title: Role of PKCα activation of Src, PI-3K/AKT, and ERK in EGF-stimulated proliferation of rat and human conjunctival goblet cells.
abstract: OBJECTIVE
  To determine the order and components of the signaling pathway utilized by epidermal growth factor (EGF) to stimulate conjunctival goblet cell proliferation.
  
  
  METHODS
  Goblet cells from rat bulbar and forniceal conjunctiva and human bulbar conjunctiva were grown in organ culture. Goblet cells (GCs) were serum starved for 24 hours and preincubated with inhibitors for 30 minutes or small interfering RNA (siRNA) for 48 hours prior to addition of EGF. Proliferation was then measured or Western blot analysis was performed using antibodies against phosphorylated protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), or the non-receptor tyrosine kinase Src. Rat GCs were also incubated with adenoviruses expressing dominant negative protein kinase Cα (DNPKCα) or constitutively activated protein kinase Cα (myrPKCα), and activation of AKT and ERK1/2 was determined by Western blot analysis.
  
  
  RESULTS
  Inhibitors of phosphoinositol-3 kinase (PI-3K)/AKT pathway blocked EGF-stimulated ERK1/2 activation and GC proliferation. Inhibitors of EGF-stimulated ERK1/2 activity did not inhibit AKT activation but blocked proliferation. DNPKCα blocked EGF-stimulated activation of AKT and ERK1/2 while myrPKCα increased activation of these kinases. Inhibitors of PI-3K, ERK1/2, and protein kinase C (PKC) blocked myrPKCα-stimulated GC proliferation. EGF and myrPKCα increased phosphorylation of Src, and inhibition of Src with the chemical inhibitor PP1 or siRNA inhibited EGF-stimulated GC proliferation.
  
  
  CONCLUSIONS
  We found that EGF activates a major pathway to stimulate goblet cell proliferation. This pathway consists of induction of phospholipase C (PLC)γ to activate PKCα. Active PKCα phosphorylates Src to induce PI-3K to phosphorylate AKT that subsequently activates the ERK1/2 cascade to stimulate goblet cell proliferation.
authors.affiliation: Schepens Eye Research Institute/Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Dayu
authors.initials: D
authors.lastname: Li
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D020397:Goblet Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009924:Organ Culture Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000502:physiology; D061251:Primary Cell Culture; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D019950:Mitogen-Activated Protein Kinase 1; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051571:Protein Kinase C-alpha; D051057:Proto-Oncogene Proteins c-akt; D016392:Proto-Oncogene Proteins pp60(c-src); D034741:RNA, Small Interfering; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; C535150:MAPK1 protein, human
keywords: EGF; cell proliferation; conjunctiva; goblet cells