ERK/p44p42 mitogen-activated protein kinase mediates EGF-stimulated proliferation of conjunctival goblet cells in culture.
OBJECTIVE
To determine whether activation of the ERK pathway by EGF leads to rat and human goblet cell proliferation.


METHODS
The conjunctiva was removed from male Sprague-Dawley rats. Human conjunctiva was removed during ocular surgery. The tissue was minced and goblet cells were grown. The cells were stimulated with EGF (10(-7) M) for 1 and 5 minutes and Western blot analysis was performed with an antibody against phosphorylated EGFR, to measure the activation of the EGF receptor (EGFR). The cells were incubated with EGF (10(-7) M) for 24 hours, and cell proliferation was measured by WST-8. Inhibitors were added either 20 minutes before EGF or 2 hours after. The cells were stimulated with EGF (10(-7) M) for 1 minute to 24 hours. The number of cells expressing phosphorylated ERK (pERK) in the nucleus and Ki-67 was determined by immunofluorescence.


RESULTS
EGF increased the activation of EGFR in rat conjunctival goblet cells. EGF-stimulated proliferation was inhibited by the EGFR inhibitor AG1478 and the MEK inhibitor U0126 in rat and human cultured goblet cells. EGF caused the translocation of pERK to the nucleus in a biphasic manner. Inhibition of the second peak with U0126 prevented proliferation. EGF-stimulated goblet cells progressed through the cell cycle expressing pERK in the nucleus.


CONCLUSIONS
EGF stimulated human and rat conjunctival goblet cell proliferation by activating the EGFR. EGFR stimulated ERK causing its biphasic translocation to the nucleus. The second peak response is responsible for cell proliferation, but the role of the first peak is not known.
Shatos
Marie A
MA
Department of Ophthalmology, Shepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.
Gu
Jian
J
Hodges
Robin R
RR
Lashkari
Kameran
K
Dartt
Darlene A
DA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D020397:Goblet Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley
D019394:Ki-67 Antigen; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; C483803:Egfr protein, rat; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3