Signaling by HGF and KGF in corneal epithelial cells: Ras/MAP kinase and Jak-STAT pathways.
OBJECTIVE
To characterize the signaling pathways used by hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in human corneal epithelial cells.


METHODS
Cultures of SV40 large T antigen-transfected human corneal epithelial cells were treated with recombinant human HGF or KGF at 50 ng/ml to 100 ng/ml for 5 to 30 minutes and harvested for protein isolation. Immunoprecipitation was performed with antisera to signal transducers and activators of transcription 1 (STAT1), STAT3, Janus kinase 1 (Jak1), Shc, Grb2, Sos1, and HGF receptor (met). Immunoprecipitated proteins were analyzed by western blot analysis. Gel retardation experiments were carried out with first-passage human corneal epithelial cells to detect binding of STATs to the high affinity c-sis (platelet-derived growth factor) inducible DNA element (hSIE). Effects of HGF, KGF, and kinase inhibitors on mitogen-activated protein kinase (MAPK) activation were evaluated by western blot analysis and enzymatic assays.


RESULTS
In human corneal epithelial cells, the phosphorylation of adapter protein Shc (52- and 66-kDa isoforms) was enhanced by HGF, but not by KGF. Phosphorylated HGF receptor coimmunoprecipitated with Shc, Grb2, and Sos1. Hepatocyte growth factor or KGF rapidly activated MAPK in corneal epithelial cells. The activation of MAPK (p42 and p44) by HGF or KGF was transient and decreased gradually within 1 hour. MAPK kinase 1 (MEK1) inhibitor PD098059 or the protein tyrosine kinase inhibitor genistein blocked MAPK activation. Activation of MAPK induced by HGF was partially inhibited by protein kinase C inhibitor calphostin C. Hepatocyte growth factor and KGF had no effect on the activation of Jak-STAT cascade components that are activated by epidermal growth factor.


CONCLUSIONS
Hepatocyte growth factor and KGF activate Ras-MAPK pathways in human corneal epithelial cells. There may be at least two routes used by HGF in transmitting signals from its receptor to the MAPK cascade. One is the receptor-Grb2/Sos complex to the Ras pathway, and the other is through protein kinase C. Hepatocyte growth factor and KGF did not activate the Jak-STAT cascade components STAT1, STAT3, or Jak1 in corneal epithelial cells.
Liang
Q
Q
Eye Institute and Department of Cell Biology, The Cleveland Clinic Foundation, Ohio 44195, USA.
Mohan
R R
RR
Chen
L
L
Wilson
S E
SE
D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051526:Fibroblast Growth Factor 10; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D005419:Flavonoids / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D053613:Janus Kinase 1; D009281:Naphthalenes / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C496453:FGF7 protein, human; D051526:Fibroblast Growth Factor 10; D005419:Flavonoids; D006133:Growth Substances; D009281:Naphthalenes; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D017228:Hepatocyte Growth Factor; D019833:Genistein; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; D053613:Janus Kinase 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); C058819:calphostin C; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one