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Tyrosine Phosphatase and Cytochrome P450 Activity Are Critical in Regulating Store-Operated Calcium Channels in Human Fibroblasts

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Tyrosine Phosphatase and Cytochrome P450 Activity Are Critical in Regulating Store-Operated Calcium Channels in Human Fibroblasts EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 38, No. 6, 703-717, December 2006 Tyrosine phosphatase and cytochrome P450 activity are critical in regulating store-operated calcium channels in human fibroblasts Kyung-Mi Lee1,4*, Sang-Wook Son2*, contrast, Ca2+ entry was unaffected by the guanylate György Babnigg3 and Mitchel L. Villereal3,4 cyclase inhibitor LY83583, or the membrane per- meant cyclic GMP analog 8-bromo-cyclic GMP 1Department of Biochemistry and (8-Br-cGMP). Neither nitric oxide donors nor phorbol 2+ Division of Brain Korea 21 Program for Biomedical Science esters affected BK-stimulated Ca entry. SOCE in 2Deaprtment of Dermatology HSWP cells is primarily regulated by tyrosine Korea University College of Medicine phosphorylation and the cytochrome P-450 pathway, Seoul 136-705, Korea but not by cyclic GMP, nitric oxide, or protein kinase 3Department of Neurobiology C. Thus, multiple pathways do operate in a single cell 2+ University of Chicago type leading to the activation of Ca entry and some Chicago, Illinois 60637, USA of these signaling pathways are more prominently 4Corresponding authors: Tel, 82-2-920-6409 (KML); involved in regulating calcium entry in different cell 1-773-702-9334 (MLV); types. Fax, 82-2-928-4853 (KML); 1-773-702-9334 (MLV); E-mail, [email protected] (KML); Keywords: bradykinin; calcium channels; protein-ty- [email protected] (MLV) rosine kinases; protein-tyrosine phosphatases *These authors contributed equally to this work. Accepted 18 November 2006 Introduction One of the early events following stimulation of many Abbreviations: BAPTA-AM, bis-(O-aminophenoxy)ethane-N,N,N',N'- G protein coupled receptors is an increase in the tetraacetic acid, tetra-acetoxymethyl ester; BK, Lys-bradykinin; 2+ 2+ 2+ intracellular Ca concentration ([Ca ]i) (Bird et al., [Ca ]i, intracellular free calcium concentration; EET, epoxyeico- 2004), which initiates a cascade of signal transduc- satrienoic acids; HSWP, human foreskin fibroblast cells; Ins tion events often leading to DNA synthesis and cell (1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphates; Ins(1,4,5)P3, inositol proliferation (Parekh and Putney, 2005). Typically, 2+ 1,4,5-trisphosphates; PI, phosphatidylinositol; PIP2, phosphati- the Ca response evoked by agonists is a biphasic dylinositol 4,5-bisphosphates one that includes a peak and a sustained plateau. The peak is due to the release of intracellular Ca2+ from Ins(1,4,5)P3 -sensitive internal stores and the Abstract sustained plateau is due to Ca2+ entry from the extracellular space. The relationship between the 2+ 2+ Diverse signaling pathways have been proposed to release of internal Ca stores and activation of Ca regulate store-operated calcium entry (SOCE) in a entry has been examined in depth and the prevailing wide variety of cell types. However, it still needs to hypothesis is that the two events are functionally be determined if all of these known pathways operate coupled. Jim Putney first defined “capacitative cal- cium entry”, also called “store-operated calcium in a single cell type. In this study, we examined 2+ involvement of various signaling molecules in SOCE entry (SOCE)”, as Ca entry initiated by a signal generated when Ca2+ stores are emptied (Putney using human fibroblast cells (HSWP). Bradykinin 2+ and McKay, 1999). But what is the signal that is (BK)-stimulated Ca entry, previously shown to be 2+ produced by a decrease in the Ca loading state of via SOCE, is enhanced by the addition of vanadate, the internal store that, in turn, communicates with an inhibitor of tyrosine phosphatases. Furthermore, Ca2+ channels in the plasma membrane? A direct SOCE is regulated by cytochrome P-450, as interaction between store-operated channels and IP3 demonstrated by the fact that the products of receptors, the insertion of sub-membrane vesicles cytochrome P-450 activity (14,15 EET) stimulated containing store-operated channels into the plasma SOCE while econazole, an inhibitor of cytochrome membrane, or a secretion-like coupling may be 2+ P450, suppressed BK-stimulated Ca entry. In involved in regulation of SOCE (Parekh and Putney, 704 Exp. Mol. Med. Vol. 38(6), 703-718, 2006 2005b). One can not ignore the wealth of evidence et al., 1993) and examined the role of various arguing for the involvement of various signal signaling pathways. Our results indicate that Ca2+ transduction pathways in the regulation of SOCE. entry into HSWP cells is dynamically regulated by Irvine proposed a model that suggested the tyrosine kinases and phosphatases and is inde- involvement of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 recep- pendent of cyclic GMP, nitric oxide, and diacy- tors in activating Ca2+ entry following depletion of lglycerol acting via a classic PKC pathway. Ca2+ stores (1991). In addition to inositol phos- phates, several other signaling molecules have been 2+ reported to play a role in Ca entry following internal Materials and Methods Ca2+ store depletion. Cyclic GMP has been im- plicated in the regulation of SOCE in pancreatic Cell culture acinar cells (Bahnson et al., 1993), in vascular endothelial cells (Kwan et al., 2000) and platelets Human foreskin fibroblast cells (HSWP) were ob- (Rosado et al., 2001). A role for cytochrome P450 tained from J. Regan, Oak Ridge National Labo- enzymes has been suggested for the regulation of ratory. Cells between the 15th and 25th passage SOCE in thymocytes (Alvarez et al., 1991), platelets were plated in 100 mm dishes in Eagle’s minimal (Alonso et al., 1991), and human neutrophils (Mon- essential medium (EMEM) containing 10% fetal tero et al., 1991). In addition, small molecular weight bovine serum. DDT1MF-2 cells were provided by G proteins have been shown to regulate SOCE in Donald Gill, University of Maryland School of Medicine, and were grown in DMEM + 10% FBS. mouse lacrimal acinar cells (Bird and Putney, 1993) o and HL-60 granulocytes (Jaconi et al., 1993). In Cells were grown at 37 C in a humidified atmo- Jurkat T cells, a small molecule named Ca2+-influx sphere containing 5% CO2 and 95% air. For single factor (CIF), generated during stimulation of cells, cell image analysis, cells were subcultured on 25 was reported to activate Ca2+ influx in several cell mm round glass coverslips. lines (Randriamampita and Tsien, 1993). The nitric oxide pathway (Favre et al., 1998) and epoxyeco- Immunoblotting of whole cell lysates with satrienoic acids (EETs) (Graier et al., 1995; Rziga- monoclonal anti-phosphotyrosine antibodies linski et al., 1999) also have been suggested for regulation of SOCE. HSWP cells were serum deprived in EMEM for 6 h o In addition to the pathways described above, we at 37 C. Following stimulation, cells were washed have provided evidence for the role of tyrosine with isotonic ice-cold Tris buffered saline (TBS, 10 kinases in the regulation of SOCE in human fib- mM Tris, 75 mM NaCl, 75 mM KCl, pH 7.4) and roblast cells (Lee et al., 1993). A number of sub- extracted in isoelectrofocusing stop solution (IEF, 8M sequent reports have supported our findings that urea, 0.5% SDS, 50 mM 2-mercaptoethanol). A tyrosine kinases are involved in regulating SOCE in fraction (10 g of protein) of each sample was many cell types (Marhaba et al., 1996; Davis and solubilized by boiling in an equal volume of 2 × Sharma, 1997; Krutetskaia et al., 1997; Taketo et al., Laemmli buffer (62 mM Tris, 1% SDS, 0.001% 1997). In addition, studies of platelets indicate that pyronin Y, 10% glycerol, 5% 2-mercaptoethanol) and depletion of Ca2+ from intracellular stores leads to separated by SDS-PAGE on 7.5% polyacrylamide the tyrosine phosphorylation of a 130 kD protein, minigel. Proteins were transferred to nitrocellulose which led to speculation that this protein is involved membranes and nonspecific binding sites were in the regulation of Ca2+ entry (Vostal et al., 1991). blocked by incubating the membranes in blocking Since the studies defining the role of these buffer (150 mM NaCl, 10 mM sodium phosphates, numerous signaling pathways in regulating SOCE pH 7.4, 2 mM EDTA, 0.2% NP-40) containing 5 involve a wide range of cell types, the question mg/ml of BSA for 2 h at room temperature. Mono- arises whether the evidence for multiple signaling clonal anti-phosphotyrosine antibodies at a concen- pathways simply reflects variations in the method of tration of 0.5 g /ml were added to the blot and o regulation between different cell and tissue types, or incubated overnight at 4 C. The blot was washed 5 whether these multiple signaling pathways are all times with blocking buffer. Then, anti-mouse IgG involved in regulating SOCE in a single cell type. F(ab')2 conjugated to horseradish peroxidase (2 g That is, do all of these signaling pathways converge /ml in blocking buffer) was added to the blot and on SOCs in most cell types or are there differences incubated for 1 h at room temperature. The blot was between cell types in how they regulate SOCE? To washed 5 times and subjected to enhanced chemilu- answer this question, we have stimulated human minescence (ECL). fibroblast (HSWP) cells with bradykinin or thapsi- gargin, both agents capable of inducing SOCE (Lee Regulation of store-operated calcium entry pathway 705 2+ Measurements of [Ca ]i by image analysis Results Image analysis was carried out as previously Vanadate enhanced tyrosine phosphorylation and described (Byron and Villereal, 1989; Lee et al., 2+ 1993). All image analysis was conducted at room Ca entry stimulated by BK temperature. Coverslips were washed twice with We showed previously that the amount of SOCE control medium (135 mM NaCl, 5 mM KCl, 1 mM stimulated by either BK or thapsigargin is decreased CaCl2, 1 mM MgCl2, 11 mM glucose, 11 mM Hepes, by tyrosine kinase inhibitors (Lee et al., 1993).
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