Thapsigargin induces expression of activating transcription factor 3 in human keratinocytes involving Ca2+ ions and c-Jun N-terminal protein kinase.
Thapsigargin is a specific inhibitor of the sarco/endoplasmic reticulum Ca(2+) ATPase of the endoplasmic reticulum. Here, we show that stimulation of human HaCaT keratinocytes with nanomolar concentrations of thapsigargin triggers expression of activating transcription factor (ATF) 3, a basic-region leucin zipper transcription factor. ATF3 expression was also up-regulated in thapsigargin-stimulated glioma cells, hepatoma cells, retinal pigment epithelial cells, and airway epithelial cells. Thapsigargin-induced up-regulation of ATF3 expression in keratinocytes was attenuated by BAPTA-acetoxymethyl ester or by expression of the Ca(2+)-binding protein parvalbumin in the cytosol of HaCaT cells but not by a panel of pharmacological agents that chelate extracellular Ca(2+) (EGTA) or inhibit either ryanodine receptors (dantrolene) or voltage-gated Ca(2+) channels (nifedipine). Hence, elevated levels of intracellular Ca(2+), released from intracellular stores, are essential for the effect of thapsigargin on the biosynthesis of ATF3. The thapsigargin-induced signaling pathway was blocked by expression of either mitogen-activated protein kinase phosphatase-1 or -5. Experiments involving pharmacological and genetic tools revealed the importance of c-Jun N-terminal protein kinase (JNK) within the signaling cascade, whereas inhibition of extracellular signal-regulated protein kinase or p38 protein kinase did not attenuate thapsigargin-induced expression of ATF3. Functional studies showed that treatment of HaCaT keratinocytes with thapsigargin led to a 2-fold induction of caspase-3/7 activity. The up-regulation of caspase-3/7 activity in thapsigargin-stimulated HaCaT cells was attenuated by inhibition of JNK. Together, these data show that stimulation of HaCaT cells with thapsigargin induces a specific signaling pathway in keratinocytes involving activation of JNK, biosynthesis of ATF3, and up-regulation of caspase-3/7 activity.
Spohn
Daniel
D
Department of Medical Biochemistry and Molecular Biology, Building 44, University of Saarland Medical Center, D-66421 Homburg, Germany.
Rössler
Oliver G
OG
Philipp
Stephan E
SE
Raubuch
Michael
M
Kitajima
Shigetaka
S
Griesemer
Désirée
D
Hoth
Markus
M
Thiel
Gerald
G
D051697:Activating Transcription Factor 1 / Q000096:biosynthesis*; D000841:Anisomycin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002413:Cations, Divalent; D002460:Cell Line; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D054637:Dual-Specificity Phosphatases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000378:metabolism; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology*; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
C531110:ATF1 protein, human; D051697:Activating Transcription Factor 1; D002413:Cations, Divalent; D019284:Thapsigargin; D000841:Anisomycin; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C120407:DUSP10 protein, human; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054638:Dual Specificity Phosphatase 1; D054637:Dual-Specificity Phosphatases; D053148:Caspase 3; D053179:Caspase 7; D000252:Calcium-Transporting ATPases; D002118:Calcium