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• W2102073119 abstract "Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression. Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression. In the mammalian system, LPA 1The abbreviations used are: LPA, lysophosphatidic acid; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; MEK, MAPK/Erk kinase; PNGase F, peptide N-glycosidase F; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; EGF, epidermal growth factor; PTH, parathyroid hormone; R, receptor; RTK, receptor tyrosine kinase; PVDF, polyvinylidene difluoride; COX-2, cyclooxygenase-2; iNOS, inducible nitric-oxide synthase; PTX, pertussis toxin; PAF, platelet-activating factor; IL-1β, interleukin-1β; RT, room temperature; RT-PCR, reverse transcription-PCR; BSA, bovine serum albumin; PBS, phosphate-buffered saline; pCMVEC, porcine cerebral microvascular endothelial cell; PM, plasma membrane; WN, whole nuclei; NE, nuclear envelope; BSAfaf, bovine serum albumin fatty acid-free; oLPA, oleoyl-lysophosphatidic acid; sLPA, stearoyl-lysophosphatidic acid; pLPA, palmitoyl-lysophosphatidic acid.1The abbreviations used are: LPA, lysophosphatidic acid; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; MEK, MAPK/Erk kinase; PNGase F, peptide N-glycosidase F; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; EGF, epidermal growth factor; PTH, parathyroid hormone; R, receptor; RTK, receptor tyrosine kinase; PVDF, polyvinylidene difluoride; COX-2, cyclooxygenase-2; iNOS, inducible nitric-oxide synthase; PTX, pertussis toxin; PAF, platelet-activating factor; IL-1β, interleukin-1β; RT, room temperature; RT-PCR, reverse transcription-PCR; BSA, bovine serum albumin; PBS, phosphate-buffered saline; pCMVEC, porcine cerebral microvascular endothelial cell; PM, plasma membrane; WN, whole nuclei; NE, nuclear envelope; BSAfaf, bovine serum albumin fatty acid-free; oLPA, oleoyl-lysophosphatidic acid; sLPA, stearoyl-lysophosphatidic acid; pLPA, palmitoyl-lysophosphatidic acid. signaling cascades regulate important cellular processes, including gene expression, cell proliferation and growth, cell survival and death, and cell motility and secretion (1Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (490) Google Scholar, 2Moolenaar W.H. Exp. Cell Res. 1999; 253: 230-238Crossref PubMed Scopus (371) Google Scholar, 3Tigyi G. Prostaglandins. 2001; 64: 47-62Crossref PubMed Scopus (91) Google Scholar). These plethora of activities are characteristic features of inflammation that occur in various physiological as well as pathological states (e.g. ontogenic change, wound healing, cancer, etc.) (1Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (490) Google Scholar, 2Moolenaar W.H. Exp. Cell Res. 1999; 253: 230-238Crossref PubMed Scopus (371) Google Scholar, 3Tigyi G. Prostaglandins. 2001; 64: 47-62Crossref PubMed Scopus (91) Google Scholar). In humans, physiological responses induced by LPA arise from specific interactions with at least three genetically identified receptors designated LPA1, LPA2, and LPA3 (formerly referred to as EDG2, EDG4, and EDG7 receptors, respectively), which belong to the heptahelical transmembrane-spanning G protein-coupled receptor (GPCR) superfamily (4Chun J. Goetzl E.J. Hla T. Igarashi Y. Lynch K.R. Moolenaar W. Pyne S. Tigyi G. Pharmacol. Rev. 2002; 54: 265-269Crossref PubMed Scopus (443) Google Scholar). These receptors show a broad, virtually distinct distribution and may couple in a cell-dependent manner to numerous heterotrimeric G proteins. In this context, LPA1 and LPA2 receptors have been shown to interact with Gi/o, Gq/11/14, and G12/13 proteins, whereas the LPA3 receptor combines with Gi/o and Gq/11/14 proteins (5Fukushima N. Ishii I. Contos J.J. Weiner J.A. Chun J. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 507-534Crossref PubMed Scopus (321) Google Scholar). Although many responses induced by extracellular LPA can result from its interaction with plasma membrane GPCRs, there is circumstantial evidence for an intracrine mode of action of LPA. For instance, putative biogenesis (e.g. secretory and cytosolic calcium-dependent and -independent phospholipase A2, phospholipase D, and monoacylglycerol kinase) and degradation (e.g. phosphohydrolase and lysophospholipase) pathways for LPA have been detected at the nuclear membrane and/or within the nucleus of targeted cells (6Baker R.R. Chang H. Biochim. Biophys. Acta. 2000; 1483: 58-68Crossref PubMed Scopus (20) Google Scholar, 7Baker R.R. Chang H.Y. Biochim. Biophys. Acta. 1999; 1438: 253-263Crossref PubMed Scopus (13) Google Scholar, 8Pagès C. Simon M.F. Valet P. Saulnier-Blache J.S. Prostaglandins. 2001; 64: 1-10Crossref Scopus (158) Google Scholar, 9Gaits F. Fourcade O. Le Balle F. Gueguen G. Gaige B. Gassama-Diagne A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. FEBS Lett. 1997; 410: 54-58Crossref PubMed Scopus (147) Google Scholar, 10D'Santos C.S. Clarke J.H. Divecha N. Biochim. Biophys. Acta. 1998; 1436: 201-232Crossref PubMed Scopus (159) Google Scholar, 11Kim Y.J. Kim K.P. Rhee H.J. Das S. Rafter J.D. Oh Y.S. Cho W. J. Biol. Chem. 2002; 277: 9358-9365Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Further support for the functionality of constitutive intracellular LPA receptors, specifically at the cell nucleus, is revealed by adjacent localization of required signaling effectors, which couple to the receptors. These accessory proteins include, among others, G proteins, ion channels, phospholipases A2, C, and D, adenylate cyclase, MAPKs, and NF-κB (see Ref. 12Gobeil Jr., F. Vazquez-Tello A. Marrache A.M. Bhattacharya M. Checchin D. Bkaily G. Lachapelle P. Ribeiro-Da-Silva A. Chemtob S. Can. J. Physiol. Pharmacol. 2003; 81: 196-204Crossref PubMed Scopus (40) Google Scholar for review). Alternatively, LPA may exert intracellular actions by generating its own formation, inferring possible active intracellular binding sites. Along this line of thought, LPA signaling at discrete subcellular domains may be provided in part by intracellular conveyors such as gelsolin and the fatty acid-binding protein (8Pagès C. Simon M.F. Valet P. Saulnier-Blache J.S. Prostaglandins. 2001; 64: 1-10Crossref Scopus (158) Google Scholar), by uptake of extracellular LPA bound to albumin (via albondin receptor; gp60) (13Tiruppathi C. Finnegan A. Malik A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 250-254Crossref PubMed Scopus (117) Google Scholar), and/or to lipocalins (14Bratt T. Biochim. Biophys. Acta. 2000; 1482: 318-326Crossref PubMed Scopus (118) Google Scholar).Lastly, several established pathways of GPCR regulation and desensitization driven by extracellular agonists have also been implicated in their intracellular relocalization, which results in delayed complementary signaling cascades (15Sarret P. Nouel D. Dal Farra C. Vincent J.P. Beaudet A. Mazella J. J. Biol. Chem. 1999; 274: 19294-19300Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 16Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). In this context, accumulating evidence suggests that nuclear translocation of peptide growth factors (e.g. angiogenin, PDGF, basic FGF, EGF, and PTH) and/or their integral membrane receptors, is mandatory for gene transcription associated with proliferating/growth events (17Re R. Hypertension. 1999; 34: 534-538Crossref PubMed Scopus (62) Google Scholar). Because most of these latter receptors (e.g. EGF, PDGF, and PTH receptors) co-localize within caveolae (18Razani B. Woodman S.E. Lisanti M.P. Pharmacol. Rev. 2002; 54: 431-467Crossref PubMed Scopus (839) Google Scholar), we speculated that translocation to this specific organelle proceeds via receptor endocytosis through the caveolar compartment. Whether this phenomenon holds true for the transmission of LPA gene responses (1Goetzl E.J. An S. FASEB J. 1998; 12: 1589-1598Crossref PubMed Scopus (490) Google Scholar, 2Moolenaar W.H. Exp. Cell Res. 1999; 253: 230-238Crossref PubMed Scopus (371) Google Scholar) is not yet known.The biochemical mechanisms by which GPCRs, including LPA-Rs, modulate gene transcription are complex and not fully understood (19Pierce K.L. Lutrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar). Some mechanisms recently uncovered to explain GPCR-mediated gene induction implicate an endocytosis-associated β-arrestin-c-Src interaction leading to downstream activation of Ras and mitogen-activated protein kinases (MAPKs) through possibly metalloprotease-dependent transactivation of receptor tyrosine kinases (RTK) involving de novo release of their ligand (19Pierce K.L. Lutrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar). Herein, we postulated that endogenous LPA stimulates gene expression, specifically the pro-inflammatory genes cyclooxygenase-2 (COX-2) and inducible nitric-oxide synthase (iNOS), through a formerly undescribed mechanism, which involves the activation of nuclear LPA receptors that pre-exist at the nuclear envelope or originate from the internalization/endocytosis of plasmalemmal LPA receptors. In the present study, we focused on endothelial cells, which are known to express predominantly LPA1R (20Lee H. Goetzl E.J. An S. Am. J. Physiol. Cell Physiol. 2000; 278: C612-C618Crossref PubMed Google Scholar) and consolidated our findings on LPA1R stably transfected HTC4 rat hepatoma cells (21An S. Bleu T. Zheng Y. Goetzl E.J. Mol. Pharmacol. 1998; 54: 881-888Crossref PubMed Scopus (149) Google Scholar) and rat liver tissue specimens. Our in vitro and in vivo findings support the existence of constitutive LPA1R at the cell nucleus, which upon stimulation mediates calcium transients and transcriptional signals of immediate-early response genes. Our results also suggest that (i) LPA-induced PLA2-dependent COX-2 expression is not reliant on prostaglandin, leukotriene, or epoxide production and (ii) contrary to common peptide growth hormone receptors, the sequestration and transcellular transport of LPA-R via caveolae to the nucleus is not a prerequisite for LPA-R activity on gene expression. This study unravels an as yet undescribed mechanism by which LPA modulates gene expression.EXPERIMENTAL PROCEDURESChemical Reagents and Antibodies—Materials and chemicals were obtained from the following sources: oleyl-, stereoyl-, palmitoyl-lysophophatidic acids (LPA); dioleoyl-phosphatidic acid, oleoyl-lysophosphatidylcholine, oleoyl-lysophosphatidylglycerol, oleoyl-lysophosphatidylserine, and oleoyl-lysophosphatidylethanolamine (Avanti Polar Lipids Inc., Birmingham, AL); (C16)-PAF (Cayman); 3-aminopropyltriethoxysilane, Nonidet P-40, filipin, thapsigargin, methyl-β-cyclodextrin, ibuprofen (Sigma); EGTA, pertussis toxin (PTX), fura-2-AM, wortmannin, ionomycin, MK886, cytidine-5-diphosphocholine, mepacrine, tyrphostin AG 1478, PD 98059, and tunicamycin (Calbiochem); SK&F96365, CV 3988, and methylcarbamyl platelet-activating factor (C-PAF) (BIOMOL); ketoconazole (ICN Biochemicals Inc.); fluo-4-AM (Molecular Probes); PNGase F assay kit (New England BioLabs); brain microvessel endothelial growth media (BioWhittaker); Dulbecco's modified Eagle's medium (Invitrogen); [3H]oLPA (PerkinElmer Life Sciences); RNA guard RNase inhibitor (Amersham Biosciences); and recombinant human interleukin-1β (IL-1β) (BIOSOURCE International). All other chemicals were analytical reagents and were purchased from Fisher Scientific (Montréal, Québec, Canada).Antibodies and their sources are: Anti-murine iNOS monoclonal antibody, anti-CD51 monoclonal antibody, and anti-human caveolin-1 monoclonal antibody (Transduction Laboratory); anti-human COX-2 polyclonal antibody (Cayman); anti-human β-actin polyclonal antibody; anti-human Von Willebrand factor polyclonal antibody (Dako, Denmark); anti-phospho-MAPK (Erk1/2) polyclonal antibody (Promega); anti-MAPK (Erk1/2) polyclonal antibody (Upstate Biotechnology); anti-phospho-Akt (Ser473) and anti-Akt polyclonal antibodies (New England BioLabs); anti-alkaline phosphatase polyclonal antibody (Abcam); anti-lamin A/C monoclonal antibody (Chemicon International); anti-cytochrome c monoclonal antibody (BD Pharmingen); horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (Pierce); rabbit polyclonal anti-human LPA1R-C antibody (raised against a C-terminal epitope; consisting of amino acids 328-344) and its cognate peptide antigen (Upstate Biotechnology); and rabbit polyclonal and mouse monoclonal anti-human LPA1R-N receptor antibodies (N-terminal epitope consisting of amino acids 6-25; Dr. E. J. Goetzl, University of California, CA). The specificity of anti-LPA1R antibodies has been fully established elsewhere (22Fukushima N. Kimura Y. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6151-6156Crossref PubMed Scopus (251) Google Scholar, 23Goetzl E.J. Dolezalova H. Kong Y. Zeng L. Cancer Res. 1999; 59: 4732-4737PubMed Google Scholar, 24Goetzl E.J. Kong Y. Voice J.K. J. Immunol. 2000; 164: 4996-4999Crossref PubMed Scopus (77) Google Scholar, 25Zheng Y. Kong Y. Goetzl E. J. Immunol. 2001; 166: 2317-2322Crossref PubMed Scopus (86) Google Scholar).Animals—Experiments were performed on endothelial cells derived from Yorkshire piglet brain microvasculature (Fermes Ménard, Quebec, Canada) and hepatocytes from adult Sprague-Dawley male rats (Charles River, Quebec, Canada). Animal housing and experimental protocols were carried out in accordance with regulations set by the Canadian Council of Animal Care Committee and were approved by the Sainte-Justine Hospital Animal Care Committee.Cell Culture and Fractionation—Primary endothelial cells obtained from porcine cerebral microvessels (26Dumont I. Hou X. Hardy P. Peri K.G. Beauchamp M. Najarian T. Molotchnikoff S. Varma D.R. Chemtob S. J. Pharmacol. Exp. Ther. 1999; 291: 627-633PubMed Google Scholar) and stably transfected, Geneticin-resistant LPA1-HTC4 rat hepatoma cells (21An S. Bleu T. Zheng Y. Goetzl E.J. Mol. Pharmacol. 1998; 54: 881-888Crossref PubMed Scopus (149) Google Scholar) were cultured and passaged, as previously reported. Cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin G, 1% streptomycin and used between passages 6-13 at ∼80% confluency unless otherwise stated. Prior to stimulation, cells were serum-starved overnight. Isolation of nuclei was achieved by cell fractionation using the hypotonic/Nonidet P-40 lysis method (27Gobeil Jr., F. Dumont I. Marrache A.M. Vazquez-Tello A. Bernier S.G. Abran D. Hou X. Beauchamp M.H. Quiniou C. Bouayad A. Choufani S. Bhattacharya M. Molotchnikoff S. Ribeiro-Da-Silva A. Varma D.R. Bkaily G. Chemtob S. Circ. Res. 2002; 90: 682-689Crossref PubMed Scopus (115) Google Scholar). Nuclear envelopes were prepared by incubating nuclear suspensions (8 × 106 nuclei/ml) with DNase I (800 units, pancreas type II, Roche Applied Science) and RNase A (32 mg/ml, Promega) for 30 min at 37 °C (28Kaufmann S.H. Gibson W. Shaper J.H. J. Biol. Chem. 1983; 258: 2710-2719Abstract Full Text PDF PubMed Google Scholar). Supernatants of homogenized cells (in lysis buffer) were sequentially centrifuged at 10,000 × g for 15 min then 120,000 × g for 60 min to obtain mitochondrial and plasma membrane fractions, respectively. Plasma and nuclear membranes and intact nuclei were stored at -80 °C unless otherwise stated. Protein concentration was determined by Bradford protein assay using BSA as standard. The morphological integrity and purity (>98%) was routinely assessed by light microscopy after trypan blue staining and confirmed by electron microscopy (27Gobeil Jr., F. Dumont I. Marrache A.M. Vazquez-Tello A. Bernier S.G. Abran D. Hou X. Beauchamp M.H. Quiniou C. Bouayad A. Choufani S. Bhattacharya M. Molotchnikoff S. Ribeiro-Da-Silva A. Varma D.R. Bkaily G. Chemtob S. Circ. Res. 2002; 90: 682-689Crossref PubMed Scopus (115) Google Scholar) (Fig. 1D, inset). The plasma membrane marker 5′-nucleotidase activity (Sigma assay kit) in nuclear versus plasma membrane fraction was less than 7%.Isolation of Subcellular Fractions of Rat Liver—Hepatocytes were harvested following digestion of liver with collagenase (type II, 0.05%, Sigma), as described (29Nanji A.A. Rahemtulla A. Maio L. Khwaja S. Zhao S. Tahan S.R. Thomas P. J. Pharmacol. Exp. Ther. 1997; 282: 1037-1043PubMed Google Scholar). Isolation of liver nuclei was carried out by ultracentrifugation through a sucrose gradient according to Nicotera et al. (30Nicotera P. McConkey D.J. Jones D.P. Orrenius S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 453-457Crossref PubMed Scopus (255) Google Scholar). Plasma membrane microsomes and nuclear envelopes were prepared, and morphological integrity and purity were ascertained as indicated above; 5′-nucleotidase activity endowed in the isolated nuclear fraction was less than 2% of that of plasma membrane fraction. Purity of subcellular fractions was further substantiated by means of immunological methods using the specific organelle marker antigens alkaline phosphatase and CD51 (plasma membrane), lamin A/C (nuclei), and cytochrome c (mitochondria, cytosol), as depicted below in Fig. 1A. Fig. 1B exhibits electron micrographs of highly purified nuclei (>98%) and prepared nuclear envelopes (right panel), appearing as intact spheres, following this isolation procedure.Radioligand Binding Assays—Cells were seeded into 24-well plates (500 μl of media/well) and allowed to reach 90% confluency (∼70,000 cells/well) before beginning experiments. Quiescent cells were washed thrice with PBS containing BSAfaf (0.1%), soybean trypsin inhibitor (100 μg/ml), and phenylmethylsulfonyl fluoride (1 mm) then incubated in the same buffer at 4 °C for 90 min with 10 nm radioactive tracer [3H]oLPA (50 Ci/mmol). Thereafter, cells were rinsed twice with cold incubation buffer (1 ml), lysed with sodium hydroxide (0.1 n), and transferred into scintillation vials. Nonspecific binding was determined in the presence of unlabeled LPA (10 μm). Radioactivity of samples was measured with a β-counter. In parallel, cells from untreated wells within the same plate were harvested with trypsin and counted thrice with a hemacytometer for cell count normalization.Subcellular Fractions—Binding assays were conducted on either whole nuclei or nuclear envelopes originating from porcine EC and rat liver using 10 nm radioactive tracer [3H]oLPA. Thawed rat liver nuclei (5 × 106 nuclei corresponding to ∼250 μg of protein) and prepared envelopes (5 × 106 nuclear envelope corresponding to ∼100 μg of protein) were resuspended in buffer consisting of Trizma-HCl (50 mm), pH 7.4, KCl (25 mm), MgCl2 (5 mm), sucrose (0.25 mm), CuSO4 (0.5 mm), BSAfaf (0.1%); pCMVEC-isolated nuclei (0.15 × 106 nuclei/100 μg of protein) in Trizma-HCl (20 mm), pH 7.4, KCl (10 mm), MgCl2 (3 mm), phenylmethylsulfonyl fluoride (0.5 mm), BSAfaf (0.1%). The reaction was terminated by diluting samples (twice with a 40× volume excess) followed by rapid filtration on GF/C filters presoaked in incubation buffer supplemented with BSAfaf (1%). In each experiment, nonspecific binding was determined in the presence of unlabeled LPA or surrogates (10 μm). Under these experimental conditions, the level of binding was directly proportional to time exposure (equilibrium time ∼ 60 min) or the amount of proteins added (50-400 μg) (not shown).Western Blot Analysis of LPA 1 R—Equal amounts (25 μg) of plasma membrane (PM), whole nuclei (WN), and derived nuclear envelope (NE) protein from pCMVEC, HTC4, and hepatocyte cells were solubilized in Laemmli buffer, separated by 12% reducing SDS-PAGE, and electro-blotted onto PVDF membranes. Membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline-Tween 20 (20 mm Tris-HCl, 150 mm NaCl, and 0.1% Tween 20) (TBS-T) and subsequently incubated overnight at 4 °C either with a monoclonal anti-LPA1R-N (1:100), a polyclonal anti-LPA1R-N (1:2000), or a polyclonal anti-LPA1R-C (1: 2000). Thereafter, membranes were washed with TBS-T and incubated for 1 h at RT with secondary antibodies (monoclonal, 1:2500; polyclonal, 1:5000) conjugated to horseradish peroxidase. Finally, membranes were thoroughly washed and developed using an enhanced plus ECL system (PerkinElmer Life Sciences). In some experiments, the effects of the N-glycosylation inhibitor, tunicamycin, and the endoglycosidase PNG-ase F were evaluated on cultured pCMVECs and an enriched plasmalemmal fraction of rat liver, respectively. These experiments were based on the presence of putative glycosylation consensus sequences (NX(S/T)) residing in rat (27NES29; 35NES37) and human (27NES29; 35NRS37) LPA1R orthologues (31Allard J. Barron S. Diaz J. Lubetzki C. Zalc B. Schwartz J.C. Sokoloff P. Eur. J. Neurosci. 1998; 10: 1045-1053Crossref PubMed Scopus (81) Google Scholar, 32Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar). For this purpose, pCMVEC were treated or not with tunicamycin (0.05, 0.5, or 1.0 μg/ml) for 24 h at 37 °C. Cells were then washed twice with cold PBS, lysed in 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 2 mm Na3VO4, 1 mm NaF, complete protease mixture inhibitor, and solubilized for 1 h at 4 °C using a rotary shaker. Cellular extracts were centrifuged (21,000 × g for 10 min at 4 °C), and the resulting supernatant was collected and proteins were measured. For deglycosylation experiments, 50 μg of rat liver PM was denatured and incubated or not with peptide N-glycosidase F (PNGase F) (500,000 units/ml; 5 μl) at 37 °C for 5 h according to the manufacturer's instructions. Samples (10-50 μg of proteins) were resolved by SDS-PAGE (9-12%), transferred onto PVDF membranes, and blotted with a polyclonal anti-LPA1R-N antibody (1:4000).Electron and Cryomicroscopic Immunohistochemistry of LPA 1 R— Male Sprague-Dawley rats (250-300 g) were used. Rat liver tissue sectioning and pre-embedding immunogold staining was done as described in detail previously (33Ribeiro-Da-Silva A. Priestley J.V. Cuello C. Cuello A.C. Immunohistochemistry II. John Wiley & Sons, New York1993: 182-210Google Scholar). Vibratome sections (50 μm) of liver were incubated with the primary antibody (a rabbit anti-LPA1R-C antibody) (1:50) overnight at 4 °C followed by another overnight incubation with goat anti-rabbit gold (10 nm)-conjugated IgG (1:50) (British Biocell International). In control sections, the primary antibody was either removed or pre-absorbed with its cognate peptide (10-fold excess by weight, Upstate Biotechnology). Thereafter, specimens were postfixed in 1% osmium tetroxide, subsequently dehydrated in graded ethanol, and embedded in Epon according to standard technique. Ultrathin sections were cut using a Reichert Ultracut ultramicrotome, mounted on Formvar-coated copper grids, stained with uranyl acetate and lead nitrate, and examined with a transmission electron microscope (Philips 410LS, Netherlands).Electron Cryomicroscopy—All procedures were based on previously reported methods (34Dahan S. Ahluwalia J.P. Wong L. Posner B.I. Bergeron J.J. J. Cell Biol. 1994; 127: 1859-1869Crossref PubMed Scopus (76) Google Scholar). Rat liver sections were cut at -100 °C in an FC4D cryochamber using a Reichert-Jung ultramicrotome and transferred to Formvar-coated copper grids. For immunolabeling assays, sections were incubated for 30 min at RT with rabbit polyclonal anti-human LPA1R-N or -C antibody (1:10) followed by incubation with a goat anti-rabbit gold (10 nm)-conjugated IgG (1:20) (Sigma Chemical Co.). Negative controls were analyzed by omitting primary antibodies. Frozen sections were contrasted and embedded as described (35Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar) and viewed through of a Philips 400T electron microscope.Indirect Immunofluorescence Staining—Isolated nuclei from pCMVECs were resuspended in blocking buffer constituted of 10 mm Tris-HCl, 20 mm NaCl, 3 mm MgCl2, 300 mm sucrose, complete protease mixture inhibitor, 5% horse serum, and 5% fetal calf serum. Double staining was achieved by incubating the nuclear suspension (1 h at RT) with a polyclonal rabbit anti-LPA1R-N (1:50) and a monoclonal mouse anti-caveolin-1 antibody (1:25). The nuclear suspension was diluted in a 5-fold excess of blocking buffer then centrifuged (800 × g, 10 min) and resuspended in similar buffer. The nuclei were subsequently incubated 60 min with a goat anti-rabbit AlexaFluor 488-conjugated IgG (2 μg/ml) and a chicken anti-mouse AlexaFluor 647-conjugated IgG (10 μg/ml) (Molecular Probes) and washed as indicated above. The nuclear suspension was then placed on poly-l-ornithine-treated glass coverslips (25 mm) and examined with a laser-scanning confocal microscope. Samples were imaged using a 60× differential interference contrast oil immersion objective lens on a Nikon TE300 microscope with a Bio-Rad Radiance 2000 confocal accessory. The two images were collected by using the same Z values and were merged using a Silicone graphic (SGI) software. Nuclear staining was realized at the end of the experiment using the DNA dye Syto-11 (100 nm) (Molecular Probes). For negative controls the primary antibodies were omitted.Immunoprecipitation of LPA 1 R and Western Blotting—Cellular and nuclear extracts (500 μg) of pCMVEC were lysed, immunoprecipitated by an anti-LPA1R-N polyclonal antibody (4 μg/ml), separated by SDS-PAGE (12%) and transferred onto nitrocellulose membrane. Immunoblotting was performed with either an anti-LPA1R-N polyclonal antibody (serving as positive control) (1:2000), anti-clathrin (1:1000), or caveolin-1 (1:1000) monoclonal antibody. Cellular and nuclear extracts of EC were subjected to extraction in Triton X-100 followed by sucrose density gradient centrifugation to isolate caveolae (35Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar). Caveolae-enriched fractions (low density and Triton X-100-insoluble materials) were verified by SDS-PAGE and probed for caveolin-1. Equal amounts of proteins from caveolar and non-caveolar membranes were then immunoblotted for LPA1R as indicated above.Western Blot of COX-2 and iNOS Proteins—pCMVEC were seeded into 10-cm dishes, rendered quiescent with starving medium, and treated for 6 h with either the vehicle (bidistilled and d" @default.
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