Crosstalk between elevation of [Ca2+]i, reactive oxygen species generation and phospholipase A2 stimulation in a human keratinocyte cell line.
The aim of the study was to explore the possible interrelationship between reactive oxygen species (ROS) formation and cPLA2 activation and the mediator role that [Ca2+]i may play in these processes in the human keratinocyte cell line, HaCaT. HaCaT cells can be invoked to transiently produce ROS by epidermal growth factor (EGF), thapsigargin (TPG) and the Ca(2+)-ionophore, A23187. These 3 agonists transiently increase [Ca2+]i with characteristic kinetics and magnitude. TPG and A23187 each activates on its own [3H]AA release from prelabeled cells, whereas EGF on its own has no effect on [3H]AA release. However, EGF augments [3H]AA release invoked by TPG or A23187 several fold. EGF activates MAP kinase cascades in HaCaT cells, leads to ROS formation and induces relatively small (1.6 fold) elevation in [Ca2+]i, whereas A23187 and TPG lead to a substantial elevation in [Ca2+]i (2.5 to 5 fold) and to ROS formation. Both have a minor effect on MAP kinase activation. The synergism in PLA2 activation by EGF and TPG or A23187, and the sensitivity of [3H]AA release to N-acetylcysteine (NAC) and dithiothreitol (DTT) (potent reducing agents) or to DPI (an inhibitor of FAD-dependent oxidases) lead to the suggestion that ROS formation, elevation of [Ca2+]i and PLA2 activation are causally related. Since we show that elevation of [Ca2+]i is a prerequisite for both ROS and PLA2 activation, it is possible that these processes contribute to the toxicity (apoptosis) exerted by chronic elevation of [Ca2+]i.
Goldman
R
R
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Moshonov
S
S
Chen
X
X
Berchansky
A
A
Fürstenberger
G
G
Zor
U
U
D001095:Arachidonic Acids / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D017382:Reactive Oxygen Species / Q000502:physiology*; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology
D001095:Arachidonic Acids; D017382:Reactive Oxygen Species; D000001:Calcimycin; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium