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1.
Biochem Pharmacol ; 81(9): 1087-97, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21338579

ABSTRACT

Our aim was to get new information about the Peroxisome Proliferator Activated Receptor gamma (PPARγ)-independent pathway involved in the antiproliferative action of PPARγ ligands in breast cancer cells. We investigated the effects of Troglitazone (TGZ), Ciglitazone (CGZ), Rosiglitazone (RGZ) and, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ(2)) on the hormone-dependent breast cancer cell line MCF7. The early transcription factor EGR1 (Early Growth Response gene 1) mRNA and protein levels peaked after 3h of incubation with 25µM TGZ, CGZ or 15d-PGJ(2) and then gradually decreased. RGZ, the most potent activator of PPARγ, did not show this effect. The PPARγ antagonist GW 9662 did not block EGR1 mRNA induction which also still occurred in case of PPARγ silencing as well as in case of treatment with the PPARγ-inactive compound Δ2-TGZ. EGR1 mRNA induction required ERK1/2 phosphorylation which was not blocked by EGF Receptor (EGFR) inhibition. The ERK1/2 pathway was also involved in Δ2-TGZ-induced EGR1 mRNA expression in the hormone-independent breast cancer cell line MDA-MB-231. Using the fluorescent dye Fura2, we showed in MCF7 that TGZ or Δ2-TGZ induced an immediate increase in cytosolic calcium which was required for ERK1/2 phosphorylation and EGR1 mRNA induction as demonstrated by calcium chelation experiments. Furthermore, in MCF7 transfected with siRNA targeting EGR1, Δ2-TGZ inhibited less efficiently cell proliferation.


Subject(s)
Breast Neoplasms/pathology , Calcium/metabolism , Cell Proliferation/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , PPAR gamma/metabolism , Prostaglandin D2/analogs & derivatives , Thiazolidinediones/pharmacology , Base Sequence , Breast Neoplasms/enzymology , Breast Neoplasms/metabolism , Cell Line, Tumor , DNA Primers , Early Growth Response Protein 1/metabolism , Female , Humans , Prostaglandin D2/pharmacology , Reverse Transcriptase Polymerase Chain Reaction
2.
Mol Pharmacol ; 78(5): 865-76, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20713550

ABSTRACT

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.


Subject(s)
Activating Transcription Factor 1/biosynthesis , Calcium-Transporting ATPases/antagonists & inhibitors , Calcium/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Keratinocytes/drug effects , Thapsigargin/pharmacology , Anisomycin/pharmacology , Apoptosis/drug effects , Caspase 3/metabolism , Caspase 7/metabolism , Cations, Divalent , Cell Line , Dual Specificity Phosphatase 1/metabolism , Dual-Specificity Phosphatases/metabolism , Enzyme Activation , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Mitogen-Activated Protein Kinase Phosphatases/metabolism , Signal Transduction , Up-Regulation , p38 Mitogen-Activated Protein Kinases/physiology
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