title: Epidermal growth factor regulates NIKS keratinocyte proliferation through Notch signaling.
abstract: BACKGROUND
  Cutaneous wound healing is a significant health issue in the US, often requiring skin grafts. StrataGraft (Stratatech Corporation, Madison, WI), a second-generation living human skin substitute created from NIKS human keratinocyte progenitors, was recently found to be a promising skin graft in phase I/II safety and efficacy clinical trial. NIKS proliferation is optimal in the presence of epidermal growth factor (EGF). Our preliminary data suggested that Notch signaling also plays a role in NIKS keratinocyte proliferation. Therefore, we hypothesized that EGF might stimulate NIKS proliferation by regulating Notch1 signaling.
  
  
  METHODS
  Notch1 messenger RNA (mRNA) levels from NIKS cells in monolayer culture were assessed by real-time polymerase chain reaction and Notch1 protein levels were detected by Western blot. To determine the role of EGF on Notch1 regulation, cells were incubated in basal media and then treated with EGF (10 ng/mL). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to test NIKS cell proliferation. Cells were grown in basal media supplemented with EGF for 72 h in the presence or absence of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (0-30 μM), an inhibitor of Notch1 signaling.
  
  
  RESULTS
  Notch1 mRNA levels were cell confluence-dependent, being more abundant in a subconfluent cell monolayer. We detected a 2-fold decrease in Notch1 mRNA expression and a reduction in active Notch1 protein level in response to EGF. EGF treatment stimulated NIKS cellular proliferation. However, co-treatment with DAPT inhibited NIKS proliferation to basal levels. Blocking Notch1 activation by DAPT alone inhibited NIKS cellular proliferation (P < 0.01%).
  
  
  CONCLUSIONS
  Our results suggest that Notch1 is an essential downstream mediator of NIKS cellular proliferation via the EGF signaling pathway.
authors.affiliation: Division of Plastic Surgery, The University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin; Department of Surgery, The University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
authors.forename: Madhuchhanda
authors.initials: M
authors.lastname: Roy
authors.forename: Timothy W
authors.initials: TW
authors.lastname: King
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000378:metabolism; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012883:Skin Ulcer / Q000378:metabolism / Q000473:pathology; D014945:Wound Healing / Q000502:physiology
chemical_list: D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; C497761:NOTCH1 protein, human; D012333:RNA, Messenger; D051881:Receptor, Notch1; D004815:Epidermal Growth Factor; D053829:Amyloid Precursor Protein Secretases
keywords: Epidermal growth factor/EGF; Keratinocytes; NIKS; Notch; Proliferation; Wound healing