12/15-Lipoxygenase Translocation Enhances Site-specific Actin Polymerization in Macrophages Phagocytosing Apoptotic Cells
2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês
10.1074/jbc.m011276200
ISSN1083-351X
AutoresYury I. Miller, Mi-Kyung Chang, Colin Funk, James R. Feramisco, Joseph L. Witztum,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells. The enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells. lipoxygenase peroxisome proliferator-activated receptor fetal bovine serum hydroxyeicosatetraenoic acid hydroxyoctadecadienoic acid fluorescein isothiocyanate 12/15-Lipoxygenase (LO)1is a member of the LO family of enzymes that insert peroxyl groups into double bonds of free and phospholipid-bound polyunsaturated fatty acids. The exact role of these enzymes in biological processes has remained elusive, but increasingly evidence has accumulated that they play important roles in specific cellular functions. For example, 15-LO activity in reticulocytes at the stage of organelle degradation may contribute to membrane destabilization and contribute to pore formation in intracellular membranes (1Kühn H. Brash A.R. J. Biol. Chem. 1990; 265: 1454-1458Abstract Full Text PDF PubMed Google Scholar, 2van Leyen K. Duvoisin R.M. Engelhardt H. Wiedmann M. Nature. 1998; 395: 392-395Crossref PubMed Scopus (255) Google Scholar). Fatty acid products of 12/15-LO are powerful agonists for the nuclear receptor PPAR-γ, which helps regulate glucose metabolism and adipocyte and macrophage differentiation and function (3Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. Witztum J.L. Funk C.D. Conrad D. Glass C.K. Nature. 1999; 400: 378-382Crossref PubMed Scopus (781) Google Scholar). A remarkable feature of 12/15-LO is that its expression is not constant during the cell life span but rather turns on at certain points during cell development. While circulating human monocytes do not express 15-LO, monocyte-derived macrophages exposed to interleukin-4 or interleukin-13 express 15-LO (4Conrad D.J. Kühn H. Mulkins M. Highland E. Sigal E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 217-221Crossref PubMed Scopus (349) Google Scholar, 5Nassar G.M. Morrow J.D. Roberts L.J. Lakkis F.G. Badr K.F. J. Biol. Chem. 1994; 269: 27631-27634Abstract Full Text PDF PubMed Google Scholar). In addition, mouse macrophages residing for a long time in the peritoneum (resident macrophages) also highly express the mouse homologue, 12/15-LO, although the pathway leading to 12/15-LO expression may differ somewhat from that which occurs with human monocytes (6Sendobry S.M. Cornicelli J.A. Welch K. Grusby M.J. Daugherty A. J. Immunol. 1998; 161: 1477-1482PubMed Google Scholar). Macrophages of atherosclerotic lesions express high levels of 15-LO (7Ylä-Herttuala S. Rosenfeld M.E. Parthasarathy S. Glass C.K. Sigal E. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6959-6963Crossref PubMed Scopus (405) Google Scholar), and recent evidence utilizing apoE−/−mice in which the 12/15-LO gene was disrupted demonstrated its importance in the pathogenesis of atherosclerosis (8Cyrus T. Witztum J.L. Rader D.J. Tangirala R. Fazio S. Linton M.F. Funk C.D. J. Clin. Invest. 1999; 103: 1597-1604Crossref PubMed Scopus (466) Google Scholar). Another characteristic of atherosclerotic tissue but not of normal vascular wall is the high abundance of apoptotic cells (9Kockx M.M. Herman A.G. Cardiovasc. Res. 2000; 45: 736-746Crossref PubMed Scopus (262) Google Scholar). This fact might reflect either an increased rate of formation of such cells, a decreased rate of clearance, for example by arterial macrophages, or both. In either case, phagocytosis and the degradation and metabolism of the ingested contents of dying cells are crucial for preventing the release of toxic cellular compounds and consequent inflammation. Inhibition of efficient phagocytosis would presumably lead to the accumulation of pro-inflammatory necrotic debris, plaque instability, and thrombogenesis. During the complex process of phagocytosis, major changes in the cytoskeleton of the cell occur leading to the formation of filopodia surrounding an apoptotic cell or a microorganism to be engulfed. Changes in actin polymerization play a vital role in this process. Remarkably, 12-LO products are found in many tumor cells and have been suggested to have effects on actin polymerization (10Rice R.L. Tang D.G. Haddad M. Honn K.V. Taylor J.D. Int. J. Cancer. 1998; 77: 271-278Crossref PubMed Scopus (26) Google Scholar) and cytoskeleton reorganization during cell transformation (10Rice R.L. Tang D.G. Haddad M. Honn K.V. Taylor J.D. Int. J. Cancer. 1998; 77: 271-278Crossref PubMed Scopus (26) Google Scholar, 11Tang D.G. Honn K.V. Adv. Exp. Med. Biol. 1997; 400: 349-361Crossref Scopus (16) Google Scholar). Therefore, it was tempting to propose that the activity of 12/15-LO in non-malignant cells, such as macrophages, was also related to a cytoskeleton function and to phagocytosis. Indeed, we now demonstrate that upon exposure to apoptotic cells, 12/15-LO translocates from the cytosol to sites of apoptotic cell binding and furthermore that actin polymerization itself is dependent on activity of 12/15-LO. Peritoneal macrophages were harvested from 8- to 10-week-old female mice, either Swiss Webster or C57BL/6 strains. The latter were strain-, age-, and sex-matched to 12/15-LO knockout mice. Resident or thioglycollate-elicited macrophages were plated in RPMI 1640 (BioWhittaker) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific). Murine fibroblast cell lines overexpressing either human 15-LO (clone 12) or β-galactosidase (LacZ) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) with 10% FBS and 0.2 mg/ml G418 (Calbiochem) to maintain selection (12Benz D.J. Mol M. Ezaki M. Mori-Ito N. Zelaan I. Miyanohara A. Friedmann T. Parthasarathy S. Steinberg D. Witztum J.L. J. Biol. Chem. 1995; 270: 5191-5197Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Thymocytes were harvested from the thymuses of 4-week-old mice of the same strain as used for macrophage isolation and treated with 1 μm dexamethasone in 10% FBS/RPMI 1640 for 4 h to induce apoptosis (13Chang M.K. Bergmark C. Laurila A. Hörkkö S. Han K.H. Friedman P. Dennis E.A. Witztum J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6353-6358Crossref PubMed Scopus (397) Google Scholar). The appearance of condensed nuclei was a marker for apoptosis. We previously demonstrated that concentrations up to 20 μm of the specific lipoxygenase inhibitor PD 146176 (a gift from J. Cornicelli of Parke-Davis) were non-toxic for macrophages (14Scheidegger K.J. Butler S. Witztum J.L. J. Biol. Chem. 1997; 272: 21609-21615Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Cytochalasin D was from Sigma, and latrunculin A was from Molecular Probes. 13(S)-Hydroxyoctadecadienoic acid (13(S)-HODE), 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), and linoleic acid were from Cayman Chemical. Cells were lysed on the plate with 5% SDS in phosphate-buffered saline. Protein content was determined with a BCA kit (Pierce), and 15–100 μg of the cell lysate was run on a pre-cast 4–12% gradient polyacrylamide gel (Novex) and then transferred to a nitrocellulose membrane (Millipore). The membrane was blocked with 5% non-fat milk and incubated with a protein A-purified polyclonal guinea pig anti-rabbit 15-LO antibody (7Ylä-Herttuala S. Rosenfeld M.E. Parthasarathy S. Glass C.K. Sigal E. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6959-6963Crossref PubMed Scopus (405) Google Scholar). This antibody cross-reacts not only with human 15-LO (7Ylä-Herttuala S. Rosenfeld M.E. Parthasarathy S. Glass C.K. Sigal E. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6959-6963Crossref PubMed Scopus (405) Google Scholar) but also with mouse 12/15-LO; it stained the band of 75 kDa, typical for 12/15-LO. It did not yield any such band when lysates from 12/15-LO−/− mice were used. Guinea pig preimmune IgG produced no specific staining. Macrophages plated overnight on coverslips were fixed with 3.7% paraformaldehyde for 10 min at 37 °C, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 0.8 μg/ml Fc block (PharMingen) in 5% non-fat milk, 0.2% Triton X-100, stained for 30 min with the guinea pig anti-rabbit 15-LO antibody and for another 30 min with a rhodamine red-X-conjugated F(ab′)2 fragment donkey anti-guinea pig Ig G (H+L) antibody (Jackson ImmunoResearch). Alternatively, a guinea pig anti-rabbit ApoA1 antibody was used as a negative control. Filamentous actin (F-actin) was stained by addition of 1.5 μm FITC-conjugated phalloidin (Sigma) to the solution of the secondary antibody. Cytosol was labeled by incubation of live cells with 0.5 μm, 5-chloromethylfluorescein diacetate (CellTracker green CMFDA from Molecular Probes) in serum-free medium for 30 min followed by a 30-min incubation in the regular culture medium. This green staining remained in fixed cells. Cell nuclei were stained blue with 1 μg/ml Hoechst 33258 (Sigma) for 15 min. The coverslips were mounted on microscopic glass slides with ProLong antifade medium (Molecular Probes). Images were captured by deconvolution microscopy (15Agard D.A. Hiraoka Y. Shaw P. Sedat J.W. Methods Cell Biol. 1989; 30: 353-377Crossref PubMed Scopus (565) Google Scholar) using a DeltaVision deconvolution microscopic system operated by SoftWorx software (Applied Precision). Pixel intensities were kept in the linear response range of the digital camera. Optical sections through the samples were taken with increments of 0.2–0.5 μm depending on magnification. The images were deconvolved and examined either section by section or volume views were generated by combining areas of maximal intensity of each optical section with SoftWorx programs. Data Inspector application was used to quantitatively analyze the images. Adobe Photoshop 6.0 software was used to design figures. The relative content of F-actin in macrophages activated by addition of apoptotic thymocytes was assessed by flow cytometry as described in Ref. 16Howard T.H. Meyer W.H. J. Cell Biol. 1984; 98: 1265-1271Crossref PubMed Scopus (274) Google Scholar with some modification. In brief, at the end of incubation of the plated macrophages with apoptotic thymocytes, 1 volume of the solution containing 1.6 μm FITC-phalloidin, 18% paraformaldehyde, and 0.8% saponin (all from Sigma) was added to 3 volumes of the culture medium and incubated for an additional 10 min. Cells were then washed, scraped from the plate, filtered through a Nitex nylon mesh (Sefar America), and analyzed on a FACScan (Becton Dickinson). To examine the expression of cell-specific CD markers, the cells attached to the plate were gently scraped, incubated in suspension for 30 min with either a FITC-conjugated anti-CD80 antibody, a FITC-conjugated anti-CD3 antibody, or a phycoerythrin-conjugated anti-CD19 antibody (all from PharMingen), washed, and analyzed on the FACScan. Assays were performed as described previously (17Higgs H.N. Blanchoin L. Pollard T.D. Biochemistry. 1999; 38: 15212-15222Crossref PubMed Scopus (242) Google Scholar). This assay is based on the measurement of fluorescence intensity of pyrene covalently linked to actin, which increases when actin polymerizes. In brief, unlabeled and pyrene-labeled monomeric G-actin from rabbit muscle (kindly provided by K. Aman from the Salk Institute) at the ratio of 95:5 were diluted in G-buffer (2 mm Tris, pH 8.0, 0.2 mm ATP, 0.1 mmCaCl2, and 0.5 mm dithiothreitol) and then converted to Mg-actin by adding 0.1 volume of 10 mm EGTA and 1 mm MgCl2. Polymerization was initiated by addition of either macrophage lysates or 0.1 volume of 10× KMEI (500 mm KCl, 10 mm MgCl2, 10 mm EGTA, and 100 mm imidazole, pH 7.0). Lysates were prepared from macrophages scraped from the plate in a lysis buffer (2 mm Tris, pH 8.0, 1 mm EGTA, 0.2 mm MgCl2, and protease inhibitors mixture (Sigma)) by sonication and centrifugation at 10,000 ×g for 30 min. Protein concentration was measured using a BCA kit from Pierce. Spectra and time courses of pyrene fluorescence were measured on an LS50B luminescence spectrophotometer (PerkinElmer Life Sciences). When non-septic inflammation in mice is induced by intraperitoneal injection of thioglycollate, many monocytes are recruited into the peritoneum, where they differentiate into macrophages. Initially, these newly recruited, "elicited" macrophages express very little 12/15-LO. This was evident from Western blots of cell lysates made from elicited and resident macrophages. Relative to total cell protein, there was 10–15-fold less 12/15-LO expressed in the elicited macrophages as compared with the enzyme content in resident macrophages (data not shown). Immunocytochemical examination of the elicited macrophage population revealed two cell populations, either positive or negative for 12/15-LO staining (Fig.1 a). The 12/15-LO-positive cells (less than 10% of total) presumably originated from resident macrophages. The 12/15-LO-negative cells were probably newly recruited monocyte-macrophages that did not yet express the enzyme. This observation is in agreement with an earlier report on heterogeneity of elicited macrophages from immunodeficient mice (6Sendobry S.M. Cornicelli J.A. Welch K. Grusby M.J. Daugherty A. J. Immunol. 1998; 161: 1477-1482PubMed Google Scholar). To ensure that the thioglycollate-elicited cells that attached to the plate overnight were indeed macrophages and not other cell types, these cells were analyzed by flow cytometry for the presence of cell-specific markers. Ninety eight percent of the attached cells were positive for the macrophage marker CD80 and negative for the T-cell marker CD3 and the B-cell marker CD19. (Splenocytes, a mixed population of all the three cell types, were used as positive controls.) Thus, the majority of the plate-attached cells harvested from peritoneum were macrophages, and the difference in the 12/15-LO expression is probably a function of the stage of macrophage differentiation. When apoptotic thymocytes were incubated with the elicited macrophage population, we noted a striking difference between 12/15-LO-positive and -negative cells in the ability to bind and engulf apoptotic thymocytes (Fig. 1 a). Counting multiple microscopic fields confirmed that following a 15-min incubation, the 12/15-LO-expressing macrophages bound 20 times more apoptotic cells than the 12/15-LO-negative cells (cross-hatched columns in Fig.1 b). This ability of the 12/15-LO-positive cells to bind apoptotic cells was significantly reduced when the elicited macrophages were pretreated with the specific 12/15-LO inhibitor PD 146176 (18Sendobry S.M. Cornicelli J.A. Welch K. Bocan T. Tait B. Trivedi B.K. Colbry N. Dyer R.D. Feinmark S.J. Daugherty A. Br. J. Pharmacol. 1997; 120: 1199-1206Crossref PubMed Scopus (159) Google Scholar). The effect of PD 146176 was dose-dependent. A statistically significant decrease in apoptotic cells binding to 12/15-LO-positive elicited macrophages was observed already at 0.5 μm PD 146176. This result corresponds well to the previously reported IC50 values of 0.8 μm for the PD 146176 inhibition of 15-LO activity in cell culture (19Bocan T.M. Rosebury W.S. Mueller S.B. Kuchera S. Welch K. Daugherty A. Cornicelli J.A. Atherosclerosis. 1998; 136: 203-216Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Following a 1-h incubation, when most of the apoptotic thymocytes were already engulfed by the macrophages, the same tendency was observed (black columns in Fig. 1 b). The correlation between the phagocytic function of elicited macrophages and the 12/15-LO activity suggests a role for 12/15-LO in phagocytosis. To explore further a potential relationship of 12/15-LO to phagocytosis, we examined the localization of 12/15-LO and F-actin in resident macrophages, resting or phagocytosing apoptotic thymocytes. Nearly all (more than 95%) resident macrophages expressed 12/15-LO. In resident macrophages not exposed to apoptotic cells (Fig.2 a, a volume view, Fig.2 b, a 2-fold magnified optical section), 12/15-LO protein (red) was evenly distributed throughout the cytosol and did not colocalize with F-actin (green) at the cell surface. The three-dimensional intensity graphs below Fig. 2 b document the very different distributions of 12/15-LO and F-actin in the highlighted area. In contrast, in resident macrophages exposed to apoptotic thymocytes, 12/15-LO concentrated on the cell surfaces in general, and this was greatly enhanced at the sites where apoptotic cells were bound (Fig. 2, c and d). To show that the translocation of 12/15-LO toward the bound apoptotic cell at the periphery of the macrophage was specific and not just following general movement of cytosol, we labeled cytosol green with CellTracker, a dye that evenly binds to thiol groups in the cell (20Poot M. Kavanagh T.J. Kang H.C. Haugland R.P. Rabinovitch P.S. Cytometry. 1991; 12: 184-187Crossref PubMed Scopus (103) Google Scholar). The presence of 12/15-LO on the cell surface (red color) close to attached apoptotic thymocytes but the absence of the yellow color (that would have represented colocalization with CellTracker) suggest the specificity of the 12/15-LO translocation (Fig. 2, c andd). Accordingly, the intensity graphs below Fig.2 d show a different distribution of 12/15-LO and CellTracker in the highlighted area. Fig. 3 presents another example in which a resident macrophage is in close or partial contact with four different apoptotic cells simultaneously. Fig. 3 bdemonstrates staining for 12/15-LO (red) and Fig.3 c staining for F-actin (green). Fig.3 a is a merged view where the yellow color demonstrates 12/15-LO colocalization with the sites of actin polymerization. The yellow color is clearly more heavily concentrated in the vicinity of bound apoptotic cells. Quantification of the effect is provided by intensity maps of the whole cell (derived from the volume view) for each color. In general, the concentrations of 12/15-LO and F-actin on the surface of macrophages phagocytosing apoptotic cells were 3–6-fold higher than in resting macrophages (compare intensity scales in Figs. 2 and 3). This supports the generalized movement of both to the cell periphery. However, the intensity graph for 12/15-LO (below Fig. 3 b) also demonstrates increased 12/15-LO concentration at the sites of apoptotic cell binding, as compared with either cytosol or even other sites of the periphery of the cell. Specific 12/15-LO intensity in the areas of cell-cell contact was 3.11 ± 0.54/voxel as compared with 1.49 ± 0.73/voxel in all the rest of the cell perimeter (p < 0.001). The intensity maps derived from the highlighted area of a specific focal plane (below Fig. 3,e and f) show nearly identical patterns of 12/15-LO and F-actin distribution in the area of contact with an apoptotic cell, confirming their specific colocalization. We next examined the relationship between activity of 12/15-LO and F-actin function. To approach this question, we inhibited either the activity of 12/15-LO or the process of actin polymerization. Formation of F-actin was inhibited by treating macrophages with either cytochalasin D or latrunculin A, toxins that bind monomeric G-actin, thereby preventing formation of filamentous F-actin (21DeFife K.M. Jenney C.R. Colton E. Anderson J.M. FASEB J. 1999; 13: 823-832Crossref PubMed Scopus (56) Google Scholar, 22Couae M. Brenner S.L. Spector I. Korn E.D. FEBS Lett. 1987; 213: 316-318Crossref PubMed Scopus (653) Google Scholar). The images in Fig. 4, a–c show a non-treated resident macrophage caught in the process of phagocytosis of an apoptotic thymocyte. 12/15-LO has translocated to the surface where it appears to be interacting with the apoptotic cell. This same site has also been greatly enriched by F-actin (seeblack-white images in b and c, and intensity graphs below). Again, although there is clear translocation of the 12/15-LO to the periphery of the cell, a formal analysis of the intensity maps shows 12/15-LO-specific intensity of 5.40 ± 0.73/voxel at the site of apoptotic cell binding versus2.40 ± 1.16/voxel in the rest of cell perimeter (p < 0.001) providing additional evidence that 12/15-LO translocation is concentrated at sites of contact with apoptotic cells. The cytochalasin D treatment partially disrupted the actin polymerization (Fig. 4, d and f). Nevertheless, 12/15-LO translocation toward the bound apoptotic thymocyte did not seem to be impaired (Fig. 4, d and e). Latrunculin A treatment almost completely disrupted F-actin formation (Fig. 4, g and i), and even under these severe conditions 12/15-LO translocation to sites of apoptotic cell binding occurred (Fig. 4, g and h). These data suggest that actin polymerization is not a prerequisite for 12/15-LO translocation. Can 12/15-LO activity in turn affect the process of actin polymerization? In order to assess the level of polymerized actin in individual cells, we used a flow cytometry assay as described under "Experimental Procedures." A shift of the cell distribution histogram to the area of higher fluorescence intensity (e.g.to the right) reflects an increase in the level of F-actin. Such a shift was observed in resident macrophages in response to incubation with apoptotic thymocytes (Fig. 5,a and c). It was not due to the F-actin of internalized apoptotic thymocytes because the latter did not show any F-actin signal by flow cytometry (green histograms barely seen in left bottom corners of Fig. 5, a andc), and no F-actin staining was observed microscopically in the apoptotic cells (Figs. Figure 1, Figure 2, Figure 3, Figure 4). The F-actin response usually reached its peak in 10–20 min and then disappeared 40–60 min after the start of incubation (data not shown). Macrophages harvested from Swiss Webster mice (Fig. 5 a) generally responded with a higher level of polymerized actin than macrophages from C57BL/6 mice (compare Fig. 5, a versus c). Treatment of the resident macrophages with the 12/15-LO inhibitor PD 146176 prior to addition of the apoptotic cells blocked actin polymerization in the macrophages (Fig. 5 b). Finally, in macrophages harvested from 12/15-LO knockout mice (12/15-LO−/−), no change in the F-actin content in response to addition of apoptotic cells was observed (Fig. 5 d). The next set of experiments directly examined the effect of the products of 12/15-LO on actin polymerization, using an in vitro polymerization assay in the presence or absence of cell lysates. In the first set of experiments, actin polymerization was initiated by addition of cell lysates (Fig. 6 a). Whole cell lysates prepared from the 12/15-LO knockout macrophages had a limited ability to promote in vitro polymerization of G-actin (Fig.6 a, dotted line). In contrast, lysates from wild type macrophage had much higher nucleating and elongating activities as seen by a shorter lag phase before the start of elongation and an increased rate of elongation (a 130 ± 27% increase, p < 0.001; Fig. 6 a, solid line). Remarkably, addition of 13(S)-HODE (the oxidation product of linoleic acid) to the 12/15-LO−/− lysates significantly increased the elongation rate but did not affect the lag phase, indicating that 13(S)-HODE does not have a nucleating activity (Fig.6 a, dashed line). The same positive effect of 13(S)-HODE on actin elongation but not nucleation was also observed in an in vitro assay conducted in the absence of cell lysates, when actin polymerization was initiated by addition of KMEI instead of cell lysate (a 50 ± 18% increase,p < 0.01; Fig. 6 b, solid line). Preincubation of G-actin with non-oxidized linoleic did not show any sizable difference from the ethanol vehicle (Fig. 6 b, dashedand dotted lines). Because light scattering from cell lysates could have interfered with fluorescence measurements, emission spectra were recorded at the beginning and the end of the time courses. A multipeak analysis of a difference spectrum (Fig. 6 a, inset) shows a peak of 405.5 nm, which is fairly close to the peak of pyrene emission (406.8 nm) from F-actin in lysate-free samples (Fig.6 b, inset). Similar results were also observed in experiments with the products of 12/15-LO oxidation of arachidonic acid, 15(S)-HETE and 12(S)-HETE, although the effects were not as pronounced (data not shown). To determine if the relationship between LO activity and F-actin formation could also be observed in another cell type, we also examined spreading of fibroblasts, another function dependent on actin polymerization. For these studies we used murine cell lines stably overexpressing either human 15-LO (clone 12) or β-galactosidase (LacZ, control cells) (12Benz D.J. Mol M. Ezaki M. Mori-Ito N. Zelaan I. Miyanohara A. Friedmann T. Parthasarathy S. Steinberg D. Witztum J.L. J. Biol. Chem. 1995; 270: 5191-5197Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Plated clone 12 cells spread much faster than LacZ cells, and the presence of the 15-LO inhibitor PD 146176 in the medium delayed spreading of the clone 12 cells (Fig.7). These observations complement the hypothesis that 12/15-LO stimulates actin polymerization and that the functions of 12/15-LO may be more pleiotropic than only assisting macrophage phagocytic function. Many lines of evidence suggest an important role for 15-LO (and its 12/15-LO homologue in mice) in atherogenesis (7Ylä-Herttuala S. Rosenfeld M.E. Parthasarathy S. Glass C.K. Sigal E. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6959-6963Crossref PubMed Scopus (405) Google Scholar, 8Cyrus T. Witztum J.L. Rader D.J. Tangirala R. Fazio S. Linton M.F. Funk C.D. J. Clin. 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Both human and rabbit atherosclerotic lesions express 15-LO mRNA, protein, and enzymatic activity (7Ylä-Herttuala S. Rosenfeld M.E. Parthasarathy S. Glass C.K. Sigal E. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6959-69
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