title: Keratinocyte growth inhibition by high-dose epidermal growth factor is mediated by transforming growth factor beta autoinduction: a negative feedback mechanism for keratinocyte growth.
abstract: 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.
authors.affiliation: Department of Dermatology, Ehime University School of Medicine, Ehime, Japan. yamasaki@m.ehime-u.ac.jp
authors.forename: Kenshi
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Nobuko
authors.initials: N
authors.lastname: Toriu
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hanakawa
authors.forename: Yuji
authors.initials: Y
authors.lastname: Shirakata
authors.forename: Koji
authors.initials: K
authors.lastname: Sayama
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takayanagi
authors.forename: Masafumi
authors.initials: M
authors.lastname: Ohtsubo
authors.forename: Shinobu
authors.initials: S
authors.lastname: Gamou
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Shimizu
authors.forename: Makiko
authors.initials: M
authors.lastname: Fujii
authors.forename: Kohei
authors.initials: K
authors.lastname: Miyazono
authors.forename: Koji
authors.initials: K
authors.lastname: Hashimoto
mesh_terms: D030201:Activin Receptors, Type I / Q000502:physiology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000008:administration & dosage*; D066246:ErbB Receptors / Q000502:physiology; D005246:Feedback; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology*; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D007371:Interferon-gamma; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human