B-Myb-Dependent Regulation of c-Myc Expression by Cytosolic Phospholipase A2

Cytosolic phospholipase A2 (cPLA2) cleaves membrane phospholipids to release arachidonic acid, initiating lipoxygenase and cyclooxygenase pathways. Mice lacking a gene for cPLA2 suggested important roles of the protein in allergic responses, fertility, and neural cell death. Here we show that cPLA2 negatively regulates c-Myc expression in a B-Myb-dependent manner. Overexpression of cPLA2 protein but not a mutant cPLA2 protein that lacks in vitro binding ability with B-Myb inhibits B-Myb-dependent c-myc gene expression. The inhibition was associated with physical interaction of B-Myb protein with cPLA2 both in the cytoplasm and the nucleus. Binding site analysis demonstrated that both the N and C termini of cPLA2 interact with B-Myb. Macrophage colony stimulating factor (MCSF) stimulated cPLA2 redistribution into the nucleus and also association with B-Myb in human monocytes. Importantly, macrophages from mice with a disrupted cPLA2 gene demonstrated significantly increased levels of c-Myc protein in the nucleus compared with cells from the wild-type mice, whereas B-Myb levels were similar in the cells from the cPLA2+/+ and cPLA2-/- mice. Moreover, an introduction of cPLA2 into cPLA2-/- mouse macrophages resulted in decreased c-Myc protein levels, and an inhibition of cPLA2 expression by small interfering RNAs or antisense RNA increased the c-myc transcription in macrophage colony stimulating factor-activated human monocytes. These findings provide new insights into the function of cPLA2 in B-Myb-dependent gene expression. Cytosolic phospholipase A2 (cPLA2) cleaves membrane phospholipids to release arachidonic acid, initiating lipoxygenase and cyclooxygenase pathways. Mice lacking a gene for cPLA2 suggested important roles of the protein in allergic responses, fertility, and neural cell death. Here we show that cPLA2 negatively regulates c-Myc expression in a B-Myb-dependent manner. Overexpression of cPLA2 protein but not a mutant cPLA2 protein that lacks in vitro binding ability with B-Myb inhibits B-Myb-dependent c-myc gene expression. The inhibition was associated with physical interaction of B-Myb protein with cPLA2 both in the cytoplasm and the nucleus. Binding site analysis demonstrated that both the N and C termini of cPLA2 interact with B-Myb. Macrophage colony stimulating factor (MCSF) stimulated cPLA2 redistribution into the nucleus and also association with B-Myb in human monocytes. Importantly, macrophages from mice with a disrupted cPLA2 gene demonstrated significantly increased levels of c-Myc protein in the nucleus compared with cells from the wild-type mice, whereas B-Myb levels were similar in the cells from the cPLA2+/+ and cPLA2-/- mice. Moreover, an introduction of cPLA2 into cPLA2-/- mouse macrophages resulted in decreased c-Myc protein levels, and an inhibition of cPLA2 expression by small interfering RNAs or antisense RNA increased the c-myc transcription in macrophage colony stimulating factor-activated human monocytes. These findings provide new insights into the function of cPLA2 in B-Myb-dependent gene expression. cPLA2 1The abbreviations used are: cPLA2, cytosolic phospholipase A2; NLS, nuclear localization signal; FBS, fetal bovine serum; MCSF, macrophage colony stimulating factor; PBS, phosphate-buffered saline; NES, nuclear export signal. is activated by cytokines, submicromolar concentrations of Ca2+ ions, and mitogen-activated protein kinase-mediated phosphorylation of serine residues in the protein (1Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar, 2Schievella A.R. Regier M.K. Smith W.L. Lin L.L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Activated cPLA2 protein distributes preferentially to the perinuclear region of the cell (3Clark J.D. Lin L.L. kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 4Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 5Perisic O. Paterson H.F. Mosedale G. Lara-Gonzalez S. Williams R.L. J. Biol. Chem. 1999; 274: 14979-14987Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), where the enzyme is thought to participate in arachidonic acid release. Consistent with this notion, 5-lipoxygenase and cyclooxygenase, two major enzymes downstream of cPLA2, reside in the nuclear envelope and oxidize arachidonic acid (6Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 7Chen X.S. Zhang Y.Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Gilroy D.W. Colville-Nash P.R. Willis D. Chivers J. Paul-Clark M.J. Willoughby D.A. Nature Med. 1999; 5: 698-701Crossref PubMed Scopus (1123) Google Scholar, 9Hanaka T. Shimizu T Izumi T. Biochem. J. 2002; 361: 505-514Crossref PubMed Scopus (38) Google Scholar, 10Woods J.W. Coffey M.J. Brock T.G. Singer I.I. Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2046Crossref PubMed Scopus (160) Google Scholar). However, the entry of cPLA2 into the nucleus does not occur in these conditions (5Perisic O. Paterson H.F. Mosedale G. Lara-Gonzalez S. Williams R.L. J. Biol. Chem. 1999; 274: 14979-14987Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). cPLA2 localizes in the nucleus of subconfluent endothelial cells (11Sierra-Honigmann M.R. Bradley J.R. Pober J.S. Lab. Invest. 1996; 74: 684-695PubMed Google Scholar). cPLA2 can regulate the NF-κB-dependent transcription (12Pawliczak R. Han C. Huang X.L. Demetris J. Shelhamer J.H. Wu T. J. Biol. Chem. 2002; 277: 33153-33163Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These findings along with the observations that more downstream enzymes such as lipoxygenase and cyclooxygenase reside in the nuclear envelope prompted us to test a hypothesis that cPLA2 can enter the nucleus under some cellular conditions. Because the cPLA2 protein does not harbor any apparent classical nuclear localization signal (NLS), cPLA2 might be directed toward the nucleus by a partner protein(s). Recent studies using mice lacking a gene for cPLA2 showed that these mice were associated with impairment of allergic responses and fertility and also suggested important contributions of cPLA2 to the pathophysiology of neural cell death (13Bonventre J.V. Huang Z. Reza Taheri M. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (760) Google Scholar, 14Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (645) Google Scholar). These studies imply that cPLA2 may play a more important role than previously thought. B-Myb is a nuclear transcriptional factor belonging to the Myb family that is ubiquitously expressed in tissues and is involved in cell growth control, differentiation, and cancer (15Oh I.H. Reddy E.P. Oncogene. 1999; 18: 3017-3033Crossref PubMed Scopus (420) Google Scholar). Furthermore, B-Myb interacts with other transcriptional factors and modulates their biological functions. Here we demonstrate so-far undefined physical associations of cPLA2 and BMyb transcriptional factor, which facilitates redistribution of cPLA2-B-Myb complexes into the nucleus. Importantly, we also show that redistribution of cytoplasmic cPLA2 after its specific binding to B-Myb transcriptional factor can regulate B-Myb-dependent c-myc gene expression. Cell Culture—African Green monkey kidney cells (CV-1) and human kidney cells (293T) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 4 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Human monocytes were isolated from the peripheral blood of healthy volunteers by Ficoll-Paque separation followed by adherence for 1 h and removal of non-adherent cells. The adherent cells were collected and washed 3 times with culture medium. The resultant cell population consisted of >90% monocytes as judged by morphological examination and α-naphthyl acetate esterase staining (16Nakamura T. Lin L.L. Kharbanda S. Knopf J. Kufe D. EMBO J. 1992; 11: 4917-4922Crossref PubMed Scopus (102) Google Scholar). Monocytes were cultured in RPMI1640 medium supplemented with 10% FBS, 4 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. The monocytes were treated with 103 units/ml MCSF (Leukoprol; Morinaga Co., Tokyo, Japan). Thioglycolate-elicited peritoneal cells were collected from cPLA2+/+ and cPLA2-/- mice and seeded at 4 × 105 cells/ml in RPMI1640 supplemented with 10% FBS. To obtain mature macrophages, the collected cells were allowed to adhere to the plastic dish bottom for 2 h in culture medium, and adherent cells were harvested after washing the dish with phosphate-buffered saline (PBS). Lung fibroblasts were isolated from cPLA2+/+ and cPLA2-/- mice by digesting the minced lung tissues for 4 h in Dulbecco's modified Eagle's medium supplemented with 0.5% collagenase and 2% FBS. Obtained cells were cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS. Plasmid Construction and Transfection—The FLAG-tagged cPLA2 fragments 1-749 (full-length), 1-524, 1-294, 1-202, and 202-749 were constructed by subcloning restriction fragments of cPLA2 into a pact vector. A FLAG-tagged C-terminal truncated mutant cPLA2-(493-749) and FLAG-tagged cPLA2 mutated in nuclear export signal (NES) were created by PCR and were also subcloned into the same vector. We carried out DNA transfection by LipofectAMINE reagents (Invitrogen) or by calcium phosphate precipitation method. We used 3 and 65 μg of plasmids, respectively, for LipofectAMINE and calcium phosphate precipitation methods. Transfection of mouse peritoneal macrophages from cPLA2-/- mice was carried out according to a commercially available protocol using a Nucleofector transfection system and Mouse Neuron Nucleofector Solution (Amaxa Biosystems). We transfected 4 × 106 peritoneal macrophages with 0.5 μg of pact plasmid containing full-length cPLA2. Immunofluorescence Staining and Confocal Microscopy—Cells were fixed for 10 min by 4% paraformaldehyde in PBS at room temperature, permeabilized by 0.1% Triton X-100 in PBS, and blocked with 1.5% skimmed milk and 1.5% bovine serum albumin in PBS. Primary antibodies (10 μg/ml) were prepared in 2% bovine serum albumin in PBS. Cells were then incubated with a fluorescein isothiocyanate-conjugated goat anti-mouse antibody (1:20; Zymed Laboratories Inc.) and Cy3-conjugated goat anti-rabbit antibody (1:100; Amersham Biosciences). Confocal imaging was performed using a Leica TCS SP2 AOBS laser-scanning microscope. Cell Fractionation—Cell were suspended in ice-cold hypotonic buffer (10 mm Hepes, pH 7.4, 1.5 mm MgCl2,10 mm KCl) supplemented with 0.25% Nonidet P-40, 0.5 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride and incubated for 5 min on ice. After centrifugation at 16,000 × g for 10 min at 4 °C, the supernatant (cytoplasmic fraction) was collected. The pellets were washed twice with ice-cold buffer. The pellets were resuspended in an appropriate volume of high salt buffer (20 mm Hepes, 25% glycerol, 0.42 m KCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride) and incubated for 15 min on ice. After centrifugation at 16,000 × g for 10 min at 4 °C, the supernatants were collected (nuclear fraction). Immunoprecipitation and Western Blotting—For the immunoprecipitation experiments we used monoclonal antibodies specific for cPLA2 (Santa Cruz) or FLAG (Eastman Kodak Co.) or polyclonal antibodies for B-Myb (Santa Cruz). Immunoprecipitates were then electrophoresed in an SDS-PAGE gel and transferred to a polyvinylidene difluoride filter (Millipore), and the protein-protein interaction was confirmed by Western blotting using the above antibodies. Reporter Gene Activity Assays—CV-1 cells were co-transfected with empty vector or a pact plasmid containing B-Myb-(1-700) or pact plasmid containing cPLA2-(1-749) or both. The reporter gene activity was measured as described previously (17Mizuguchi G. Nakagoshi H. Nagasa T. Nomura N. Date T. Ueno Y. Ishii S. J. Biol. Chem. 1990; 265: 9280-9284Abstract Full Text PDF PubMed Google Scholar). The chloramphenicol acetyltransferase (CAT) reporter plasmid pmycCAT was used for the c-myc transcription assays in CV-1 cells expressing B-Myb and/or wild-type or mutant cPLA2. The amount of cell extracts used for chloramphenicol acetyltransferase assays were normalized with respect to cotransfected pRL-SV40 plasmid (Promega). The radioactivity of either [14C]chloramphenicol or its acetylated form was measured using a BAS 1500 imager (Fuji Film). Data were shown as averages of three experiments. PLA2 Assay—In vitro PLA2 assay was performed on cell lysates after subcellular fractionation into the nuclear and cytoplasmic components using the same protocol for Western blotting as described above. The PLA2 assay was performed as described in the previous publication (16Nakamura T. Lin L.L. Kharbanda S. Knopf J. Kufe D. EMBO J. 1992; 11: 4917-4922Crossref PubMed Scopus (102) Google Scholar). PLA2 activity was assessed in the presence of 4 mm Ca2+ by measuring labeled arachidonic acid release from the sn-2 position of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. The radioactive spots on TLC plates, each corresponding to released arachidonic acid, were visualized by a BAS 1500 imager (Fuji Film) and were quantitated by densitometric scanning of the spots. Generation of cPLA2-/- Mice—cPLA2-/- mice were generated by using homologous recombination as described previously (14Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (645) Google Scholar). The mouse strains used (C57BL/6J and 129/Ola) are congenitally defective in type II PLA2, which is a secretory PLA2 participating in the release of arachidonate in the inflammatory lesions (18Bingham C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Reverse Transcription-PCR—c-myc expression in MCSF-activated human monocytes was assessed by reverse transcription-PCR using a commercially available kit (RT-PCR high-Plus, Toyobo). Primers used were human c-myc 5′-GCCAAGCTCGTCTCAGAGAAG and 5′-CAGAAGGTGATCCAGACTCTG, spanning exon 2 and exon 3 of the human c-myc genome. Real-time PCR was performed with a LightCycler instrument (Roche Diagnostics) using a commercially available kit (LightCycler RNA amplification kit SYBR Green I, Roche Diagnostics). The level of c-myc mRNA was expressed as a relative value to glyceraldehydes 3-phosphate dehydrogenase mRNA. cPLA2 Ablation by RNA-mediated Interference and Antisense Oligonucleotide—The RNA-mediated ablation of endogenous cPLA2 in MCSF-activated human monocytes was performed essentially described as previously (19Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschi T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar). A 21-nucleotide small interfering RNA duplex with 3′-dTdT overhangs corresponding to human cPLA2 mRNA (5′-CCUGACGUUUCAGAGCUGATT and 5′-TTGGACUGCAAAGUCUCGACU) was synthesized (Japan Bio Services). RNA-mediated interference transfection was performed using Oligofectamine reagent (Invitrogen). Harvested human peripheral blood monocytes were stimulated with MCSF for 24 h and then transfected once using the manufacturer's protocol (Invitrogen). The ablation of endogenous cPLA2 in human monocytes was also performed using antisense oligonucleotide, which was complementary to nucleotide 219-238 of human cPLA2 (5′-GTGCTGGTAAGGATCTAT) (20Locati M. Lamorte G. Luini W. Introna M. Bernasconi S. Mantovani A. Sozziani S. J. Biol. Chem. 1996; 271: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The mis-sense oligomer was used for the control (5′-GTGCTCCTAAGTTTCTAT). The phosphorothioate-modified oligonucleotides were synthesized and purified by high performance liquid chromatography (Sigma). Subcellular Localization of cPLA2—To identify the sequence(s) regulating subcellular distribution of cPLA2, we created serial deletion mutants and assayed their localization by indirect immunostaining and confocal microscopy. Deletions of the C-terminal amino acids up to but not including the putative NES (PLLLLTP), which resides in the N-terminal region of the PH-like domain, did not affect the distribution of cPLA2 (Fig. 1, A-D). Further deletion of the C-terminal 547 amino acids, cPLA2-(1-202), resulted in nuclear localization of the protein (Fig. 1, A and E). cPLA2 distributed diffusely in the nucleus and cytoplasm when CV-1 cells were transfected with a C-terminal-truncated cPLA2 (amino acids 493-749) (Fig. 1, A and F). Extension of the truncation to the amino acid 202, cPLA2-(202-749), however, resulted in exclusively cytoplasmic localization (Fig. 1, A and G). We obtained similar results with 293T cells. Replacement of all four leucines with alanines in amino acids 263-269 in a truncated (1-294) (Fig. 1, A and H) and a full-length (1-794) (Fig. 1, A and I) cPLA2 or complete deletion of this sequence (not shown) resulted in predominant nuclear localization of the proteins in CV-1 cells. Collectively, these findings suggest that the mid-portion of cPLA2 protein directs the protein to the cytoplasm, probably via the nuclear export signal (PLLLLTP). In Vivo Interaction of cPLA2 and B-Myb—Because the cPLA2 protein does not harbor any apparent classical NLS, cPLA2 might be directed toward the nucleus by a partner protein(s). We, therefore, investigated a possible adapter protein(s) that binds to cPLA2 and drives the protein into the nucleus. Physical or functional interactions have been suggested between cPLA2 and proteins involved in transcriptional regulation (12Pawliczak R. Han C. Huang X.L. Demetris J. Shelhamer J.H. Wu T. J. Biol. Chem. 2002; 277: 33153-33163Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21Flati V. Haque S.J. Williams B.R.G. EMBO J. 1996; 15: 1566-1571Crossref PubMed Scopus (53) Google Scholar). Therefore, we tested the possibility that B-Myb, another protein that regulates transcription of several genes, may play a role as an adopter protein for nuclear transport of cPLA2. B-Myb is a potential candidate for this adapter protein, since it is ubiquitously expressed in tissues and is involved in cell growth control, differentiation, and cancer (15Oh I.H. Reddy E.P. Oncogene. 1999; 18: 3017-3033Crossref PubMed Scopus (420) Google Scholar). Furthermore, B-Myb interacts with other transcriptional factors and modulates their biological functions (22Tashiro S. Takemoto Y. Handa H. Ishii S. Oncogene. 1995; 10: 1699-1707PubMed Google Scholar). To test this hypothesis we first determined the subcellular distribution of cPLA2 after co-transfection with vectors containing either wild-type or mutant B-Myb protein. As described above, the wild-type full-length cPLA2-(1-749) predominantly localizes in the cytoplasm (Fig. 1B). Surprisingly, cPLA2 redistributes into the nucleus and colocalizes with B-Myb in the nucleus when the wild-type B-Myb is co-expressed (Fig. 2A). In contrast, the wild-type cPLA2 remains in the cytoplasm of cells expressing NLS-deleted B-Myb (B-Myb NLS) (Fig. 2B), suggesting that the NLS of B-Myb is required for the nuclear entry of cPLA2-B-Myb complexes. Similarly, truncated cPLA2 that is cytoplasmic when expressed alone translocates into the nucleus after co-expression with the wild-type B-Myb but not with NLS-mutated B-Myb (not shown). In contrast, co-expression of c-Myb, another member of the Myb family functioning as a transcriptional transactivator, does not direct cPLA2 into the nucleus (Fig. 2C), indicating that interaction between cPLA2 and B-Myb is specific. We next examined in vivo interaction between cPLA2 and B-Myb by immunoprecipitating cPLA2 followed by Western blotting with monoclonal antibody specific for B-Myb after transfection of 293T cells with the wild-type cPLA2 tagged with FLAG (Fig. 2F). In this condition, cPLA2 coimmunoprecipitated with endogenous B-Myb. A reciprocal experiment using FLAG-specific antibodies for immunoprecipitation of the full-length cPLA2 followed by Western blotting for B-Myb confirmed in vivo interaction of cPLA2 and B-Myb (Fig. 2G). We detected B-Myb protein in cPLA2 immunoprecipitates from 293T cells overexpressed with the N- or C-terminal-truncated cPLA2 (Fig. 2H), suggesting that the B-Myb protein can interact with both the N- and C-terminal regions of cPLA2. Nuclear Colocalization of cPLA2 and B-Myb in Human Macrophages—We ask next whether the specific interaction between cPLA2 and B-Myb and subsequent entry of cPLA2 into the nucleus might have any biological relevance. To address this question, we examined cPLA2-B-Myb interaction and the subcellular distributions of these proteins in human monocytes. MCSF up-regulates cPLA2 protein levels and intrinsic enzymatic activity in human monocytes (16Nakamura T. Lin L.L. Kharbanda S. Knopf J. Kufe D. EMBO J. 1992; 11: 4917-4922Crossref PubMed Scopus (102) Google Scholar). In resting human monocytes, cPLA2 was exclusively cytoplasmic, and the endogenous B-Myb protein level was very low in the nucleus (Fig. 3, A and C). MCSF treatment resulted in a significant nuclear redistribution of cPLA2, which colocalizes with B-Myb (Fig. 3, B and C). The interaction was confirmed by Western blotting using antibodies specific for cPLA2 of immunoprecipitates isolated by antibodies specific for B-Myb (Fig. 3D). A reciprocal experiment using antibodies specific for cPLA2 with immunoprecipitation and antibodies specific for B-Myb with Western blotting also showed an in vivo interaction of these proteins (not shown). In vitro cPLA2 enzyme assay showed that the nuclear fraction of resting monocytes contained a greater activity than the cytoplasmic fraction (Fig. 3E), indicating that a significant part of cPLA2 resides in the cytoplasmic fraction. The nuclear redistribution of cPLA2 was associated with a 2.6-fold increase in cPLA2 enzymatic activity in the nuclear fraction from MCSF-activated monocytes. In contrast, MCSF treatment resulted in only a 40% increase in cPLA2 activity in the cytoplasmic fraction, consistent with the notion that MCSF-induced terminal differentiation of human monocytes is associated with selective induction of cPLA2 activity in the nucleus. Regulation of B-Myb-dependent Gene Expression by cPLA2—B-Myb participates in regulation of several genes, each of which has an important role in cell growth. For example, the c-myc gene bears a putative B-Myb-responsive element on its promoter (23Nakagoshi H. Kanei-Ishii C. Sawazaki T. Mizuguchi G. Ishii S. Oncogene. 1992; 7: 1233-1240PubMed Google Scholar). Therefore, we tested whether nuclear cPLA2 could affect the B-Myb-dependent c-myc gene expression. To this end we measured the B-Myb-dependent reporter gene activity in CV-1 cells transiently expressing the pmycCAT reporter in the presence or absence of coexpressed wild-type cPLA2 (Fig. 4A). As expected, transient expression of B-Myb activated the reporter gene activity. Interestingly, coexpression of the full-length cPLA2-(1-749) greatly inhibits the B-Myb-dependent reporter gene activity to the control level. These findings suggest that cPLA2 negatively regulates c-myc gene expression in a B-Myb-dependent manner. However, it is unclear how the interaction between cPLA2 and B-Myb would be effective. To explore this, we next asked whether the action of B-Myb would be influenced by changes in levels of cPLA2. To test this, we transfected CV-1 cells with varying doses of plasmid containing full-length cPLA2 and then monitored the B-Myb-dependent activation of c-myc reporters. We found that cPLA2 inhibited B-Myb-dependent c-myc expression in a dose-dependent manner (Fig. 4B). These findings suggest that cPLA2 can inhibit the transcriptional activity of M-Myb. Up-regulation of c-Myc in Macrophages from cPLA2-null Mice—To investigate further the biological relevance of cPLA2-B-Myb interaction we examined c-Myc protein levels in cells that lack cPLA2 activity. To this end we used mice with disrupted cPLA2 gene (cPLA2-/- mice) (14Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (645) Google Scholar). Peritoneal macrophages were isolated from these mice and were then monitored for the c-Myc protein levels in the nucleus and cytoplasm. Peritoneal macrophages from cPLA2+/+ mice showed substantial amounts of cPLA2 in the cytoplasm and much smaller but detectable levels of cPLA2 in the nuclei (Fig. 5A). The absence of immunoreactive cPLA2 protein and cPLA2 transcripts (not shown) of peritoneal macrophages confirmed the disruption of the cPLA2 gene in the cells from cPLA2-/- mice. Peritoneal macrophages from cPLA2+/+ and cPLA2-/- mice contained equivalent quantities of B-Myb protein, suggesting that disruption of the cPLA2 gene does not affect the B-Myb protein levels in these cells. However, peritoneal macrophages from cPLA2-/- mice showed a substantial level of c-Myc protein in the nucleus, whereas c-Myc protein was barely detectable in macrophages from cPLA2+/+ mice. Next, we tested whether the restoration of cPLA2 expression could affect the c-Myc protein level in cPLA2-/- mouse peritoneal macrophages. We obtained low levels of cPLA2 after transfection in these primary cells, whereas the c-Myc expression was inhibited to 69% of the levels in non-transfected cPLA2-/- cells (Fig. 5B). We further examined the effects of cPLA2 inhibition in normal human peripheral blood monocytes. When the cPLA2 expression was specifically inhibited by using antisense oligonucleotide or small interfering RNA in MCSF-activated human monocytes, the c-myc expression was up-regulated in these cells (Fig. 5, C-E). Collectively, these results suggest that cPLA2 interacts directly with B-Myb to enter into the nucleus and thereby functions as an inhibitory factor for the c-Myc activity. A causative relationship was found between cPLA2 activation and UV irradiation and apoptosis (24Gresham A. Masferrer J. Chen X. Leal-Khouri S. Pentland A.P. Am. J. Physiol. 1996; 270: C1037-C1050Crossref PubMed Google Scholar, 25Pilane C.M. LaBelle E.F. J. Cell. Physiol. 2002; 191: 191-197Crossref PubMed Scopus (18) Google Scholar). Therefore, we assessed the lack of cPLA2 on cell survival after DNA-damaging agents using lung fibroblasts isolated from cPLA2+/+ and cPLA2-/- mice. The growth rate was similar between cPLA2+/+ and cPLA2-/- cells without treatment (data not shown). However, compared with cPLA2+/+ cells, cPLA2-/- cells showed decreased numbers of cells 48 h after treatment with UV irradiation or H2O2 (Table I). In contrast, cPLA2-/- cells were more resistant to ionizing radiation than cPLA2+/+ cells, as assessed 96 h after irradiation. Taken together these results suggest that the lack of cPLA2 may affect cell growth and/or apoptosis after DNA damage, depending on the types of cells and damaging.Table ISurvival and growth rates after treatment with UV irradiation, H2O2, or X irradiationPercent survivalUVH2O2X irradiation%cPLA2+/+24.951.244.7cPLA2−/−3.514.4100.0 Open table in a new tab Our report has demonstrated thus far undefined physical associations of cPLA2 and B-Myb transcriptional factor that facilitates redistribution of cPLA2-B-Myb complexes into the nucleus. cPLA2 protein does not harbor any apparent intrinsic NLS, whereas B-Myb protein has multiple functional NLSs (26Takemoto Y. Tashiro S. Handa H. Ishii S. FEBS Lett. 1994; 350: 55-60Crossref PubMed Scopus (17) Google Scholar). Furthermore, a deletion of the NLS from the B-Myb protein results in a mutant protein that cannot direct cPLA2 protein into the nucleus. Therefore, B-Myb protein may serve as an adapter protein for the nuclear entry of cPLA2. We also found that the physical interaction of cPLA2 and B-Myb proteins is specific and c-Myb, which is another Myb family transcriptional factor, does not direct cPLA2 into the nucleus. More importantly, our results suggest that, after binding to B-Myb protein, nuclear cPLA2 may participate in the transcriptional regulation of B-Myb-dependent expression of c-myc gene. It is surprising that cPLA2 is not confined to the cytoplasm but is redistributed into the nucleus to serve as a regulator for B-Myb-dependent gene expression. Originally, cPLA2 was considered to translocate onto the cytoplasmic membrane after appropriate stimuli to release arachidonic acid from membrane phospholipids (3Clark J.D. Lin L.L. kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar). However, accumulating evidence suggests that cPLA2 can translocate from the cytosolic compartment to the nuclear membrane (2Schievella A.R. Regier M.K. Smith W.L. Lin L.L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 4Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The ATP depletion may contribute to the nuclear redistribution of cPLA2 (27Sheridan A.M. Sapirstein A. Lemieux N. Martin B.D. Kim D.K. Bonventre J.V. J. Biol. Chem. 2001; 276: 29899-29905Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Although the localization of downstream enzyme 5-lipoxygenase and cyclooxygenase in the nuclear membrane (6Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 7Chen X.S. Zhang Y.Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Gilroy D.W. Colville-Nash P.R. Willis D. Chivers J. Paul-Clark M.J. Willoughby D.A. Nature Med. 1999; 5: 698-701Crossref PubMed Scopus (1123) Google Scholar, 9Hanaka T. Shimizu T Izumi T. Biochem. J. 2002; 361: 505-514Crossref PubMed Scopus (38) Google Scholar, 10Woods J.W. Coffey M.J. Brock T.G. Singer I.I. Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2046Crossref PubMed Scopus (160) Google Scholar) may explain in part the physiologic relevance of the redistribution of cPLA2 to the nuclear membrane, the definite role of the “nuclear” cPLA2 is not well understood. Recent findings that an acetyltransferase Tip60 interacted and colocalized with cPLA2 in the nucleus and that the introduction of protein complexes into the nucleus was associated with stimulation of apoptosis (28Sheridan A.M. Force T. Yoon H.J. O'Leary E. Choukroun G. Taheri M.R. Bonventre J.V. Mol. Cell. Biol. 2001; 21: 4470-4481Crossref PubMed Scopus (57) Google Scholar) strongly support the notion that cPLA2 plays additional roles in the nucleus other than its enzymatic activity at the nuclear membrane. Our results suggest that the c-myc gene is one of the major targets of cPLA2-B-Myb complexes in the nucleus. We also foundthatcPLA2-B-MybcomplexesinthenucleusinhibitB-Myb-dependent gene expressions of cdc25C, but the effect was minimal (data not shown). cPLA2 was reported to be involved in the induction of apoptosis caused by tumor necrosis factor (29Adam-Klages S. Schwandner R. Lüschen S. Ussat S. Kreder D. Krönke M. J. Immunol. 1998; 161: 5687-5694PubMed Google Scholar). Tumor necrosis factor is considered to transduce apoptotic signals from the cytoplasmic membrane to mitochondria. During these processes the 100-kDa cPLA2 is cleaved in a caspase 3-dependent fashion to a 70-kDa proteolytic fragment that loses its catalytic activity but contributes to apoptosis (30Wissing D. Mouritzen H. Egeblad M. Poirier G.G. Jaäättela M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5073-5077Crossref PubMed Scopus (183) Google Scholar, 31Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Although we have shown in the present study that the nuclear cPLA2 exhibited in vitro enzyme activity, the enzyme activity may be inhibited or masked in the nucleus by an adaptor protein such as B-Myb and Tip60. The c-myc protooncogene encodes a transcriptional factor that participates in the regulation of cellular proliferation and apoptosis (32Adachi S. Obaya A.J. Han Z. Ramos-Desimone N. Wyche J.H. Sedivy J.M. Mol. Cell. Biol. 2001; 21: 4929-4937Crossref PubMed Scopus (87) Google Scholar, 33Amati B. Nat. Cell Biol. 2001; 3: 112-113Crossref PubMed Scopus (36) Google Scholar). In particular, the Myc protein can induce S-phase entry and apoptosis by independent mechanisms (34Leone G. Sears R. Huang E. Rempel R. Nuckolls F. Park C.H. Giangrande R. Wu L. Saavedra H.I. Field S.J. Thompson M.A. Yang H. Fujiwara Y. Greenberg M.E. Orkin S. Smith C. Nevins J.R. Mol. Cell. 2001; 8: 105-113Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). As shown in cells from cPLA2-/- mice, the absence of cPLA2 may lead to an increased expression of c-Myc protein. Therefore, cPLA2 might exert an antiproliferative function in the nucleus by inhibiting aberrant expression of c-Myc and other proteins whose expressions are B-Myb-dependent. Most recently, Pawliczak et al. (12Pawliczak R. Han C. Huang X.L. Demetris J. Shelhamer J.H. Wu T. J. Biol. Chem. 2002; 277: 33153-33163Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) raised possibility that cPLA2 may regulate peroxisome proliferator activated receptor-mediated gene transcription, implying further a role in the regulation of genes that participate in differentiation and apoptosis. However, cPLA2 may exert its regulatory function for peroxisome proliferator-activated receptor-dependent gene transcription in the cytoplasm, since this property was apparent only after stimulation by calcium ionophore A23187, where cPLA2 did not enter the nucleus. In this report we showed that lack of cPLA2 affects cell survival after DNA damages (Table I). In this context it is noteworthy that Haq et al. (35Haq S. Kilter H. Michael A. Tao J. O'Leary E. Sun X.M. Walters B. Bhattacharya K. Chen X. Cui L. Andreucci M. Rosenzweig A. Guerrero J.L. Patten R. Liao R. Molkentin J. Picard M. Bonventre J.V. Force T. Nat. Med. 2003; 9: 944-951Crossref PubMed Scopus (76) Google Scholar) show that deletion of cPLA2 sustained activation of insulin-like growth factor signaling and promoted striated muscle growth. Therefore, we postulate the possibility that cPLA2 has separate roles for cell proliferation and apoptosis in the different compartment of the cell; the protein participates in stimulation of cell death in the cytoplasm, whereas the protein plays a role in the inhibition of proliferation and cell death in the nucleus.