed by 60 °C (HB-EGF, AR, EGF and GAPDH) or 64 °C (epiregulin and TGF-α) for 20 s for annealing, and 72 °C for 20 s for extension. All PCR reactions were performed by OPticon2 DNA Engine (BioRad, Hercules, CA), and each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of primer dimers. The comparative cycle threshold (Ct) was calculated for all samples to quantify the relative expression of each mRNA with standardization using that of GAPDH mRNA . All experiments were performed in duplicate for each datum point.
Cadaveric corneas were fixed in methanol, embedded in paraffin, and cut into 5 μm sections. Immunohistochemical staining was carried out with anti-epiregulin antibody (R & D Systems, 1:50 dilution, 16 h at 4 °C) using the VECTASTAIN Elite ABC kit (Vector Lab, Burlingame, CA), according to the manufacturer's protocol for paraffin sections. All sections were developed with AEC and counterstained with hematoxylin. For control, sections were treated with normal goat immunoglobulin G (IgG).
Expression of epiregulin in human cornea and corneal epithelial cells
To determine whether epiregulin is expressed in human corneal epithelial cells, we used RT-PCR to detect epiregulin mRNA in the corneal epithelium collected from the cadaveric eyes and from cultured HCECs. Epiregulin mRNA was detected in both corneal epithelial cells and cultured HCECs (Figure 1). Immunohistochemical staining showed that immunoreactivity was detected in all layers of the corneal epithelium, however, strong staining was detected in the basal and superficial layers of the limbal epithelium and in the basal layer of peripheral corneal epithelium. Immunoreactivity was very weakly detected in the central cornea (Figure 2). Immunoreactivity was also detected in the endothelium and weakly detected in keratocytes. Other than the cornea, epiregulin was detected strongly in the conjunctival epithelium, blood vessel endothelium, and weakly in the subconjunctival fibroblasts.
Recombinant human epiregulin stimulates proliferation of HCECs
To determine whether epiregulin stimulates the proliferation of HCECs, HCECs were cultured in serum-free medium with serially diluted concentrations of epiregulin, and the total number of cells and the number of BrdU-labeled cells were counted. The results showed that epiregulin stimulated the proliferation of HCECs in a dose-dependent manner with an 8.0 fold increase in cell numbers at 10 ng/ml (Figure 3). The number of BrdU labeled cells was also increased by epiregulin in a dose dependent manner with an increase up to 3.0 fold at 10 and 20 ng/ml (Figure 4).
Phosphorylation of ErbB1 and ERK1/2 by epiregulin
Epiregulin is known to bind to ErbB1 and to activate MAP kinase pathways . In the next study, we investigated whether the tyrosine residues of ErbB1 and ERK1/2 in HCECs were phosphorylated in the presence of epiregulin. Western blot analysis revealed that epiregulin enhanced the phosphorylation of ErbB1 and ERK1/2 in a dose-dependent manner (Figure 5). The phosphorylation by 100 ng/ml of epiregulin was suppressed by a 1 μM or higher concentration of AG1478 (Figure 6).
Autoinduction of epiregulin and cross-induction of other EGF family members by epiregulin
We examined whether epiregulin up-regulated the transcription of its own mRNA in HCECs, i.e., an auto-induction mechanism of epiregulin. The results from real-time PCR showed that the expression of epiregulin increased as early as 0.5 h after incubation with 10 ng/ml of epiregulin, reached a peak (4.8 fold increase from 0 h) at 3 h after incubation (Figure 7), and gradually returned to its original level by 12 h. This up-regulation was completely inhibited by AG1478 (Figure 8).
Because EGF-related autocrine growth factors were able to induce other growth factors of the EGF family in a variety of epithelial cells , we examined whether epiregulin up-regulated the other members of the EGF family, i.e., a cross-induction mechanism of epiregulin, in HCECs. Real time PCR analysis showed that epiregulin exposure significantly increased the mRNA levels of AR at 2 h and of HB-EGF between 0.5 and 2 h, while the mRNA levels of EGF and TGF-α remained essentially unchanged (Figure 9).
Lastly, we investigated whether the expression of epiregulin was enhanced by other EGF family members, i.e., a cross-induction mechanism by other EGF family members, using real-time PCR analysis. The results showed that the expression of mRNA of epiregulin increased around 2 or 3 h after treatment with all of the EGF family members including TGF-α, HB-EGF, EGF, and AR (Figure 10).
The integrity of the EGF receptor-ligand signaling system is of great importance in regulating epithelial cell differentiation and proliferation. Among the members of the EGF family (EGF, TGF-α, AR, and HB-EGF), ligands for ErbB1 are known to be responsible for the migration and proliferation of HCECs and for the synthesis of extracellular matrix during corneal epithelial wound healing [13-16,34,35].
Epiregulin is a relatively new member of the EGF family, and in a previous report on the distribution of epiregulin in human tissues, northern blot analysis showed that epiregulin mRNA was found predominantly in the placenta and peripheral blood leukocytes and to a lesser extent in the heart. A very weak expression was also detected in normal adult bone marrow, ovaries, small intestine, colon, lungs, and liver . Similar to other growth factors belonging to the EGF family, epiregulin has been reported to promote the proliferation of other human epithelial cells such as the urothelium and epidermis [43,44].
We have shown that epiregulin mRNA was expressed in human corneal epithelial cells. Immunohistochemical staining for epiregulin revealed that immunoreactivity was strongly detected in limbal epithelial cells, and in the basal layer of peripheral corneal epithelia in which transient amplifying cells are supposed to exist. Similarly, Zhou et al.  reported that the mRNA of epiregulin was preferentially expressed in limbal basal cells of mouse corneal epithelia. The differences in expression patterns may be due to the differences in species and experimental methods. However, in both studies epiregulin expression was strongly detected in the progenitor or proliferating corneal epithelial cells. These results would suggest that epiregulin may contribute to the proliferative capacity of corneal epithelial cells.
We have shown that epiregulin was able to enhance the proliferation of HCECs. It has been suggested that the proliferation of corneal epithelial cell by members of the EGF family was induced through the ErbB1-MAPK pathway . Our results showed that epiregulin was also able to activate ErbB1 following ERK1/2 phosphorylation. Thus, like other EGF family members, epiregulin may induce corneal epithelial cell proliferation as, at least, a ligand for ErbB1.
Most of the EGF-related autocrine growth factors are capable of promoting the expression of other EGF family growth factors in a variety of epithelial cells [47,48], and an increase in the level of EGF family growth factors (EGFR ligand transcripts) is dependent upon signals from their own receptors, "EGFR" [48-51]. Shirakata et al. reported that epiregulin is part of an auto-induction and cross-induction mechanism involving HB-EGF, AR, and TGF-α in keratinocyte proliferation . In this study, we have shown that epiregulin stimulated the induction of its own mRNA as well as the phosphorylation of ErbB1. The significant inhibition of the induction of epiregulin expression by AG1478, a specific inhibitor of EGFR kinase activity, suggests the possible existence of an auto-induction of epiregulin, and phosphorylated ErbB1 may be part of the pathway of the auto-induction mechanism. These results also indicate that epiregulin can be listed as one of the auto- or paracrine growth factors for corneal epithelial cells interaction with ErbB1.
Our results also showed that epiregulin served as an effector of similar auto- and cross-induction mechanisms in corneal epithelial cells. Although an up-regulation by epiregulin was limited to HB-EGF and AR in the case of HCECs, the epiregulin mRNA was up-regulated by EGF, HB-EGF, TGF-α, and AR. These findings suggest that epiregulin most likely operates in corneal epithelial proliferation in concert with other EGF family members.
The extent of cross-induction differs among the members of the EGF family. It has been reported that each EGF family growth factor has distinct biological functions. The tissue distribution, molecular characteristics, receptor binding, preference to dimerize, and receptor affinity differ among the growth factors [44,47]. Interestingly, Barnard et al. reported results similar to ours that HB-EGF and AR were rapidly induced by EGF-related peptides in the intestinal epithelial cell line, and speculated that differences in the post-receptor processing may contribute to the heterogeneity in the biological responses . TGF-α, HB-EGF, and AR have been reported to be up-regulated on corneal epithelial cells, although the levels of EGF are at trace levels and do not change during the corneal wound healing process [16,52]. Although TGF-α mRNA was not induced by epiregulin under the conditions we employed, it is consistent that HB-EGF and AR were induced in corneal epithelial cells, and these results might be a molecular characteristic of the corneal epithelial cells. Consistent with other reports, EGF was not induced by epiregulin; however, EGF induced the expression of epiregulin in HCECs.
It is known that EGF exists in tear fluid along with HB-EGF and TGF-α, and corneal wounds increase the expression of EGF supplied from lacrimal glands through the sensory nerves [53,54]. Thus, EGF may be able to stimulate corneal epithelial cells, but not be induced in corneal epithelial cells. Together with previous reports, our results suggested that epiregulin may function together with not only endogenously produced EGF family members, but also with those in the tear fluid through cross-induction mechanisms during corneal epithelial wound-healing.
The presence of autocrine or paracrine feedback loops involving EGFR have been documented for the members of the EGF family in different tissues . When it comes to these pathways, an increase in the level of EGFR ligand transcripts is dependent upon the signal from their own receptors, "EGFR" [48-51]. As shown in this study, the phosphorylation of ErbB1 by epiregulin can lead to an increase in transcripts of the other EGF family members. It would be highly efficient for corneal epithelial cells to utilize a common pathway when the integrity of the corneal epithelium is seriously compromised.
In conclusion, epiregulin is present in human corneal epithelial cells and is able to induce HCECs to proliferate as an autocrine growth factor. The intricate cross-induction mechanism involving epiregulin certainly plays a role in a variety of events which occur in the corneal epithelium.
The authors thank all participants of this investigation and the authors also thank T. Tsuda for technical help.
1. Lu L, Reinach PS, Kao WW. Corneal epithelial wound healing. Exp Biol Med (Maywood) 2001; 226:653-64.
2. Liu JJ, Kao WW, Wilson SE. Corneal epithelium-specific mouse keratin K12 promoter. Exp Eye Res 1999; 68:295-301.
3. Wen D, Peles E, Cupples R, Suggs SV, Bacus SS, Luo Y, Trail G, Hu S, Silbiger SM, Levy RB, Koski RA, Lu HS, Yarden Y. Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 1992; 69:559-72.
4. Shoyab M, Plowman GD, McDonald VL, Bradley JG, Todaro GJ. Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science 1989; 243:1074-6.
5. Shing Y, Christofori G, Hanahan D, Ono Y, Sasada R, Igarashi K, Folkman J. Betacellulin: a mitogen from pancreatic beta cell tumors. Science 1993; 259:1604-7.
6. Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science 1991; 251:936-9.
7. Chang H, Riese DJ 2nd, Gilbert W, Stern DF, McMahan UJ. Ligands for ErbB-family receptors encoded by a neuregulin-like gene. Nature 1997; 387:509-12.
8. Carraway KL 3rd, Weber JL, Unger MJ, Ledesma J, Yu N, Gassmann M, Lai C. Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases. Nature 1997; 387:512-6.
9. Derynck R, Roberts AB, Winkler ME, Chen EY, Goeddel DV. Human transforming growth factor-alpha: precursor structure and expression in E. coli. Cell 1984; 38:287-97.
10. Cohen S. Isolation and biological effects of an epidermal growth-stimulating protein. Natl Cancer Inst Monogr 1964; 13:13-37.
11. Marquardt H, Hunkapiller MW, Hood LE, Todaro GJ. Rat transforming growth factor type 1: structure and relation to epidermal growth factor. Science 1984; 223:1079-82.
12. Toyoda H, Komurasaki T, Uchida D, Takayama Y, Isobe T, Okuyama T, Hanada K. Epiregulin. A novel epidermal growth factor with mitogenic activity for rat primary hepatocytes. J Biol Chem 1995; 270:7495-500.
13. Schultz G, Chegini N, Grant M, Khaw P, MacKay S. Effects of growth factors on corneal wound healing. Acta Ophthalmol Suppl 1992; 202:60-6.
14. Bennett NT, Schultz GS. Growth factors and wound healing: biochemical properties of growth factors and their receptors. Am J Surg 1993; 165:728-37.
15. Gipson IK, Inatomi T. Extracellular matrix and growth factors in corneal wound healing. Curr Opin Ophthalmol 1995; 6:3-10.
16. Zieske JD, Takahashi H, Hutcheon AE, Dalbone AC. Activation of epidermal growth factor receptor during corneal epithelial migration. Invest Ophthalmol Vis Sci 2000; 41:1346-55.
17. Harris RC, Chung E, Coffey RJ. EGF receptor ligands. Exp Cell Res 2003; 284:2-13.
18. Yamamoto T, Ikawa S, Akiyama T, Semba K, Nomura N, Miyajima N, Saito T, Toyoshima K. Similarity of protein encoded by the human c-erb-B-2 gene to epidermal growth factor receptor. Nature 1986; 319:230-4.
19. Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, Downward J, Mayes EL, Whittle N, Waterfield MD, Seeburg PH. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 1984; 309:418-25.
20. Plowman GD, Culouscou JM, Whitney GS, Green JM, Carlton GW, Foy L, Neubauer MG, Shoyab M. Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family. Proc Natl Acad Sci U S A 1993; 90:1746-50.
21. Kraus MH, Issing W, Miki T, Popescu NC, Aaronson SA. Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Natl Acad Sci U S A 1989; 86:9193-7.
22. Riese DJ 2nd, Bermingham Y, van Raaij TM, Buckley S, Plowman GD, Stern DF. Betacellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin-beta. Oncogene 1996; 12:345-53.
23. Komurasaki T, Toyoda H, Uchida D, Morimoto S. Epiregulin binds to epidermal growth factor receptor and ErbB-4 and induces tyrosine phosphorylation of epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4. Oncogene 1997; 15:2841-8.
24. Elenius K, Paul S, Allison G, Sun J, Klagsbrun M. Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation. EMBO J 1997; 16:1268-78.
25. Miettinen PJ, Berger JE, Meneses J, Phung Y, Pedersen RA, Werb Z, Derynck R. Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 1995; 376:337-41.
26. Sibilia M, Wagner EF. Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 1995; 269:234-8. Erratum in: Science 1995; 269:909.
27. Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D, LaMantia C, Mourton T, Herrup K, Harris RC, Barnard JA, Yuspa SH, Coffey RJ, Magnuson T. Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 1995; 269:230-4.
28. Zhang D, Sliwkowski MX, Mark M, Frantz G, Akita R, Sun Y, Hillan K, Crowley C, Brush J, Godowski PJ. Neuregulin-3 (NRG3): a novel neural tissue-enriched protein that binds and activates ErbB4. Proc Natl Acad Sci U S A 1997; 94:9562-7.
29. Lee KF, Simon H, Chen H, Bates B, Hung MC, Hauser C. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 1995; 378:394-8.
30. Meyer D, Birchmeier C. Multiple essential functions of neuregulin in development. Nature 1995; 378:386-90. Erratum in: Nature 1995; 378:753.
31. Gassmann M, Casagranda F, Orioli D, Simon H, Lai C, Klein R, Lemke G. Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 1995; 378:390-4.
32. Erickson SL, O'Shea KS, Ghaboosi N, Loverro L, Frantz G, Bauer M, Lu LH, Moore MW. ErbB3 is required for normal cerebellar and cardiac development: a comparison with ErbB2-and heregulin-deficient mice. Development 1997; 124:4999-5011.
33. Tao W, Liou GI, Wu X, Abney TO, Reinach PS. ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells. Invest Ophthalmol Vis Sci 1995; 36:2614-22. Erratum in: Invest Ophthalmol Vis Sci 1996; 37:1937.
34. Xu KP, Ding Y, Ling J, Dong Z, Yu FS. Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells. Invest Ophthalmol Vis Sci 2004; 45:813-20.
35. Block ER, Matela AR, SundarRaj N, Iszkula ER, Klarlund JK. Wounding induces motility in sheets of corneal epithelial cells through loss of spatial constraints: role of heparin-binding epidermal growth factor-like growth factor signaling. J Biol Chem 2004; 279:24307-12. Erratum in: J Biol Chem. 2004; 279:36166.
36. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000; 103:211-25.
37. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 2001; 410:37-40.
38. Kang SS, Wang L, Kao WW, Reinach PS, Lu L. Control of SV-40 transformed RCE cell proliferation by growth-factor-induced cell cycle progression. Curr Eye Res 2001; 23:397-405.
39. Xu KP, Yin J, Yu FS. SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells. Invest Ophthalmol Vis Sci 2006; 47:2832-9.
40. Toyoda H, Komurasaki T, Ikeda Y, Yoshimoto M, Morimoto S. Molecular cloning of mouse epiregulin, a novel epidermal growth factor-related protein, expressed in the early stage of development. FEBS Lett 1995; 377:403-7.
41. Toyoda H, Komurasaki T, Uchida D, Morimoto S. Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family. Biochem J 1997; 326:69-75.
42. Shelly M, Pinkas-Kramarski R, Guarino BC, Waterman H, Wang LM, Lyass L, Alimandi M, Kuo A, Bacus SS, Pierce JH, Andrews GC, Yarden Y. Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes. J Biol Chem 1998; 273:10496-505.
43. Varley C, Hill G, Pellegrin S, Shaw NJ, Selby PJ, Trejdosiewicz LK, Southgate J. Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro. Exp Cell Res 2005; 306:216-29.
44. Shirakata Y, Komurasaki T, Toyoda H, Hanakawa Y, Yamasaki K, Tokumaru S, Sayama K, Hashimoto K. Epiregulin, a novel member of the epidermal growth factor family, is an autocrine growth factor in normal human keratinocytes. J Biol Chem 2000; 275:5748-53.
45. Zhou M, Li XM, Lavker RM. Transcriptional profiling of enriched populations of stem cells versus transient amplifying cells. A comparison of limbal and corneal epithelial basal cells. J Biol Chem 2006; 281:19600-9.
46. Rutledge RG, Cote C. Mathematics of quantitative kinetic PCR and the application of standard curves. Nucleic Acids Res 2003; 31:e93.
47. Barnard JA, Graves-Deal R, Pittelkow MR, DuBois R, Cook P, Ramsey GW, Bishop PR, Damstrup L, Coffey RJ. Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family. J Biol Chem 1994; 269:22817-22.
48. Hashimoto K, Higashiyama S, Asada H, Hashimura E, Kobayashi T, Sudo K, Nakagawa T, Damm D, Yoshikawa K, Taniguchi N. Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes. J Biol Chem 1994; 269:20060-6.
49. Dong J, Opresko LK, Dempsey PJ, Lauffenburger DA, Coffey RJ, Wiley HS. Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor. Proc Natl Acad Sci U S A 1999; 96:6235-40.
50. Shvartsman SY, Hagan MP, Yacoub A, Dent P, Wiley HS, Lauffenburger DA. Autocrine loops with positive feedback enable context-dependent cell signaling. Am J Physiol Cell Physiol 2002; 282:C545-59.
51. Singh AB, Harris RC. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell Signal 2005; 17:1183-93.
52. Wilson SE, Chen L, Mohan RR, Liang Q, Liu J. Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding. Exp Eye Res 1999; 68:377-97.
53. Wilson SE, Liang Q, Kim WJ. Lacrimal gland HGF, KGF, and EGF mRNA levels increase after corneal epithelial wounding. Invest Ophthalmol Vis Sci 1999; 40:2185-90.
54. Dartt DA. Interaction of EGF family growth factors and neurotransmitters in regulating lacrimal gland secretion. Exp Eye Res 2004; 78:337-45.