Epidermal growth factor-induced cytoprotection in human intestinal cells involves intracellular calcium signaling.
BACKGROUND
The mechanism(s) whereby epidermal growth factor (EGF) protects against cellular injury remains poorly understood. Previous data in our laboratory have suggested that EGF-induced cellular proliferation in human colonic carcinoma cells (Caco-2) may involve changes in intracellular calcium content ([Ca(2+)](i)). Our current objective was to determine if a similar process was involved with EGF-induced cytoprotection.


METHODS
Postconfluent Caco-2 cells were employed for all experimentation. [Ca(2+)](i) was measured with Fluo-3 fluorescence. Injury was measured employing Ethidium homodimer 1 uptake and lactate dehydrogenase (LDH) release.


RESULTS
Caco-2 cells pretreated, but not concomitantly treated, with EGF (10-100 ng/ml, 30-60 min) significantly attenuated cellular injury induced subsequently by 500 microM deoxycholate (DC). Cells exposed to 100 ng/ml EGF demonstrated an initial increase in [Ca(2+)](i) (1-5 min) which was blocked with neomycin, an inhibitor of inositol 1,4,5-trisphosphate (IP(3)) generation, and the phospholipase C (PLC) inhibitor U73122, but not U73343 (inactive control). This was followed by sustained extracellular Ca(2+) influx (5-20 min), which was attenuated with calcium-free buffer and the store operated Ca(2+) channel blocker La(3+). [Ca(2+)](i) then returned to baseline (20-30 min), a process blocked with the Ca(2+)-ATPase inhibitors quercetin and vanadate. The above treatments, which in and of themselves did not induce cellular injury, were repeated and cells were subsequently exposed to DC. All groups exposed to 500 microM DC demonstrated significant increases in both Ethidium Homodimer 1 uptake and LDH release. Both indices of injury were significantly decreased when cells were pretreated with EGF +/- the inactive PLC inhibitor U73343. However, protection induced by EGF was lost when any of its effects on changes in [Ca(2+)](i) were prevented: internal Ca(2+) store release via PLC and IP(3), sustained Ca(2+) influx through store operated Ca(2+) channels, or subsequent Ca(2+) efflux.


CONCLUSIONS
Taken together, these data strongly suggest that the cytoprotective effects of EGF may involve Ca(2+) signaling.
Kokoska
E R
ER
Theodore Cooper Surgical Research Institute, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104, USA.
Wolff
A B
AB
Smith
G S
GS
Miller
T A
TA
D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D019610:Cytoprotection; D003840:Deoxycholic Acid / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D007422:Intestines / Q000187:drug effects*
D003840:Deoxycholic Acid; D004815:Epidermal Growth Factor; D002118:Calcium