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Endogenous EGF-Family Growth Factors are Necessary for the Progression from the G1 to S Phase in Human Keratinocytes

1998; Elsevier BV; Volume: 111; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.1998.00331.x

1523-1747

Teruaki Kobayashi, Koji Hashimoto, Hidenobu Okumura, Hideo Asada, Kunihiko Yoshikawa,

Wnt/β-catenin signaling in development and cancer

Recently several endogenous epidermal growth factor (EGF)-family growth factors (transforming growth factor-α, amphiregulin, and heparin-binding EGF-like growth factor) have been identified in human keratinocytes. These factors are known to play an important role in the regulation of cell proliferation. Here we show that the interaction between these factors and EGF receptor are key factors in the progression from the G1 phase to the S phase (the G1/S progression) in human keratinocytes. In this study, human keratinocytes were cultured in serum-free MCDB153 medium and then partially synchronized by isoleucine deprivation. After synchronization, the number of S phase cells increased and reached a maximum after 18–24 h. The immediate addition of anti-EGF receptor blocking antibody (1 μg per ml) to synchronized cells decreased S phase cells by 42.5% compared with untreated keratinocytes at 18 h. By contrast, the addition of anti-EGF receptor antibodies at 12 h or later did not alter the percentage of S phase cells. Northern blot analysis of synchronized cells demonstrated that mRNA expression of transforming growth factor-α, amphiregulin, heparin-binding EGF-like growth factor, and EGF receptor reached a maximum within 0.5–3 h after synchronization, when many cells initiated progression from the G1 to the S phase. The results show that anti-EGF receptor antibodies block the G1/S progression and the rapid increase of mRNA expression of endogenous EGF-family growth factors and EGF receptor during G1/S progression. These findings indicate that growth factor binding and EGF receptor activation are involved in the G1/S cell cycle progression of human keratinocytes. Recently several endogenous epidermal growth factor (EGF)-family growth factors (transforming growth factor-α, amphiregulin, and heparin-binding EGF-like growth factor) have been identified in human keratinocytes. These factors are known to play an important role in the regulation of cell proliferation. Here we show that the interaction between these factors and EGF receptor are key factors in the progression from the G1 phase to the S phase (the G1/S progression) in human keratinocytes. In this study, human keratinocytes were cultured in serum-free MCDB153 medium and then partially synchronized by isoleucine deprivation. After synchronization, the number of S phase cells increased and reached a maximum after 18–24 h. The immediate addition of anti-EGF receptor blocking antibody (1 μg per ml) to synchronized cells decreased S phase cells by 42.5% compared with untreated keratinocytes at 18 h. By contrast, the addition of anti-EGF receptor antibodies at 12 h or later did not alter the percentage of S phase cells. Northern blot analysis of synchronized cells demonstrated that mRNA expression of transforming growth factor-α, amphiregulin, heparin-binding EGF-like growth factor, and EGF receptor reached a maximum within 0.5–3 h after synchronization, when many cells initiated progression from the G1 to the S phase. The results show that anti-EGF receptor antibodies block the G1/S progression and the rapid increase of mRNA expression of endogenous EGF-family growth factors and EGF receptor during G1/S progression. These findings indicate that growth factor binding and EGF receptor activation are involved in the G1/S cell cycle progression of human keratinocytes. anti-EGF receptor antibodies amphiregulin epidermal growth factor receptor heparin-binding EGF-like growth factor The progression of the cell cycle can be regulated either at control points in the G1 phase that precedes the S phase or at points in the G2 phase prior to mitosis. Thus, progression from the G1 to the S phase (the G1/S progression) is a crucial step in the cell cycle. In order to study the G1/S progression, synchronized populations of cells are required. We adapted the isoleucine-deprivation method for cell synchronization to normal human keratinocytes (Ashihara and Baserga, 1979Ashihara T. Baserga R. Cell synchronization.Methods of Enzymology. Vol. LVIII. Academic Press, San Diego1979: 248-262Google Scholar;Ayusawa, 1992Ayusawa D. Cell culture and its application current methods for a synchronous culture of mammalian cells.Jpn J Cancer Chemother. 1992; 19: 1935-1940PubMed Google Scholar;Kobayashi et al., 1998Kobayashi T. Okumura H. Hashimoto K. Asada H. Inui S. Yoshikawa K. Synchronization of normal human keratinocyte in culture – its application to the analysis of 1,25-dihydroxyvitamin D3 effects on cell cycle.J Dermatol Sci. 1998; 17: 108-114Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), because (i) this method exerts little or no effect on the metabolic processes of the cell, and (ii) it enables us to harvest synchronized cells in large quantities. This method allowed us to study in greater detail the cell cycle of normal human keratinocytes in culture. Recent studies have shown that human keratinocytes produce endogenous epidermal growth factor (EGF)-family growth factors such as transforming growth factor-α (TGF-α) (Coffey et al., 1987Coffey Jr., W. Derynck R. Wilcox J.N. Bringman T.S. Goustin A.S. Moses H.L. Pittelkow M.R. Production and auto-induction of transforming growth factor-a in human keratinocytes.Nature. 1987; 328: 817-820Crossref PubMed Scopus (691) Google Scholar), amphiregulin (AR) (Shoyab et al., 1989Shoyab M. Piowman G.D. McDonald V.L. Bradley J.G. Todaro G.J. Structure and function of human amphiregulin: a member of the epidermal growth factor family.Science. 1989; 243: 1074-1076Crossref PubMed Scopus (487) Google Scholar;Cook et al., 1991Cook P.W. Mattox P.A. Keeble W.W. et al.A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.Mol Cell Biol. 1991; 11: 2547-2557Crossref PubMed Scopus (208) Google Scholar), and heparin-binding EGF-like growth factor (HB-EGF) (Higashiyama et al., 1991Higashiyama S. Abraham J.A. Miller J. Fiddes X. Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.Science. 1991; 251: 936-939Crossref PubMed Scopus (1044) Google Scholar;Hashimoto et al., 1994Hashimoto K. Higashiyama S. Asada H. et al.Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes.J Biol Chem. 1994; 269: 20060-20066Abstract Full Text PDF PubMed Google Scholar). In human keratinocytes, TGF-α is synthesized, and its production is induced by EGF and TGF-α (auto-induction mechanism) (Coffey et al., 1987Coffey Jr., W. Derynck R. Wilcox J.N. Bringman T.S. Goustin A.S. Moses H.L. Pittelkow M.R. Production and auto-induction of transforming growth factor-a in human keratinocytes.Nature. 1987; 328: 817-820Crossref PubMed Scopus (691) Google Scholar). AR competes with EGF for cell surface binding, exerts at least some of its biologic effects through the EGF receptor (EGFR), and, thus, represents an autocrine growth factor for keratinocytes (Shoyab et al., 1988Shoyab M. McDonald V.L. Bradley J.G. Todaro G.J. Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate treated human breast adenocarcinoma cell line MCF-7.Proc Natl Acad Sci USA. 1988; 85: 6528-6532Crossref PubMed Scopus (295) Google Scholar,Shoyab et al., 1989Shoyab M. Piowman G.D. McDonald V.L. Bradley J.G. Todaro G.J. Structure and function of human amphiregulin: a member of the epidermal growth factor family.Science. 1989; 243: 1074-1076Crossref PubMed Scopus (487) Google Scholar;Cook et al., 1991Cook P.W. Mattox P.A. Keeble W.W. et al.A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.Mol Cell Biol. 1991; 11: 2547-2557Crossref PubMed Scopus (208) Google Scholar). HB-EGF is another new member of the EGF family that was initially purified from conditioned medium of the U-937 macrophage-like cell line (Higashiyama et al., 1991Higashiyama S. Abraham J.A. Miller J. Fiddes X. Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.Science. 1991; 251: 936-939Crossref PubMed Scopus (1044) Google Scholar). HB-EGF and AR share structural characteristics (Higashiyama et al., 1992Higashiyama S. Lau K. Besner G.E. Abraham J.A. Klagsbrun M. Structure of heparinbinding EGF-like growth factor. Multiple forms, primary structure, and glycosylation of the mature protein.J Biol Chem. 1992; 267: 6205-6212Abstract Full Text PDF PubMed Google Scholar). A recent report has shown that HB-EGF is an autocrine growth factor for human keratinocytes (Hashimoto et al., 1994Hashimoto K. Higashiyama S. Asada H. et al.Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes.J Biol Chem. 1994; 269: 20060-20066Abstract Full Text PDF PubMed Google Scholar). These factors display equivalent affinity for EGFR in an intestinal epithelial cell line (Barnard et al., 1994Barnard J.A. Graves-deal R. Pittelkow M.R. et al.Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family.J Biol Chem. 1994; 269: 22817-22822Abstract Full Text PDF PubMed Google Scholar). The EGFR is a 170 kDa transmembrane glycoprotein consisting of an extracellular binding domain and an intracellular domain that exhibits tyrosine kinase activity. The binding of physiologic ligands to the external domain of the receptor stimulates tyrosine kinase activity and promotes the activation of other signaling molecules such as phospholipase C, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Ultimately, activation of the EGFR leads to mitosis (Blenis, 1993Blenis J. Signal transduction via the MAP kinases: Proceed at your own RSK.Proc Natl Acad Sci USA. 1993; 90: 5889-5892Crossref PubMed Scopus (1157) Google Scholar). EGF, TGF-α, and EGFR play an important role in the growth regulation of many normal and malignant cell types (Ennis et al., 1989Ennis B.W. Valverius E.M. Bates S.E. et al.Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.Mol Endocrinol. 1989; 3: 1830-1838Crossref PubMed Scopus (137) Google Scholar;Bates et al., 1990Bates S.E. Valverius E.M. Ennis B.W. et al.Expression of the transforming growth factor-alphalepidermal growth factor receptor pathway in normal human breast epithelial cells.Endocrinology. 1990; 126: 596-607Crossref PubMed Scopus (113) Google Scholar;Crew et al., 1992Crew A.J. Langdon S.P. Miller E.P. Miller W.R. Mitogenic effects of epidermal growth factor and transforming growth factor-alpha on EGF-receptor positive human ovarian carcinoma cell lines.Eur J Cancer. 1992; 28: 337-341Abstract Full Text PDF PubMed Scopus (36) Google Scholar;Dong et al., 1992Dong X.F. Berthois Y. Dussert C. Isnardon D. Palmari J. Martin P.M. Mode of EW action on cell cycle kinetics in human breast cancer cell line IVICF-T some evidence that EW acts as a "progression factor".Anticancer Res. 1992; 12: 2085-2092Google Scholar;Rabiasz et al., 1992Rabiasz G.J. Langdon S.P. Bartlett J.M. et al.Growth control by epidermal growth factor and transforming growth factor alpha in human lung squamous carcinoma cells.Br J Cancer. 1992; 66: 254-259Crossref PubMed Scopus (28) Google Scholar). Monoclonal anti-EGFR neutralizing antibody (aEGFR) blocks binding of EGF-family growth factors to the receptor, inhibits cellular proliferation, and provides a tool to examine cell cycle regulation in keratinocytes (Sunada et al., 1986Sunada H. Magun B.E. Mendelsohn J. MacLeod C.L. Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation.Proc Natl Acad Sci USA. 1986; 83: 3825-3829Crossref PubMed Scopus (219) Google Scholar;Ennis et al., 1989Ennis B.W. Valverius E.M. Bates S.E. et al.Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.Mol Endocrinol. 1989; 3: 1830-1838Crossref PubMed Scopus (137) Google Scholar,Ennis et al., 1991Ennis B.W. Lippman M.E. Dickson R.B. The EW receptor system as a target for antitumor therapy.Cancer Invest. 1991; 9: 553-562Crossref PubMed Scopus (128) Google Scholar;Pittelkow et al., 1993Pittelkow M.R. Cook P. Shipley G.D. Derynck R. Coffey Jr., Rj Autonomous growth of human keratinocytes requires epidermal growth factor receptor occupancy.Cell Growth Differ. 1993; 4: 513-521PubMed Google Scholar), similar to several other cell types where proliferation is inhibited (Kawamoto et al., 1983Kawamoto T. Sato J.D. Le A. Polikoff J. Sato G.H. Mendelsohn J. Growth stimulation of A431 cells by EGR identification of high affinity receptors for epidermal growth factor by an anti-receptor monoclomal antibody.Proc Natl Acad Sci USA. 1983; 80: 1337-1341Crossref PubMed Scopus (690) Google Scholar;Sato et al., 1983Sato J.D. Kawamoto T. Le A. Mendelsohn J. Polikoff J. Sato G.H. Biological effects in vitro of monoclomal antibodies to human EGF receptors.Mol Biol Med. 1983; 1: 511-529PubMed Google Scholar;Ennis et al., 1989Ennis B.W. Valverius E.M. Bates S.E. et al.Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.Mol Endocrinol. 1989; 3: 1830-1838Crossref PubMed Scopus (137) Google Scholar;Bates et al., 1990Bates S.E. Valverius E.M. Ennis B.W. et al.Expression of the transforming growth factor-alphalepidermal growth factor receptor pathway in normal human breast epithelial cells.Endocrinology. 1990; 126: 596-607Crossref PubMed Scopus (113) Google Scholar;Pittelkow et al., 1993Pittelkow M.R. Cook P. Shipley G.D. Derynck R. Coffey Jr., Rj Autonomous growth of human keratinocytes requires epidermal growth factor receptor occupancy.Cell Growth Differ. 1993; 4: 513-521PubMed Google Scholar). Here we show that the interaction between EGFR and endogenous EGF-family growth factors plays an important role in the G1/S progression of synchronized keratinocytes. Normal human keratinocytes were cultured as described previously (Wille et al., 1984Wille Jr., Jj Pittelkow M.R. Shipley G.I.D. Scott R.E. Integrated control of growth and differentiation of normal human keratinocytes cultured in serum-free medium: clonal analysis, growth kinetics, and cell cycle studies.J Cell Physiol. 1984; 121: 31-44Crossref PubMed Scopus (350) Google Scholar). Briefly, skin was cut into pieces 3–5 mm square, and floated on dispase solution (500 U per ml) overnight at 4°C. After separation of epidermis from dermis by forceps, the epidermal sheets were rinsed with Ca2+ and Mg2+-free phosphate-buffered saline and incubated in 0.25% trypsin solution for 10 min at 37°C, and then the epidermis was teased free by forceps. Cells were suspended and cultured in MCDB153 medium (Kyokuto, Tokyo, Japan) supplemented with insulin (5 μg per ml), hydrocortisone (5 × 10–7 M), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), and bovine hypothalamic extract (150 μg per ml) ("complete" MCDB153 medium). EGF was not added. Second-passage cells were used for synchronization. The growing cells were synchronized by transfer to MCDB153 medium deprived of isoleucine, insulin, hydrocortisone, and bovine hypothalamic extract. After 60 h, they were detached from the dish by trypsin treatment and were replated in complete MCDB153 medium. The cell cycle analysis of human keratinocytes was performed by the two color cell cycle analysis method (Dolbeare et al., 1983Dolbeare F. Gratzner H. Pallavicini M.G. Gray J.W. Flowcytometric measurement of total DNA content and incorporated bromodeoxyuridine.Proc Natl Acad Sci USA. 1983; 80: 5573-5577Crossref PubMed Scopus (907) Google Scholar). Synchronized cells were pulse-labeled with 0.01 mM 5-bromodeoxyuridine for 120 min prior to each time point. 5-Bromodeoxyuridine incorporated into the nucleus was immunochemically stained using anti-5-bromodeoxyuridine monoclonal antibody (Becton Dickson, San Jose, CA), and DNA was stained with propidium iodide. These stained cells were analyzed by flow cytometry using a FACScan (Becton Dickson). Ten thousand cells were used for each FACScan analysis. aEGFR (Ab-3; Oncogene Research Products, Calbiochem-Novabiochem International, San Diego, CA) is a mouse monoclonal antibody (IgG1) produced by immunization of mice with partially purified EGFR from A431 cells. aEGFR binds to A431, HeLa cells, and other human cells. It inhibits ligand binding to receptor and acts as an antagonist of EGF-stimulated tyrosine kinase activity (Gill et al., 1984Gill G.N. Kawamoto T. Cochet C. et al.Monoclomal and-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor stimulated tyrosine protein kinase activity.J Biol Chem. 1984; 259: 7755-7760Abstract Full Text PDF PubMed Google Scholar;Kawamoto et al., 1984Kawamoto T. Mendelsohn J. Le A. Sato G.H. Lazar C.S. Gill G.N. Relation of. epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells.J Biol Chem. 1984; 259: 7761-7766Abstract Full Text PDF PubMed Google Scholar). The antibody was used at 1 μg per ml in these experiments. This concentration is enough to inhibit cell growth in complete MCDB153 medium (Inui et al., 1997Inui S. Higashiyama S. Hashimoto K. Higashiyama M. Yoshikawa K. Taniguchi N. Possible role of coexpression of CD9 with membrane-anchored heparin-binding EGF-like growth factor and amphiregulin in cultured human keratinocyte growth.J Cell Physiol. 1997; 71: 291-298Crossref Scopus (70) Google Scholar). Normal preimmune mouse IgG at the same concentration was used as control. Total RNA from cultured human keratinocyte was prepared by the acid guanidium thiocyanate/phenol/chloroform method (Chorriczynski and Sacchi, 1987Chorriczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Google Scholar'). Twenty micrograms of total RNA per lane was fractionated on a 1.8% formaldehyde-agarose gel and transferred to GeenScreen (DuPont NEN, Boston, MA) in 20 × sodium citrate/chloride buffer (150 mM sodium chloride, 15 mM sodium citrate) according to standard procedure (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory, New York1989: 7.3-7.52Google Scholar). Ultraviolet cross-linking was performed by ultraviolet radiation in a Stratalinker (Stratagene, La Jolla, CA). Prehybridization was carried out in sealed plastic bags for 6 h at 65°C in a hybridization buffer (5 × sodium citrate/chloride buffer, 10 × Denhardt's solution, 10 mM NaHPO4, pH 6.5, 0.5% sodium dodecyl sulfate, 50% formamide, 0.1 mg sonicated herring sperm DNA per ml). Hybridization was performed using hybridization buffer supplemented by 1000 ng of digoxigenin (DIG)-labeled single-stranded RNA probe per ml of buffer for 16 h at 65°C. The hybridized filters were then washed at 65°C once in 2 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate, and twice in 0.2 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate. DIG-labeled nucleic acids were detected in the following way (During, 1991During K. Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA, and proteins in plant molecular biology.Anal Biochem. 1991; 196: 433-438Crossref PubMed Scopus (29) Google Scholar;Lanzillo, 1991Lanzillo J.J. Cherniluminescent nucleic acid detection with digoxigenin-labeled probes: a model system with probes for angiotensin converting enzyme which detect less than one attomole of target DNA.Anal Biochem. 1991; 194: 45-53Crossref PubMed Scopus (80) Google Scholar). All the steps were carried out at room temperature. Reagents were a part of the Boehringer DIG DNA detection kit. The filter was briefly rinsed twice in DIG buffer 1 (0.1 M maleic acid, 0.15 M NaCI, pH 7.5), and subsequently nonspecific binding sites were blocked by incubating them for 60 min in DIG buffer 1 containing 1.5% (wt/vol) Boehringer blocking regent, a specially purified fraction of dried milk powder. Anti-DIG Fab fragment, conjugated to alkaline phosphatase diluted 1:10,000 in DIG buffer 1 containing 0.2% Tween 20, was applied for 30 min. Excess antibody was washed off twice in DIG buffer 1 containing 0.2% Tween 20 for 20 min, and this buffer was then exchanged for DIG buffer 3 (0.1 M Tris-HCI, 0.1 M NaCl, 50 mM MgCl2, pH 9.5) for 3 min. The filter was washed twice with the assay buffer (100 mM diethanolamine, 2 mM MgCl2, 0.02% NaN3) for 10 min and then incubated with 3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)-phenyl-1,2-dioxetane solution (0.1 mg per ml in the assay buffer) in a plastic bag for 15 min as described elsewhere. The filter was then transferred and sealed in a new plastic bag and exposed to Kodak X-Omat AR film at room temperature for periods ranging from 30 min to 2 h. For all of the northern blots, the rRNA (28 and 18 S) in the gels was stained with ethidium bromide and photographed before being transferred to a nylon membrane to confirm the presence of equivalent amounts of RNA in each lane. The relative signal intensities of mRNA levels were quantitated by densitometric scanning using a dual wavelength flying spot scanner (CS-9000; Shimadzu, Tokyo, Japan). We evaluated cell cycle synchronization using flow cytometry. Second-passage cells were cultured in complete MCDB153 medium for several days. Cells were switched to an isoleucine-deprived MCDB153 medium for 60 h, as described in Materials and Methods. This treatment decreased the percentage of cells in the S phase from 32.0 ± 3.7% to 4.2 ± 1.0% and increased the cell percentage in the G1/G0 and G2 + M phases from 49.1 ± 4.3% to 68.2 ± 4.4% and from 18.9 ± 2.7% to 27.6 ± 4.7%, respectively. Cells preferentially arrest in the G1/G0 phase. Next, we examined the change of cell cycle distribution over time after cells were replated in complete MCDB153 medium following isoleucine-deprivation. Cell cycle distribution was measured every 3 h up to 30 h (Figure 1). The percentage of cells in the S phase remained ≤ 2% until 6 h, when the S phase percentage started to increase. The peak percentage of 28.1 ± 5.5% was reached at 18 h and then started to decrease again after 24 h. Concomitantly, the cell percentage in the G1/G0 phase decreased from 64.9 ± 4.2% at 0 h to a low point of 36.6% at 24 h, and then started to increase. The percentage in G2 + M was 31.3 ± 4.1% at 0 h and increased gradually over time. Immediate addition of aEGFR (1 μg per ml) following synchronization decreased S phase cells by 42.5%, compared with untreated keratinocytes at 18 h (Figure 2). By contrast, the addition of aEGFR at 12 h or later after switching to complete medium did not alter the percentage of S phase cells (data not shown). We next examined expression of EGF-related ligands and EGFR during keratinocyte cell cycle progression. We analyzed the time-dependent mRNA expression of EGF-family growth factors (TGF-α, AR, and HB-EGF) by northern blot analysis and densitometry (Figure 3). mRNA expression was examined at 0.5, 1, 3, 6, 21, and 33 h after replating in complete medium. Maximum expression of all ligands was observed after between 0.5 and 3 h. Levels of TGF-α mRNA increased slowly following synchronization and release, compared with AR and HB-EGF. Peak expression was observed between 1 h (2.3-fold) and 3 h (2.2-fold), followed by a marked decrease in level of expression. The increase in levels of AR mRNA was the most marked. The maximum level was reached between 30 min (2.2-fold) and 1 h (2.6-fold). By 3 h, the AR mRNA level decreased to baseline level. HB-EGF mRNA levels changed the least. An increase of only 1.9-fold at 30 min and of 1.8-fold at 1 h was observed, followed by a gradual decrease over time. Increased expression of these EGF-related growth factors preceded the cell cycle progression of the synchronized and reflected cells. We analyzed and quantitated the levels of mRNA expression of EGFR by northern blot and densitometry (Figure 4). mRNA expression of EGFR was examined at 0.5, 1, 3, 6, 21, and 33 h after release from synchronization. EGFR mRNA did not show the more marked changes observed for mRNA levels of the EGF-related ligands, and increased only 1.4, 1.5, and 1.3-fold at 30 min, 1 h, and 3 h, respectively. EGFR mRNA levels subsequently decreased markedly to one half the initial level at 21 h, at a time when the percentage of S phase cells were maximal. In this report, we demonstrate that the interaction between EGFR and endogenous EGF-family growth factors plays an important role in the G1/S progression of synchronized keratinocytes. The discovery of several new EGF-family growth factors has made it more challenging to understand the mechanisms of cell cycle regulation and growth factor interactions in keratinocytes that express these ligands. Previous work (Coffey et al., 1987Coffey Jr., W. Derynck R. Wilcox J.N. Bringman T.S. Goustin A.S. Moses H.L. Pittelkow M.R. Production and auto-induction of transforming growth factor-a in human keratinocytes.Nature. 1987; 328: 817-820Crossref PubMed Scopus (691) Google Scholar) identified upregulation of TGF-α mRNA and protein by EGF, and described the phenomenon of autoregulation of TGF-α gene expression in cultured keratinocytes. Other studies (Barnard et al., 1994Barnard J.A. Graves-deal R. Pittelkow M.R. et al.Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family.J Biol Chem. 1994; 269: 22817-22822Abstract Full Text PDF PubMed Google Scholar;Hashimoto et al., 1994Hashimoto K. Higashiyama S. Asada H. et al.Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes.J Biol Chem. 1994; 269: 20060-20066Abstract Full Text PDF PubMed Google Scholar) have reported the cross-regulation of EGF-family growth factor gene expression by these same factors in several epithelial cell types, suggesting complexity in the molecular mechanisms that are involved in induction of these factors. Therefore, these growth factors may act not only by autoinduction but also through mutual amplification mechanisms. By contrast, it has been recently reported that inhibition of ligand binding and kinase activation of EGFR in keratinocytes is coupled to growth arrest, cell commitment, and induction of terminal differentiation (Peus et al., 1997Peus D. Hamacher L. Pittelkow M.R. EGF-Receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation.J Invest Dermatol. 1997; 109: 751-756Abstract Full Text PDF PubMed Scopus (152) Google Scholar). These findings point to EGFR in regulating growth control mechanisms as well as the expression of endogenous EGF-related ligands that mediate cell cycle progression in keratinocytes. We have shown that the addition of aEGFR inhibited the increase of S phase cells of keratinocytes synchronized in serum- and growth factor-free culture medium. This observation implicates a role for EGFR and endogenous ligands in G1/S progression of keratinocytes. We also examined the levels of mRNA expression of endogenous EGF-related ligands (TGF-α, AR, and HB-EGF). Though the mRNA levels of each of these ligands increased during G1/S progression, the profile of change for each was distinct. As can be seen in Figure 3 (e), AR and HB-EGF increased during the early phase, and HB-EGF tended to decrease quickly at 1 h. At 1 h, AR further increased and TGF-α expression began to increase. At 3 h, AR and HB-EGF declined to base line levels. We speculate from these results that AR and HB-EGF may play important roles in the early stages of G1/S progression. TGF-α continued to increase to over twice the initial level at 3 h. TGF-α may play a role in sustaining G1/S progression that has been initiated by AR and HB-EGF. Barnard et al. have drawn similar conclusions regarding the status of AR and HB-EGF as immediate early genes (Barnard et al., 1994Barnard J.A. Graves-deal R. Pittelkow M.R. et al.Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family.J Biol Chem. 1994; 269: 22817-22822Abstract Full Text PDF PubMed Google Scholar). They have reported that AR and HB-EGF were rapidly and intensely induced by EGF-family growth factor treatment, and that the induction occurred through a transcriptional mechanism. Our studies show that AR and HB-EGF may be regarded as an immediate early gene in the G1/S progression; however, it is still unclear why keratinocytes express these EGF-family growth factors. We hypothesize that the cooperation of HB-EGF, AR, and TGF-α in a time-dependent manner make it possible for keratinocytes to progress from the G1 to the S phase. We also considered the possibility that the relative expression of EGFR could regulate the G1/S progression in conjunction with expression of the cognate ligands. EGFR mRNA increased slightly in the early phases; however, the level of mRNA expression declined to one half the initial level at 21 h (when S phase cells were maximum), at a time when the EGF-related ligands had also decreased. These results indicate that EGFR–endogenous ligand interactions might not be required at later times once the cell has entered the S phase. There are few reports that have identified any cell cycle regulatory proteins upon which EGF-family growth factors act. Recently it has been shown that the growth of HPV 16-immortalized human keratinocytes can be blocked by a selective EGFR kinase inhibitor, which induces a significant increase in the Cdk2 protein inhibitors p27 and p21, and counteracts the activation of Cdk2 (Ben-bassat et al., 1997Ben-bassat H. Rosenbaum-mitrani S. Hartzstark Z. et al.Inhibitors of EGF-Receptor kinase and of cycline dependent kinase 2 activation induce growth arrest, differentiation and apotosis of human papilloma virus 16-immortalized human keratinocytes.Cancer Res. 1997; 57: 3741-3750PubMed Google Scholar). The EGFR-endogenous EGF-related ligand interactions are critical regulators of the keratinocyte cell cycle. Further investigations are necessary to fully elucidate the complex mechanisms involved in autocrine growth and differentiation control of keratinocytes and epidermis. These findings demonstrate a role for endogenous EGF-related ligands and the relevant receptor, EGFR, in controlling G1/S cell cycle progression. The authors thank Sumi Hayakawa and Kaori Sudo for their excellent technical assistance. We also thank M.R. Pittelkow for critical readings of the manuscript.