EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 32, No. 3, 170-177, September 2000 Dexamethasone enhances phospholipase D activity in M-1 cells Won-Jin Kim1, Min-Jung Lee1, are present in M-1 cells and PLD plays a role in the Myung-Ae Park1, Jin-Sup Jung2, corticosteroid-mediated response of cortical collect- David J. Uhlinger3 and Jong-Young Kwak1,4 ing duct cells. Keywords: Phospholipase D, dexamethasone, M-1 cell, 1 Department of Biochemistry, Dong-A University College of oleate Medicine, Pusan 602-103, Korea 2 Department of Physiology, College of Medicine, Pusan National University, Pusan 602-739, Korea 3 Department of Drug Discovery, R. W. Johnson Pharmaceutical Introduction Research Institute, Raritan, New Jersey 08869, USA Mammalian phospholipase D (PLD) activity is very low 4 Corresponding author: Tel, +82-51-240-2928; and becomes up-regulated transiently when cells are Fax, +82-51-241-6940; E-mail, [email protected] stimulated by a variety of hormones, growth factors, and other extracellular signals (Exton, 1997). PLD hydroly- Accepted 14 August 2000 ses phosphatidylcholine (PC), the major component of membrane phospholipid to phosphatidic acid (PA) and γ Abbreviations: ARF, ADP-ribosylation factor; GTP S, guanosine 5'- choline (Bocckino et al., 1987). PA is used as a messen- O-(3-thiotriphosphate); PA, phosphatidic acid; PC, phosphatidylcho- ger for signal transduction and can be further metaboliz- line; PEth, phosphatidylethanol; PIP2, phosphatidylinositol 4,5-bis- ed by PA phosphohydrolase to form diacylglycerol and phosphate; PKC, protein kinase C; PLD, phospholipase D; PMA, by phospholipase A2 to form lysophosphatidic acid, which phorbol 12-myristate 13-acetate have important physiological functions (Cross et al., 1996; Ktistakis et al., 1996; Jones et al., 1999). Two PLDs (PLD1 and PLD2) have been cloned and Abstract characterized (Hammond et al., 1995; Colley et al., 1997b). PLD1 and PLD2 differ with respect to both regulatory Phospholipase D (PLD) is an enzyme involved in and subcellular localization (Colley et al., 1997b). PLD1 signal transduction and widely distributed in mam- localizes to the perinuclear region (endoplasmic reticulum, malian cells. The signal transduction pathways and Golgi apparatus, and late endosomes) but PLD2 locali- role for phospholipid metabolism during hormonal zes primarily to the plasma membrane. PLD1 has a low response in cortical collecting duct remain partly basal activity that is increased by regulators including undefined. It has been reported that dexamethasone protein kinase C (PKC), tyrosine kinase, Ca2+, and low increases transepithelial transport in M-1 cells that molecular weight GTP-binding proteins such as ADP- are derived from the mouse cortical collecting duct. ribosylation factor (ARF) and RhoA (Hammond et al., We investigated the expression and activity of PLD 1997). In contrast, PLD2 exhibits a high basal activity in M-1 cells. Basal PLD activity of M-1 cells cultured that can be further increased by addition of phosphatid- in the presence of dexamethasone (5 µM) was higher ylinositol 4,5-bisphosphate (PIP2) and oleate but not than in the absence of dexamethasone. Dexametha- further activated by PKC, ARF, or Rho in vitro (Lopez et sone and ATP activated PLD in M-1 cells but phorbol al., 1998; Exton, 1999; Kim et al., 1999a). In addition, ester did not stimulate PLD activity. Vasopressin, several groups reported another form of PLD that is bradykinin, dibutyryl cyclic AMP, and ionomycin were activated by detergent including oleate (Chalifour and ineffective in activating PLD of the cells. The PLD2 Kanfer, 1982; Massenburg et al., 1994; Okamura and isotype was detected by immunoprecipitation but Yamashita, 1994). Oleate-dependent PLD has been puri- PLD1 was not detected in M-1 cells. Addition of fied from pig lung membranes but it requires further GTPγS and ADP-ribosylation factor or phosphati- characterization (Okamura and Yamashita, 1994). dylinositiol 4,5-bisphosphate to digitonin-permeabi- M-1 cells have been developed from microdissected lized cells did not augment PLD activity. In intact cortical collecting duct of a mouse transgenic for the cells PLD activity was increased by sodium oleate early region of simian virus 40 (Stoos et al., 1991). M- but there was no significant change between dex- 1 cells, which preserve functional properties typical for amethasone treated- and untreated cells by oleate. cortical collecting duct principal cells in vivo, reabsorb These results suggest that at least two types of PLD sodium and secrete potassium through a corticosteroid Elevated phospholipase D activity by dexamethasone 171 regulated Na+ channel (Chalfant et al., 1996). Recent Measurement of PLD activity studies have demonstrated that dexamethasone stimu- Assay of PLD activity was based upon the unique ability + lates Na transport in M-1 cells (Nakhoul et al., 1998). of this enzyme to stimulate transphosphatidylation in Hormones are able to regulate a variety of ion channels the presence of ethanol to form the corresponding PEth indirectly by cytoplasmic pathways involving second (Kang et al., 1998; Park et al., 1999). M-1 cells were messengers (Breyer and Ando, 1994). labeled with [3H]myristic acid (1 µCi/ml) for 16 h. The It has been reported that both PLD1 and PLD2 are cells were washed twice by phosphate buffered saline present in mouse kidney but only PLD2 can be detected (PBS) and resuspended in assay buffer (20 mM Hepes, in human kidney tissue (Colley et al., 1997a; Meier et pH 7.4, 137 mM NaCl, 2.7 mM KCl, 3 mM MgCl2, 2 mM al., 1999). Activation of PLD and subsequent production CaCl2, 2 mM EGTA, and 1 mg/ml bovine serum albu- of PA are known to be key early event and regulatory in min). After incubation for 20 min at 37oC in the pre- intracellular vesicle trafficking and exocytosis (Jones et sence of 1.6% ethanol, reactions were stopped by al., 1999; Roth, 1999). In epithelial cells, rapid changes addition of 1 ml of CHCl3/CH3OH/concentrated HCl in ion transport involve exocytosis and fusion of intra- (50 : 50 : 0.3, volume/volume), and 0.35 ml of 1 M HCl/ cellular vesicles containing transport protein and ion 5 mM EGTA. Lipids were extracted and separated on channels with the plasma membrane (Denker and Nigam, Silica gel 60 TLC plates in a solvent system consisting of 1998). Vesicle trafficking also plays an important role on ethyl acetate/trimethyl pentane/acetic acid/H2O (13:2: water reabsorption and secretion of acid or base in 3 : 10, volume/volume). The plates were exposed to cortical collecting duct (Brown, 1989). iodine vapor and [3H]PEth was identified by comigration However, it is not known whether these hormonal effects with PEth carrier. Radioactive [3H]PEth was scraped off involve metabolism and signaling machinery of phospho- the plates and quantitated in a liquid scintillation counter lipid by PLD. The purpose of the present investigation (Beckman LS 5801). The amount of radioactivity in PEth was to determine PLD activity and the effect of agents was expressed as percentage of total counts in each modulating ion transport on the activity in M-1 cells. lane. Immunoprecipitation and Western blot of PLD pro- Materials and Methods teins The cells were lysed in buffer containing 20 mM Tris- Materials HCl, pH 7.4, 50 mM NaCl, 1% Triton X-100, 1% deoxy- [9,10-3H(N)]myristic aicd (10-60 Ci/mmol) was purchased cholic acid, 1 µg leupeptin, 1 µg pepstatin A, 1 µg apro- from Dupont-New England Nuclear (Boston, USA). Dexa- tinin, and 1 mM PMSF. The lysates (10 mg) were methasone, phorbol 12-myristate 13-acetate (PMA), incubated with 2 µg anti-PLD antibody bound to protein guanosine 5’-gamma-thio-triphosphate (GTPγS), vaso- A agarose for 1 h (Lee et al., 1997). The beads were pressin, ATP, dibutyryl cyclic AMP (dbcAMP), iono- washed with PBS containing 1% Triton X-100 three mycin, digitonin, protein A-agarose, culture media and times and further washed with the PBS three times. Cell sodium oleate were from Sigma. Silica gel 60 TLC plate lysates (100 µg) and immunoprecipiated beads were was purchased from Merck. Authentic phosphatidyleth- subjected to 7.5% sodium dodecyl sulfate-polyacryl- anol (PEth) was from Avanti polar-Lipids Inc. (Alabaster, amide gel electrophoresis and then transferred to nitro- USA). Recombinant ARF1 was prepared from Escheri- cellulose membranes. The membranes were blocked chia coli expressing human ARF1 and yeast myristoyl- for 1 h at 25oC with blocking buffer (10 mM Tris HCl, CoA: protein N-myristoyltransferase (Lambeth et al., 0.15 M NaCl, 0.1% sodium azide and 5% skim milk) and 1995). Anti-PLD1 antibody was a generous gift from Dr. incubated with a mixture of primary polyclonal anti- Sung Ho Ryu at Pohang University of Science and bodies directed against PLD1 and PLD2 (1 : 1000) in Technology (Lee et al., 1997). Polyclonal rabbit anti-sera blocking buffer overnight at 4°C. Secondary antibody against PLD2 were produced by immunization with a directed against rabbit IgG conjugated to HRP was synthetic peptide encoding the 13 amino acids of mouse diluted 1 : 10,000 in blocking buffer and incubated for PLD2 (DRPFEDFIDRETT). The anti-sera recognized 1 h. The signal was detected by enhanced chemilu- mouse PLD1 and PLD2, respectively. minescence on Hyperfilm-ECL from Amersham Inter- national. Cell culture M-1 cells were grown in culture dishes in DMEM/Ham’s Statistical analysis F-12 (1 : 1 mixture) containing 10% fetal bovine serum, Results were expressed as mean values±standard devi- 100 U/ml penicillin, 100 µg/ml streptomycin. In appropri- ation of the mean (SD). Student’s t-test was used to ate experiments, dexamethasone was added to a final compare the mean PLD activity. A P value < 0.05 was concentration of 5 µM. considered significant. 172 Exp. Mol.