Internalized Group V Secretory Phospholipase A2 Acts on the Perinuclear Membranes
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m110987200
ISSN1083-351X
AutoresYoung Jun Kim, Kwang Pyo Kim, Hae Jin Rhee, Sudipto Das, John D. Rafter, Youn Sang Oh, Wonhwa Cho,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoMammalian secretory phospholipases A2 (sPLA2) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA2 mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA2 acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid,N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA2s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA2hydrolysis products or cytokines, are necessary for the internalization of sPLA2 into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA2 internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA2 induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA2 acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis. Mammalian secretory phospholipases A2 (sPLA2) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA2 mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA2 acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid,N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA2s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA2hydrolysis products or cytokines, are necessary for the internalization of sPLA2 into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA2 internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA2 induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA2 acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis. phospholipase A2 arachidonic acid bovine serum albumin group VI cytosolic PLA2 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate Dulbecco's modified Eagle's medium human embryonic kidney human group IIa PLA2 heparan sulfate proteoglycan human group V PLA2 5-lipoxygenase phosphate-buffered saline phosphatidylcholine N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine secretory PLA2 interleukin Phospholipases A2 (PLA2)1catalyze the hydrolysis of membrane phospholipids, the products of which can be transformed into potent inflammatory lipid mediators, platelet activating factor and eicosanoids that include prostaglandins, thromboxanes, leukotrienes, and lipoxins. Multiple forms of secretory PLA2s (sPLA2) and intracellular PLA2s have been found in mammalian tissues (1Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1220) Google Scholar). Recent cell studies have indicated that some sPLA2 isoforms work in concert with group IV cytosolic PLA2 (cPLA2) to induce immediate and delayed eicosanoid formation (2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 3Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). At present, the identity of proinflammatory sPLA2, the spatiotemporal dynamics of sPLA2 mobilization, and the signaling mechanism that links sPLA2, cPLA2, and other enzymes involved in eicosanoid biosynthesis are not fully understood. It has been reported that the heparan sulfate proteoglycan (HSPG)-mediated internalization of sPLA2 is an important step in sPLA2 actions on mammalian cells (3Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar); however, functional consequences of sPLA2 internalization remain controversial. In agonist-induced human embryonic kidney 293 (HEK293) cells transfected with various sPLA2s, the sPLA2 internalization resulted in arachidonic acid (AA) release and prostaglandin synthesis (3Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), whereas in human neutrophils (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and mast cells (8Enomoto A. Murakami M. Kudo I. Biochem. Biophys. Res. Commun. 2000; 276: 667-672Crossref PubMed Scopus (23) Google Scholar) the sPLA2internalization led to protein degradation. This study was undertaken to clarify the effect of sPLA2 internalization on the cellular eicosanoid biosynthesis and determine the location of sPLA2 lipolytic actions. Results described herein provide the first experimental evidence that the internalized sPLA2liberates fatty acids from the phospholipids in the nuclear envelope at which other eicosanoid-producing enzymes are localized during the cellular eicosanoid biosynthesis. 1,1′-Didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC12),N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt (PED6), and Texas RedTMC2 maleimide were purchased from Molecular Probes, Inc. (Eugene, OR). Cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Phospholipid concentrations were determined by phosphate analysis (9Kates M. Techniques of Lipidology.2nd Ed. Elsevier, Amsterdam1986: 114-115Google Scholar). Dublecco's modified Eagle's medium (DMEM) and inactivated fetal bovine serum were from Invitrogen (Grand Island, NY). HEK293 cells and Zeocin were from Invitrogen (San Diego, CA). Fatty acid-free bovine serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). Arachidonyl trifluoromethyl ketone was from Calbiochem(San Diego, CA). Recombinant human group V PLA2(hVPLA2) (10Han S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (53) Google Scholar), its mutants (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and human group IIa PLA2 (hIIaPLA2) (12Snitko Y. Koduri R. Han S.-K. Othman R. Baker S.F. Molini B.J. Wilton D.C. Gelb M.H. Cho W. Biochemistry. 1997; 36: 14325-14333Crossref PubMed Scopus (110) Google Scholar) were expressed and purified as described previously. HEK293 cells were treated with 100 nm of hVPLA2-W79A, W79A/W31A, W79A/R100E/K101E, and hIIaPLA2 for the indicated period, and the incubation was quenched by adding a solution of ice-cold 0.6 m NaCl in DMEM. After washing with the same solution, the pellet was collected by scrapping and centrifugation, then lysed in 70 μl of lysis buffer (20 mm Tris-HCl, 30 mmNa4P2O7, 50 mm NaF, 40 mm NaCl, 5 mm EDTA, pH 7.4) containing 1% Nonidet P-40, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 2 mmNa3VO4, and 0.5% deoxycholic acid. After 10 min on ice, the cell lysates were centrifuged at 12,000 ×g for 3 min to remove the cell debris. The supernatants were then mixed with 14 μl of gel loading buffer (0.125 mTris-HCl, pH 6.8, 20% (v/v) glycerol, 4% sodium dodecyl sulfate, 0.005% bromphenol blue), and the mixtures were boiled for 5 min. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions using 16% acrylamide gels. The electrotransfer of proteins from the gels to polyvinylidene fluoride membrane was achieved using a semidry system (400 mA, 120 min). The membrane was blocked with 2% BSA for 60 min, then incubated with 1 μg/ml of either the anti-hVPLA2 monoclonal antibody 3G1 (14Muñoz N.M. Kim K. Han S.-K. Boetticher E. Sperling A.I. Sano H. Zhu X. Cho W. Leff A.R. Hybridoma. 2000; 19: 171-176Crossref PubMed Scopus (14) Google Scholar) or a commercial hIIaPLA2 antibody (Upstate Biotechnology) diluted in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) overnight. The membranes were washed three times for 20 min with TBS-T. Goat anti-mouse IgG conjugated with horseradish peroxidase was diluted 3000-fold in TBS-T and incubated with polyvinylidene fluoride membrane for 60 min. The membrane was washed three times with TBS-T and assayed with an ECL chemiluminescence system (Amersham Biosciences, Inc.). Radiolabeling of human neutrophils cells with [3H]AA was performed as described previously (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Radiolabeling of HEK293 cells was achieved by incubating the cells (106) with 0.05 μCi/ml [3H]AA for 4 h at 37 °C. Unincorporated [3H]AA was removed by washing the cells three times with DMEM containing 0.2% BSA. The reaction was quenched by adding 3 ml of ice-cold DMEM and the cell and the medium were separated by centrifugation, then the radioactivity of pellet and supernatant, respectively, was measured by liquid scintillation. To create a single free cystein for chemical labeling, the W79C mutation was performed as described previously (11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). W79C was expressed, refolded and purified according to the protocol used for hVPLA2(10Han S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (53) Google Scholar). Purified W79C (0.5 mg) was dissolved in 1 ml of 25 mmTris-HCl, pH 7.5, containing 0.5 m guanidinium chloride and treated with 10-fold molar excess of Texas RedTMC2 maleimide for 2 h at room temperature. The labeled protein was fractionally precipitated with 50% ammonium sulfate on ice, collected by centrifugation at 50,000 × g and at 4 °C for 15 min, and resuspended in 1 ml of 25 mmTris-HCl buffer, pH 7.5, containing 0.2 m guanidinium chloride. The labeled protein was purified using a HitrapTMheparin column (Amersham Biosciences, Inc.) that was attached to aÄkta FPLC system (Amersham Biosciences, Inc.) and equilibrated in the same buffer. Labeled protein was eluted with the linear gradient of NaCl to 0.5 m in the same buffer. The fractions corresponding to a major protein peak were dialyzed against 25 mm Tris-HCl, pH 8.0, for 24 h at 4 °C and then stored at −20 °C. The labeling of cell membranes by PED6 was performed as described previously (13Farber S.A. Olson E.S. Clark J.D. Halpern M.E. J. Biol. Chem. 1999; 274: 19338-19346Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) with some modifications. A mixture of POPS/cholesterol/POPG/PED6 (107:31:20:1 in molar ratio, 300 nmol total) in chloroform was dried under N2 and resuspended in ethanol (10 μl), followed by the addition of DMEM (10 μl). The solution was dried again under N2 until the volume was reduced to ∼7 μl to ensure that most of ethanol was evaporated. Additional 10 μl of DMEM was added to the mixture and vesicles were prepared by sonication of the mixture on ice (20 min). HEK293 cells (3–5 × 104 cells) were seeded into each of eight wells on a sterile Nunc Lak-TeK IITM chambered cover glass filled with the DMEM supplemented with 10% fetal bovine serum and 250 μg/ml ZeocinTM, and incubated at 37 °C with 5% CO2 for 48 h. The vesicle solution (10 μl) was then added to each of eight wells and incubated with HEK293 cells for 25–50 min at 37 °C. HEK293 cells were rinsed with phosphate-buffered saline (PBS) five times, resuspended in 300 μl of DMEM media, and 150 nm (or higher) sPLA2 and 2 mmCaCl2 (final concentration) were added. Imaging was done with a Zeiss LSM510 laser scanning confocal microscope with the detector gain adjusted to eliminate the background autofluorescence. The signal from the Texas RedTM attached to W79C was observed directly upon excitation with a 568-nm argon/krypton laser and a 650-nm line pass filter whereas the BODIPYTM signal from the hydrolyzed PED6 was visualized with a 488-nm argon/krypton laser and a 530-nm band pass filter. A ×63 (1.2 numerical aperture) water immersion objective was used for all experiments. Images were analyzed using the analysis tools provided in the Zeiss biophysical software package. Using these tools, regions of interest in the cytosol and the membranes were defined, and the average fluorescence intensity in a square (1 mm × 1 mm) was obtained as a function of time. To label HEK293 cell membranes with DiIC12, each of 1 μl of dye solution in ethanol (2 mg/ml) was added rapidly to 400 μl of HEK293 cells that were cultured for 48 h in DMEM supplemented with 10% fetal bovine serum and 250 μg/ml ZeocinTM in each of eight wells on a sterile Nunc Lak-TeK IITMchambered cover glass. Unbound dye was removed by washing four times with PBS. Washed HEK293 cells were imaged in 400 μl of DMEM without phenol red. The preparation was placed on the stage of a Zeiss Pascal laser scanning confocal microscope fitted with a 570-nm line pass filter and a 543-nm He/Ne laser. 150 nm sPLA2and 2 mm CaCl2 (final concentration) were added and the imaging was performed as described above. HEK293 cells were plated onto a sterile cover glass and incubated at 37 °C with 5% CO2. The stable HEK293 cell line expressing 5-lipoxygenase (5-LO) was generated by transfecting the cells with pcDNA3.1-human 5-LO plasmid using LipofectAMINE (Invitrogen), followed by selection of clones in the presence of geneticin (800 μg/ml) for 3–4 weeks. The cells were treated with 150 nm (final concentration) of hVPLA2 in DMEM for 5, 10, and 30 min in a 37 °C, 5% CO2 humidified incubator. At the given time, cells were washed twice with cold PBS, and then were fixed at room temperature with 3.6% paraformaldehyde in PBS for 10 min. After fixation, the cells were washed six times with PBS and placed in a blocking solution (10% normal goat serum and 100 μm goat IgG in PBS) at room temperature for 3 h. The cells were then permeabilized with PBS containing 0.1% Triton X-100 and 2% BSA for 1 h at room temperature, washed four times with PBS, and incubated with the monoclonal antibodies raised against hVPLA2 (2 μg/ml) (14Muñoz N.M. Kim K. Han S.-K. Boetticher E. Sperling A.I. Sano H. Zhu X. Cho W. Leff A.R. Hybridoma. 2000; 19: 171-176Crossref PubMed Scopus (14) Google Scholar) and human 5-LO polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (500-fold diluted), respectively, in the presence of 2% BSA. After 2 h incubation at room temperature, the antibodies were removed, and cells were washed six times with PBS. A secondary antibody, Alexa488 donkey anti-goat antibody (Molecular Probes) diluted in PBS containing 2% BSA, was applied for 1 h at room temperature followed by washing and incubation with another secondary antibody, Alexa568 goat anti-mouse antibody (Molecular Probes) diluted in PBS containing 2% BSA for 1 h at room temperature. After washing six times with PBS, the slide was mounted with Fluoromount-G (Southern Biotech Associates, AL). Imaging was done with a Zeiss LSM510 laser scanning confocal microscope. The sPLA2-catalyzed hydrolysis of PED6 in the mixed vesicles of POPS/cholesterol/POPG/PED-6 (107:31:20:1) was carried out at 37 °C in 2 ml of 10 mm Tris-HCl, pH 7.4, containing 0.16 m KCl, 0.01 mm EDTA, 2.5 mm Ca2+. The progress of hydrolysis was monitored as an increase in fluorescence emission at 520 nm using a Hitachi F4500 fluorescence spectrometer with the excitation wavelength set at 488 nm. Spectral band width was set at 10 nm for both excitation and emission. Values of specific activity were determined from the initial rates of hydrolysis. The internalization of sPLA2 to mammalian cells has been observed when the cells were treated with exogenously added sPLA2 (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 8Enomoto A. Murakami M. Kudo I. Biochem. Biophys. Res. Commun. 2000; 276: 667-672Crossref PubMed Scopus (23) Google Scholar) or the cells expressing sPLA2s were stimulated with agonists, such as interleukin-1 (IL-1) (3Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). To rigorously and systematically determine the requirements for sPLA2 internalization, we selected the exogenous addition method that allows the use of the protein chemically labeled with a fluorescent probe for real-time monitoring. The chemical labeling was preferred to the genetic incorporation of a green fluorescence protein tag because the latter significantly altered either enzymatic activity or HSPG affinity (data not shown). First, we measured the internalization of hVPLA2 (W79A), its mutants (W79A/W31A and W79A/R100E/K101E), and hIIaPLA2 to unstimulated HEK293 cells by Western blotting analysis of cell extracts after sPLA2 treatment. For hVPLA2, the W79A mutant is used in place of wild type because it is fully active and gives a higher yield of refolding than wild type (11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). We previously showed that hVPLA2 (and W79A) has much higher activity on mammalian cells than hIIaPLA2 because of its ability to effectively bind and hydrolyze PC (11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). We also showed that W79A/W31A of hVPLA2 has ∼50 times lower activity on PC membranes than W79A (11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and that W79A/R100E/K101E has full PC activity but has much reduced affinity for HSPG (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). As shown in Fig.1, only hVPLA2-W79A showed a significant degree of internalization at 20 min. Even after 60 min, W79A/R100E/K101E and hIIaPLA2 did not show detectable internalization (data not shown). W79A/W31A was internalized at a greatly reduced rate: a faint band appeared at 20 min, which only after 60 min became comparable to that of wild type measured at 20 min (Fig.1 C). Interestingly, when HEK293 cells were treated with W79A/W31A in the presence of Naja naja naja PLA2 that was shown to have high PC activity (15Sumandea M. Das S. Sumandea C. Cho W. Biochemistry. 1999; 38: 16290-16297Crossref PubMed Scopus (54) Google Scholar), W79A/W31A was internalized as well as wild type (Fig.1 D). The dark 14-kDa band was not due to internalizedN. naja naja PLA2 because our hVPLA2monoclonal antibodies do not cross-react with N. naja najaPLA2 (14Muñoz N.M. Kim K. Han S.-K. Boetticher E. Sperling A.I. Sano H. Zhu X. Cho W. Leff A.R. Hybridoma. 2000; 19: 171-176Crossref PubMed Scopus (14) Google Scholar) and because N. naja najaPLA2 owing to its extremely low HSPG affinity was not internalized under our experimental conditions. Thus, both HSPG affinity and the ability to hydrolyze the outer plasma membrane (i.e. PC membranes) are required for a sPLA2 to enter unstimulated HEK293 cells. In contrast, W79A, W79A/W31A, and hIIaPLA2 (Fig. 1 F), but not W79A/R100E/K101E, were internalized when HEK293 cells were primed with IL-1β. This indicates that HSPG affinity is both necessary and sufficient for the internalization of sPLA2 into IL-1β-primed HEK293 cells. The internalized sPLA2 remained intact after several hours in HEK293 cells, which is in sharp contrast to the rapid degradation of internalized sPLA2 in neutrophils (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Taken together, these results indicate that although HSPG affinity is a critical factor for the internalization of sPLA2 into HEK293 cells, other factors, such as PLA2 hydrolysis products and cytokines, are also necessary for the internalization. Although several reports have suggested that sPLA2s might act intracellularly, whether they are intracellularly localized (16Bingham 3rd, C.O. Fijneman R.J. Friend D.S. Goddeau R.P. Rogers R.A. Austen K.F. Arm J.P. J. Biol. Chem. 1999; 274: 31476-31484Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) or re-internalized after secretion (3Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), no direct experimental evidence for the notion has been documented. To determine the correlation between the internalization of sPLA2 and its intracellular lipolytic activities, we treated [3H]AA-labeled human neutrophils and HEK293 cells with W79A and W79A/R100E/K101E and measured the time courses of AA release. As we reported previously (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), the liberation of AA from human neutrophils by W79A reached a plateau after ∼15 min, whereas the AA release by non-internalizing W79A/R100E/K101E continued to proceed even after 1 h (Fig. 2 A). The saturation of the AA release by W79A is due to its internalization into neutrophils and subsequent degradation (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Interestingly, the AA release from HEK293 cells by W79A and W79A/R100E/K101E showed similar biphasic patterns and the slower second phases lasted for more than an hour (Fig. 2 B). In view of the different fate of the internalized hVPLA2 in neutrophils and HEK293 cells (i.e. degradation versus retention), these data imply that the second phase of W79A-induced AA release from HEK293 cells is due to the action of internalized enzyme on intracellular membranes. To corroborate the notion that the internalized sPLA2 is active on intracellular membranes and to determine the intracellular location of sPLA2 lipolytic action, we labeled HEK293 cells with a fluorogenic phospholipid, PED6, which has been used for in vivo PLA2 assays (17Hendrickson H.S. Hendrickson E.K. Johnson I.D. Farber S.A. Anal. Biochem. 1999; 276: 27-35Crossref PubMed Scopus (65) Google Scholar,18Farber S.A. Pack M. Ho S.Y. Johnson I.D. Wagner D.S. Dosch R. Mullins M.C. Hendrickson H.S. Hendrickson E.K. Halpern M.E. Science. 2001; 292: 1385-1388Crossref PubMed Scopus (272) Google Scholar). In this lipid, the fluorescent BODIPYTM moiety in thesn-2 position is quenched by the dinitrophenyl group in the head group, which is relieved when the PLA2-catalyzed hydrolysis releases the BODIPYTM-labeled fatty acid. As summarized in Table I, all sPLA2s used in these studies showed relatively high activity on PED6 in the in vitro vesicle assay; however, cPLA2 had less than 0.1% of hVPLA2-W79A activity. Since the BODIPYTM fluorescence in PED6 is not completely quenched, the cellular distribution of intact PDE6 can be monitored if cells were illuminated with a higher laser power. As shown in Fig. 3 A, PDE6 was primarily localized in the plasma membrane within the first 20 min of incubation but more evenly distributed among various cellular membranes after 25 to 50 min of incubation under our experimental conditions.Table IRelative activities of PLA2 on PED6 substrateEnzymeRelative activityhVPLA2W79A1.001-aThe absolute specific activity value for W79A was 20 ± 2 μmol/ min/mg.hVPLA2 W79A/W31A0.25hVPLA2 W79A/R100E/K101E0.70hVPLA2W79C0.95Labeled hVPLA2 W79C0.90hIIaPLA20.54cPLA20.0008Each activity value was determined as an average of triplicate measurements. The vesicle composition was POPS/cholesterol/POPG/PED6 = 107:31:20:1 in mole ratio.1-a The absolute specific activity value for W79A was 20 ± 2 μmol/ min/mg. Open table in a new tab Each activity value was determined as an average of triplicate measurements. The vesicle composition was POPS/cholesterol/POPG/PED6 = 107:31:20:1 in mole ratio. We first treated PED6-labeled (unstimulated) HEK293 cells with hIIaPLA2, hVPLA2-W79A, and mutants. As shown in Fig. 3 B, the addition of W79A to the cells resulted in the appearance of BODIPYTM fluorescence, first at the plasma membrane and then intracellularly with a clear annular pattern around the nucleus. The time lapse relative fluorescence intensity profiles of the region of interest clearly show that the signal at the plasma membrane peaks at ∼2 min and the signal at the nuclear envelope reaches the plateau at ∼4 min (Fig. 3 C). The cytoplasmic signal that is much weaker than nuclear envelope signal initially (i.e. <4 min) continued to rise until up to 6 min. Thus, this relatively diffuse cytoplasmic signal seems to reflect the diffusion of the short-chain BODIPY fatty acid from the nuclear membranes. Consistent with our Western blotting data, hIIaPLA2 did not induce any app
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