Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid.
This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.
Denys
Anne
A
Département de Physiologie, UPRES Lipides et Nutrition, Université de Bourgogne, Faculté des Sciences de la Vie, 6 Boulevard Gabriel, 21000 Dijon, France.
Aires
Virginie
V
Hichami
Aziz
A
Khan
Naim Akhtar
NA
D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology*
D004075:Diglycerides; D005231:Fatty Acids, Unsaturated; D019837:Ryanodine Receptor Calcium Release Channel; D004281:Docosahexaenoic Acids; D019284:Thapsigargin; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010739:Phospholipase D; D002118:Calcium