ABSTRACT
1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D3, has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo. 1alpha,25(OH)2D3 acts through two different mechanisms. In addition to regulating gene transcription via its specific intracellular receptor (vitamin D receptor, VDR), 1alpha,25(OH)2D3 induces rapid, non-transcriptional responses involving activation of transmembrane signal transduction pathways, like growth factors and peptide hormones. The mechanisms that mediate the antiproliferative effects of 1alpha,25(OH)2D3 in breast cancer cells are not fully understood. Particularly, there is no information about the early non-genomic signal transduction effectors modulated by the hormone. The present study shows that 1alpha,25(OH)2D3 rapidly inhibits serum induced activation of ERK-1 and ERK-2 MAP kinases. The tyrosine kinase Src is involved in the pathway leading to activation of ERK 1/2 by serum. Furthermore, 1alpha,25(OH)2D3 increases the tyrosine-phosphorylated state of Src and inhibits its kinase activity, while induces the association of the VDR with Src, either in the presence or absence of serum. In parallel, the hormone rapidly increases the amounts of VDR associated to plasma membranes (PM). Pretreatment with the tyrosine phosphatase inhibitors orthovanadate or bpV (phen) prevented mitogen-activated protein kinase (MAPK) inhibition by 1alpha,25(OH)2D3. These data altogether suggest that 1alpha,25(OH)2D3 inhibits the MAPK cascade by inactivating Src tyrosine kinase through a mechanism mediated by the VDR and tyrosine phosphatases.
Subject(s)
Breast Neoplasms/enzymology , Calcitriol/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Serum , Cell Line, Tumor , Cell Membrane/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Protein Binding/drug effects , Protein Transport/drug effects , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Proto-Oncogene Proteins pp60(c-src)/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Calcitriol/metabolism , Signal Transduction/drug effectsABSTRACT
1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], the hormonally active form of Vitamin D(3), has been shown to be a potent negative growth regulator of breast cancer cells both in vitro and in vivo. 1alpha,25(OH)(2)D(3) acts through two different mechanisms. In addition to regulating gene transcription via its specific intracellular receptor (Vitamin D receptor, VDR), 1alpha,25(OH)(2)D(3) induces, rapid, non-transcriptional responses involving activation of transmembrane signal transduction pathways. The mechanisms that mediate the antiproliferative effects of 1alpha,25(OH)(2)D(3) in breast cancer cells are not fully understood. Particularly, there is no information about the early non-genomic signal transduction effectors modulated by the hormone. The present study shows that 1alpha,25(OH)(2)D(3) rapidly inhibits serum induced activation of ERK-1 and ERK-2 MAP kinases. The non-receptor tyrosine kinase Src is involved in the pathway leading to activation of ERK 1/2 by serum. Furthermore, 1alpha,25(OH)(2)D(3) increases the tyrosine-phosphorylated state of Src as well as it inhibits its kinase activity and induces the association of the VDR with Src. These data suggest that 1alpha,25(OH)(2)D(3) inhibits MAPK by inactivating Src tyrosine kinase through a so far unknown mechanism that seems to be mediated by the VDR.
Subject(s)
Calcitriol/pharmacology , Enzyme Inhibitors/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Proto-Oncogene Proteins pp60(c-src)/metabolism , Receptors, Calcitriol/metabolism , Amino Acid Sequence , Blotting, Western , Cell Line, Tumor , Humans , Precipitin TestsABSTRACT
Little is known about the regulation of cytosolic calcium Ca(2+) levels ([Ca(2+)](i)) in breast cancer cells. We investigated the existence of capacitative calcium entry (CCE) in the tumorigenic cell line MCF-7 and its responsiveness to ATP. MCF-7 cells express purinergic receptors as well as estrogen receptors (ER). Depletion of calcium stores with thapsigargin (TG, 500 nM) or ATP (10 microM) in the absence of extracellular Ca(2+), resulted in a rapid and transient elevation in [Ca(2+)](i). After recovery of basal levels, Ca(2+) readmission (1.5 mM) to the medium increased Ca(2+) influx (twofold over basal), reflecting pre-activation of a CCE pathway. Cells pretreated with TG were unable to respond to ATP, thus indicating that the same Ca(2+) store is involved in their response. Moreover, IP(3)-dependent ATP-induced calcium mobilization and CCE were completely blocked using compound U-73122, an inhibitor of phospholipase C. Compound 2-APB (75 microM) and Gd(3+) (10 microM), antagonists of the CCE pathway, completely prevented ATP-stimulated capacitative Ca(2+) entry. CCE in MCF-7 cells was highly permeable to Mn(2+) and to the Ca(2+) surrogate Sr(2+). Mn(2+) entry sensitivity to Gd(3+) matched that of the Ca(2+) entry pathway. 17Beta-estradiol blocked ATP-induced CCE, but was without effect on TG-induced CCE. Besides, the estrogen blockade of the ATP-induced CCE was completely abolished by preincubation of the cells with an ER monoclonal antibody. ER alpha immunoreactivity could also be detected in a purified plasma membrane fraction of MCF-7 cells. These results represent the first evidence on the operation of a ATP-responsive CCE pathway in MCF-7 cells and also indicate that 17beta-estradiol interferes with this mechanism by acting at the cell surface level.