Lipid Phosphate Phosphatase-1 and Ca2+ Control Lysophosphatidate Signaling through EDG-2 Receptors
2000; Elsevier BV; Volume: 275; Issue: 36 Linguagem: Inglês
10.1074/jbc.m003211200
ISSN1083-351X
AutoresJames Xu, Lana M. Love, Indrapal N. Singh, Qiuxia Zhang, Jay Dewald, De–An Wang, David J. Fischer, Gábor Tigyi, Luc G. Berthiaume, David W. Waggoner, David N. Brindley,
Tópico(s)Lysosomal Storage Disorders Research
ResumoThe serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 °C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca2+, activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K d andB max for LPA binding at 4 °C, increasing Ca2+ from 0 to 50 μm increased theK d from 40 to 900 nm. Decreasing extracellular Ca2+ from 1.8 mm to 10 μm increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca2+ dependence of the apparent K d values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca2+levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation. The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 °C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca2+, activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K d andB max for LPA binding at 4 °C, increasing Ca2+ from 0 to 50 μm increased theK d from 40 to 900 nm. Decreasing extracellular Ca2+ from 1.8 mm to 10 μm increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca2+ dependence of the apparent K d values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca2+levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation. Retraction: Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors.Journal of Biological ChemistryVol. 278Issue 39PreviewVol. 275 (2000) 27520–27530 Full-Text PDF Open Access lysophosphatidate bovine serum albumin Dulbecco's minimum essential medium endothelial cell differentiation genes epidermal growth factor extracellular-signal-regulated kinase green fluorescent protein HEPES-buffered saline lipid phosphate phosphatase (also known as phosphatidate phosphohydrolase 2A) mouse mitogen-activated protein kinase phosphatidate phosphate-buffered saline platelet-derived growth factor phospholipase D reverse transcription polymerase chain reaction LPA1 is one of the most potent growth factors in blood serum. Thrombin-activated platelets release LPA that in turn stimulates cell division and regeneration in different tissues (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 2Liliom K. Guan Z. Tseng J.T. Desiderio D.M. Tigyi G. Watsky M.A. Am. J. Physiol. 1998; 274: 1065-1074Crossref PubMed Google Scholar). LPA is implicated in promoting cell regeneration of injured corneal tissue (2Liliom K. Guan Z. Tseng J.T. Desiderio D.M. Tigyi G. Watsky M.A. Am. J. Physiol. 1998; 274: 1065-1074Crossref PubMed Google Scholar). Stimulation of α2-adrenergic receptors in adipocytes releases LPA, and this represents an autocrine/paracrine mechanism for regulating preadipocyte growth, which may have implications for the pathogenesis of obesity (3Valet P. Pagès C. Jeanneton O. Daviaud D. Barbe P. Record M. Saulnier-Blanche J.B. Lafontan M. J. Clin. Invest. 1998; 101: 1431-1438Crossref PubMed Scopus (127) Google Scholar). LPA production, through secretory phospholipase A2, may also represent a pro-inflammatory pathway (4Fourcade O. Simon M.F. Viodé C. Rugani N. Leballe F. Ragab A. Fournié B. Sarda L. Chap H. Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (500) Google Scholar). LPA is released by ovarian epithelial cancer cells, and it is found in high concentrations in ascites fluid (5Shen Z. Wu M. Wiper D.W. Morton R.E. Kennedy A.W. Belinson J. Markman M. Casey G. Xu Y. Clin. Chem. 1997; 43: S577Google Scholar, 6Xu Y. Shen Z. Wiper D.W. Wu M. Morton R.E. Elson P. Kennedy A.W. Belinson J. Markman M. Casey G. J. Am. Med. Assoc. 1998; 280: 719-723Crossref PubMed Scopus (576) Google Scholar). The LPA serves as an important growth factor for ovarian tumors and protects against apoptosis induced by cis-diamminedichloroplatinum, an important chemotherapeutic agent (7Frankel A. Mills G.B. Clin. Cancer Res. 1996; 2: 1307-1313PubMed Google Scholar). These latter observations suggest that the putative paracrine effects of LPA contribute to the poor prognosis associated with ovarian cancer. LPA concentrations are also raised in the blood of patients with multiple myeloma (8Sasagawa T. Okita M. Murakami J. Kato T. Watanabe A. Lipids. 1999; 34: 17-21Crossref PubMed Scopus (88) Google Scholar). Therefore, the role of LPA as a growth factor is of considerable biological and clinical importance. The effects of LPA in cell activation are mediated by G protein-coupled cell-surface receptors (9Guo Z. Liliom K. Fischer D.J. Bathurst I.C. Tomei L.D. Kiefer M.C. Tigyi G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14367-14372Crossref PubMed Scopus (178) Google Scholar, 10Hecht J.H. Weinerm J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (667) Google Scholar, 11Fukushima N. Kimura Y. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6151-6156Crossref PubMed Scopus (252) Google Scholar, 12An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 13An S. Blue T. Zheng Y. Goetzl E.J. Mol. Pharmacol. 1998; 54: 881-888Crossref PubMed Scopus (151) Google Scholar, 14Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (491) Google Scholar, 15Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murofushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). These receptors, encoded by theEDG-2, EDG-4, and EDG-7 genes, are related to the endothelial differentiation gene-1 and are specifically activated by LPA. Exogenous LPA stimulates signaling cascades that decrease cAMP concentrations and increase the activities of tyrosine kinases, intracellular Ca2+ mobilization, activation of phospholipase D (PLD), the Ras-Raf-ERK pathway and the stimulation of cell division (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 9Guo Z. Liliom K. Fischer D.J. Bathurst I.C. Tomei L.D. Kiefer M.C. Tigyi G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14367-14372Crossref PubMed Scopus (178) Google Scholar, 11Fukushima N. Kimura Y. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6151-6156Crossref PubMed Scopus (252) Google Scholar, 12An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 13An S. Blue T. Zheng Y. Goetzl E.J. Mol. Pharmacol. 1998; 54: 881-888Crossref PubMed Scopus (151) Google Scholar, 14Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (491) Google Scholar, 15Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murofushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 16van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 17Jalink K. Hengeveld T. Mulder S. Postma F.R. Simon M.F. Chap H. van der Marel G.A. van Boom J.H. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1995; 307: 609-616Crossref PubMed Scopus (112) Google Scholar, 18Shahrestanifar M. Fan X. Manning D.R. J. Biol. Chem. 1999; 274: 3828-3833Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). There are only a very few cell types that do not respond to LPA (17Jalink K. Hengeveld T. Mulder S. Postma F.R. Simon M.F. Chap H. van der Marel G.A. van Boom J.H. van Blitterswijk W.J. Moolenaar W.H. Biochem. J. 1995; 307: 609-616Crossref PubMed Scopus (112) Google Scholar). There is, therefore, considerable interest in understanding the mechanisms by which LPA metabolism is regulated and how its growth stimulating effects can be controlled. So far, receptor desensitization is the only mechanism implicated in the regulation of LPA receptors (19Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1990; 265: 12232-12239Abstract Full Text PDF PubMed Google Scholar, 20Fischer D.J. Liliom K. Guo Z. Nusser N. Viráge T. Murakami- Murofushi K. Kobayashi S. Erickson J.R. Sun G. Miller D.D. Tigyi G. Mol. Pharmacol. 1998; 54: 979-988Crossref PubMed Scopus (109) Google Scholar). Here we describe a new mechanism that involves LPP-1 (phosphatidate phosphohydrolase-2A) in regulating LPA signaling through EDG2 receptors. LPP-1 is a member of a family of LPPs that belong to a phosphatase superfamily (for reviews, see Refs. 21Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar and 22Waggoner D.W. Xu J. Singh I. Jasinska R. Zhang Q.X. Brindley D.N. Biochim. Biophys. Acta. 1999; 1439: 299-316Crossref PubMed Scopus (115) Google Scholar). The LPPs degrade a variety of lipid phosphates with similar efficiencies when these are presented in Triton X-100 micelles. We showed that LPP-1 is present in plasma membranes of Rat2 fibroblasts with its active site exposed on the outer surface (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar, 24Zhang Q.X. Pilquil C.S. Dewald J. Berthiaume L.G. Brindley D.N. Biochem. J. 2000; 345: 181-184Crossref PubMed Scopus (99) Google Scholar). These findings are compatible with the ability of LPP-1 to dephosphorylate exogenous lipid phosphate esters (21Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 22Waggoner D.W. Xu J. Singh I. Jasinska R. Zhang Q.X. Brindley D.N. Biochim. Biophys. Acta. 1999; 1439: 299-316Crossref PubMed Scopus (115) Google Scholar, 23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar, 24Zhang Q.X. Pilquil C.S. Dewald J. Berthiaume L.G. Brindley D.N. Biochem. J. 2000; 345: 181-184Crossref PubMed Scopus (99) Google Scholar). In intact cells, mouse (m) LPP-1 dephosphorylated albumin-bound LPA 10 times faster than phosphatidate. Therefore, LPP-1 expressed at the cell surface exhibits greater specificity for LPA than is determined in the Triton micelle assay. We proposed that LPP-1 functions partly to degrade extracellular LPA. Preliminary results showed that increasing LPP-1 activity could decrease the activation of ERK1 and ERK2 and DNA synthesis by LPA (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar). However, in the latter work the specificity of the LPA signaling effects, the receptor that was involved, and the mechanisms through which LPP-1 affected cell activation by LPA were not established. The present studies we designed to answer these outstanding problems by addressing two hypotheses. First, that LPP-1 controls the net interaction of LPA with cells thus regulating the activation of EDG receptors under conditions where the bulk concentration of LPA remains relatively constant. The alternative hypothesis is that LPP-1 simply depletes the bulk concentration of LPA in the external medium and thus the concentration of LPA that can interact directly with EDG receptors. The present work employed Rat2 fibroblasts that were transduced with cDNA for mLPP-1 to increase LPP-1 activity or treated with an antisense oligodeoxynucleotide to decrease LPP-1 expression. Changes in LPA binding and signaling were determined under conditions where less than 10% of the exogenous LPA was lost from the incubation medium. LPP-1 indeed controlled the net specific association of LPA with the fibroblasts and thereby regulated signaling through EDG-2 receptors with minimum changes in the bulk concentration of LPA. These effects are particularly significant in physiological terms and demonstrate a novel level of regulation for EDG-2 and perhaps other EDG receptors. The effects of LPP-1 on LPA signaling were specific, since no changes in cell activation were observed with other agonists (EGF, PDGF, and forskolin) that do not signal though EDG-2 receptors. While studying LPA binding to fibroblasts, it became obvious that exogenous Ca2+ decreased both LPA dephosphorylation and the net specific binding of LPA to the fibroblasts. The latter event considerably decreased the ability of LPA to activate ERK1 and ERK2. These observations provide an explanation for the long-standing problem of why the reported affinities of LPA for its EDG receptors, in the absence of Ca2+, are in the nanomolar range (12An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 14Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (491) Google Scholar, 15Bandoh K. Aoki J. Hosono H. Kobayashi S. Kobayashi T. Murakami-Murofushi K. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1999; 274: 27776-27785Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar,25An S. Dickens M.A. Blue T. Hallmark O.G. Goetzl E.J. Biochem. Biophys. Res. Commun. 1997; 231: 619-622Crossref PubMed Scopus (212) Google Scholar), whereas micromolar concentrations of LPA are normally required to activate cell signaling in cultured cells (2Liliom K. Guan Z. Tseng J.T. Desiderio D.M. Tigyi G. Watsky M.A. Am. J. Physiol. 1998; 274: 1065-1074Crossref PubMed Google Scholar, 16van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 20Fischer D.J. Liliom K. Guo Z. Nusser N. Viráge T. Murakami- Murofushi K. Kobayashi S. Erickson J.R. Sun G. Miller D.D. Tigyi G. Mol. Pharmacol. 1998; 54: 979-988Crossref PubMed Scopus (109) Google Scholar, 23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar, 26Gómez-Muñoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Abstract Full Text PDF PubMed Google Scholar). Our work therefore establishes novel mechanisms whereby both LPP-1 and extracellular Ca2+ regulate the net interaction of LPA with EDG-2 receptors and that this in turn controls cell activation and division. Dulbecco's minimum essential medium (DMEM), penicillin, streptomycin, and fetal bovine serum were purchased from Life Technologies, Inc. Forskolin and 3-isobutyl-1-methylxanthine were from Calbiochem. BSA, LPA, Fura-2/AM, aprotinin, and leupeptin were purchased from Sigma. EGF was from Biomedical Technology and PDGF was from Intergen. Ready-to-go T-primed first strand synthesis kits, donkey anti-rabbit IgG conjugated to horseradish peroxidase, enhanced chemiluminescence (ECL) Western blotting detection reagents, and [3H]myristate were purchased from Amersham Pharmacia Biotech. [3H]Thymidine was from ICN Biomedicals. cAMP125I radioimmunoassay kits were from NEN Life Science Products. BCA Protein Assay kits were from Pierce. RNAlater TM was from Ambion Inc., andTaq DNA polymerase was from Promega Life Sciences. [32P]LPA was synthesized as described previously (27Waggoner D.W. Gómez-Muñoz A. Dewald J. Brindley D.N. J. Biol. Chem. 1996; 271: 16506-16509Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and polyclonal antibodies against recombinant GFP were raised in rabbits (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar). Sense and antisense oligodeoxynucleotides for LPP-1 were synthesized by the DNA Core Facility Laboratory at the University of Alberta with phosphorothioate modification (28Tabata M.J. Kim K. Liu J.G. Yamashita K. Matsumura T. Kato J. Iwamoto M. Wakisaka S. Matsumoto K. Nakamura T. Kumegawa M. Kurisu K. Development ( Camb. ). 1996; 122: 1243-1251PubMed Google Scholar, 29Yazaki T. Ahmad S. Chahlavi A. Zylber-Katz E. Dean N.M. Rabkin S.D. Martuza R.L. Glazer R.I. Mol. Pharmacol. 1996; 50: 236-242PubMed Google Scholar). The sequence of the antisense oligodeoxynucleotides was 5′-AGGGCCACGTACGGCAGCCGCGTCTTGTCG-3′ (35-6), which is complementary to the control sense oligodeoxynucleotides containing 5′-CGACAAGACGCGGCTGCCGTACGTGGCCCT-3′. The sense sequence corresponds to nucleotides 6–35 of the mLPP-1 DNA coding sequence. There is only one difference between the mLPP-1 and rat LPP-1 coding sequences in this region, C at deoxynucleotide 32 for mLPP-1 and T for rLPP-1. The predicted coding sequences for LPP-2 and LPP-3 in this region differ markedly from LPP-1 (21Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 22Waggoner D.W. Xu J. Singh I. Jasinska R. Zhang Q.X. Brindley D.N. Biochim. Biophys. Acta. 1999; 1439: 299-316Crossref PubMed Scopus (115) Google Scholar). Rat2 fibroblasts, that were thymidine kinase-positive, were obtained as described previously (26Gómez-Muñoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Abstract Full Text PDF PubMed Google Scholar, 30Gómez-Muñoz A. Duffy P.A. Martin A. O'Brien L. Byun H.S. Bittman R. Brindley D.N. Mol. Pharmacol. 1995; 47: 883-889Google Scholar). These fibroblasts were also transduced stably with cDNA for mLPP-1 or for mLPP-1-GFP by using a retrovirus vector (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar, 24Zhang Q.X. Pilquil C.S. Dewald J. Berthiaume L.G. Brindley D.N. Biochem. J. 2000; 345: 181-184Crossref PubMed Scopus (99) Google Scholar). Puromycin-resistant clones were pooled and propagated as average cell populations. Cells expressing mLPP-1-GFP were used in immunoprecipitation studies and to provide additional evidence for the effects of increasing LPA dephosphorylation. Fibroblasts were seeded at about 2 × 103 cells/cm2 of culture dish and cultured for 2–3 days in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg of streptomycin/ml in a humidified atmosphere of 5% CO2, 95% air at 37 °C until confluence. For experiments with antisense oligodeoxynucleotides, subconfluent Rat2 fibroblasts expressing GFP alone or mLPP-1-GFP were incubated with 10 μm sense or antisense oligodeoxynucleotides for 72 h. Oligodeoxynucleotides were replenished after 48 h and included in the subsequent experimental media. LPP activity in cell lysates or immunoprecipitates was determined by measuring32Pi production from 32P-labeled LPA dispersed in 8 mm Triton X-100 (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar). For the determination of LPP activity against exogenous LPA, fibroblasts were incubated in DMEM containing 0.1% fatty acid-free BSA for at least 18 h, then rinsed twice with PBS and incubated in the same medium for 2 h. Cells were then incubated for 20 min with 0.8 ml of the same medium containing 32P-labeled LPA (approximately 3 × 105 cpm/35-mm dish), dispersed by sonication in 0.1% BSA. 32Pi production was determined as described previously (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar). For studying the effects of Ca2+ on LPA binding, Rat2 cells transduced with cDNA for GFP alone or mLPP-1-GFP were washed in the absence of Ca2+ with 20 mm HEPES, pH 7.4, 125 mm NaCl, 10 mm glucose, plus 0.1% fatty acid-free BSA (HBS) and incubated for 20 min at 4 or 37 °C in the presence of different Ca2+ concentrations in HBS containing various concentration of [32P]LPA. A portion of the binding buffer was withdrawn to measure the degradation of LPA (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar). The cells were then washed twice with ice-cold Ca2+-free HBS containing 0.1% BSA and twice with PBS without BSA. Cells were recovered by scraping, sonicated, and protein concentrations were determined using the BCA kit. LPA bound to the cells was extracted three times with H2O-saturated butan-1-ol, and32P-labeled LPA in the butan-1-ol phase was measured by liquid scintillation counting. The identity of the bound [32P]LPA was confirmed by chromatography on silica gel plates developed in chloroform/methanol/acetic acid (8:2:1, v/v/v). Total and nonspecific associations of LPA were evaluated in incubations in the absence or presence of 100 μm nonradioactive LPA, respectively. The difference between total and nonspecific association was defined as specific LPA association. In other experiments, LPA binding to Rat2 fibroblasts was measured in cells incubated at 37 °C with DMEM that contained 1.8 mm Ca2+. Cells were washed three times with ice-cold PBS and lysed with buffer containing 1% Nonidet P-40, 10% glycerol, 50 mm HEPES, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 2 mmNa3VO4, 10 mmNa4P2O7, 100 mm NaF, 5 μg/ml aprotinin, 1 mmphenylmethylsulfonylfluoride, and 1 μg/ml leupeptin. mLPP-1-GFP was immunoprecipitated from cell lysates (200–400 μg of protein) by adding 2.5 mg of anti-GFP antibodies and incubating for 6 h at 4 °C with constant rocking. Fifty μl of a 50% slurry of protein A-Sepharose in PBS was added, and the tubes were incubated overnight at 4 °C. Immunoprecipitates were then washed three times with buffer containing 50 mm HEPES, 150 mmNaCl, 100 mm NaF, 1 mmphenylmethylsulfonylfluoride, 2 mmNa3VO4, 1% Triton X-100, and 0.1% SDS. Protein obtained from equivalent amounts of cell lysates was resolved by SDS-polyacrylamide gel electrophoresis (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (208369) Google Scholar) and transferred to nitrocellulose membranes. The membranes were blocked overnight with Tris-buffered saline, pH 7.5, containing 1% BSA and 1% (w/v) dry skimmed milk at 4 °C and then incubated with anti-GFP antibody at room temperature. Anti-GFP binding was detected using donkey anti-rabbit IgG conjugated to horseradish peroxidase developed with ECL reagent and by exposure to x-ray films for various times. For the measurement of intracellular cAMP concentrations, subconfluent fibroblasts were incubated in DMEM containing 0.1% fatty acid-free BSA for at least 18 h. Cells were then incubated for 30 min with the same medium containing 0.5 mm 3-isobutyl-1-methylxanthine. Half of the dishes were incubated with the same medium containing 1 mm forskolin for 15 min. Cells were then treated for 10 min with 10 μmLPA, washed twice with ice-cold PBS, and lysed in 10% trichloroacetic acid. Lysates were boiled for 5 min, centrifuged at 12,000 ×g for 10 min, and cAMP concentrations were determined using an radioimmunoassay kit (26Gómez-Muñoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Abstract Full Text PDF PubMed Google Scholar). PLD activity was measured in fibroblasts prelabeled in with [3H]myristate by determining [3H]phosphatidylethanol formation (26Gómez-Muñoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Abstract Full Text PDF PubMed Google Scholar). Changes in intracellular Ca2+ concentrations were measured essentially as described elsewhere (32Picotto G. Vazquez G. Boland R. Biochem. J. 1999; 339: 71-77Crossref PubMed Scopus (51) Google Scholar) following a preincubation with 2 μm Fura-2/AM for 45 min at room temperature in the dark. LPA (10 μm) and PDGF (50 ng/ml) were added directly into the cuvettes at the times indicated. Total RNA was isolated using the RNAlater TM kit. cDNA was obtained by reverse transcription using the Ready-to-go T-primed first strand synthesis kit. The oligonucleotide primers, based on the rat homologues of the different receptor sequences, were as follows: EDG-2 forward primer, 5′-65AGATCTGACCAGCCGACTCAC-3′; reverse primer, 5′-GTTGGCCATCAAGTAATAAATA422-3′; EDG-4 forward primer, 5′-634CTGCTCAGCCGCTCCTATTTG-3′; reverse primer, 5′-AGGAGCACCCACAAGTCATCAG1185-3′; EDG-7 forward primer, 5′-91AGCAACACTGATACTGTCGATG-3′; reverse primer, 5′-GCATCCTCATGATTGACATGTG446-3′. mRNA for β-actin was used as a control as described previously (33Rizza C. Leitinger N. Giese M. Tyner T. Yue J. Wang D. Fischer D.J. Tigyi G. Berliner J.A. Lab. Invest. 1999; 79: 1227-1235PubMed Google Scholar). After a 3-min predenaturation at 94 °C,Taq DNA polymerase was added, and 30 amplification cycles, each consisting of a 50-s denaturation at 94 °C, annealing for 1 min at 58 °C, and elongation for 2 min at 72 °C, were performed. The PCR products were analyzed in 1% agarose gels. ERK1 and ERK2 activities (p42 and p44 MAP kinases) and DNA synthesis were measured as described previously (26Gómez-Muñoz A. Martin A. O'Brien L. Brindley D.N. J. Biol. Chem. 1994; 269: 8937-8943Abstract Full Text PDF PubMed Google Scholar,30Gómez-Muñoz A. Duffy P.A. Martin A. O'Brien L. Byun H.S. Bittman R. Brindley D.N. Mol. Pharmacol. 1995; 47: 883-889Google Scholar). For cell proliferation assays, cells were seeded at the starting density of 1.4–1.7 × 103 cells/cm2 of the culture dish in DMEM containing 10% fetal bovine serum for 24 h. Cells were then incubated with DMEM containing 0.1% BSA for 24 h. Agonists, including 5 μm LPA, 10 ng/ml EGF, or 1 ng/ml PDGF were then added. LPA was replenished by changing the medium containing LPA every day. Media containing EGF and PDGF were changed every other day. Fibroblasts were detached from the dishes by trypsinization at the days indicated, and cell numbers were determined with a hemacytometer. To investigate the role of LPP-1 in regulating cell signaling by LPA, we first used retrovirus delivery to transduce Rat2 fibroblasts with cDNA for mLPP-1 or mLPP-1 linked at the C terminus to green fluorescent protein (mLPP-1-GFP). LPP activity was measured in the cell lysates using the Triton micelle assay. Cells expressing mLPP-1 (23Jasinska R. Zhang Q.X. Pilquil C.S. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (131) Google Scholar) and mLPP-1-GFP (Fig.1 A) showed 3–5-fold increases in total LPP activity compared with cells transduced with empty vector or with cDNA for GFP alone. To provide additional evidence that LPP-1 regulates the cellular effects of exogenous of LPA, we also decreased LPP-1 expression by treating fibroblasts that stably expressed GFP or mLPP-1-GFP with the antisense oligodeoxynucleotide for LPP-1. This decreased LPP activity in cell lysates from both cell lines by as much as 90% (Fig. 1 A). As a control, we showed that treating fibroblasts with sense oligodeoxynucleotide for LPP-1 did not decrease LPP activity. We do not have an antibody against the endogenous rat LPP-1 and, therefore, could not determine its level of expression directly. Instead, we precipitated mLPP-1-GFP from the cell lysates with anti-GFP. The precipitated LPP activity was decreased by about 85% after treating transduced cells with antisense, but not with sense oligodeoxynucleotide (Fig. 1 B). To determine whether the decrease in mLPP-1-GFP activity was caused by specific suppression of its synthesis, we performed Western blotting on immunoprecipitates (Fig. 1 C). mLPP-1-GFP fusion proteins from untreated cells and those treated with sense oligodeoxynucleotides appeared in the region of about 62–65 kDa, since GFP adds about 27 kDa to the mass of mLPP-1, which is approximately 35 kDa. The two bands result from different levels of LPP-1 glycosylation (22Waggone
Referência(s)