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Low density lipoprotein receptor-dependent prostaglandin synthesis in Swiss 3 T 3 cells stimulated by platelet-derived growth factor ( prostaglandin E 2 / acetylated low density lipoprotein / fluorescent low density lipoprotein / chloroquine )
| Content Provider | Semantic Scholar |
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| Author | Habenicht, Andreas J. R. Dresel, Hans Alois Goerig, Matthias Weber, J. John, Annamma Ross, Russell T. Schettler, G. |
| Abstract | We studied the effects of human plasma lipoproteins on the synthesis of prostaglandin (PG) E2 in Swiss 3T3 mouse fibroblasts. Quiescent cells, maintained in medium deficient in both platelet-derived growth factor (PDGF) and lipoproteins, synthesized less than 8 ng ofPGE2 per 106 cells per 22 hr, and this rate did not change in response to the addition of lipoproteins. In contrast, PDGF-stimulated cells, incubated in medium deficient in lipoproteins, synthesized 45-110 ng of PGE2 per 106 cells during the same period of time, and this rate increased 2to 5-fold in the presence of added low density lipoproteins (LDL). This stimulatory effect of LDL seemed to depend on LDL receptor-mediated binding, uptake, and degradation of the lipoproteins because: (s) both LDL and very low density lipoproteins were active, whereas high density lipoproteins were not; (il) low concentrations of LDL were effective; (iii) the effect of native LDL was blocked by acetylation of the LDL; (iv) PDGF increased both the expression of LDL receptors and the cellular uptake ofLDL; (v) chloroquine blocked the effect ofLDL but not that ofexogenous arachidonic acid. These results provide evidence that the LDL pathway is critically linked to PG synthesis in PDGF-stimulated cells. In recent studies we observed stimulatory effects of human platelet-derived growth factor (PDGF) on the metabolism of arachidonic acid and prostaglandins (PGs) in Swiss 3T3 mouse fibroblasts (1-3). Early PDGF-dependent activation of the phospholipase C/diacylglycerol lipase pathway caused liberation of arachidonic acid from cellular phospholipids (1). Concomitantly the growth factor activated cyclooxygenase, the key enzyme of PG synthesis (3). Both arachidonic acid liberation (1) and activation of cyclooxygenase (3) appeared to be required for PG synthesis. In our earlier studies (3) we also noticed that PDGF had stimulatory effects on PG synthesis from exogenous unesterified arachidonic acid. We therefore decided to determine whether Swiss 3T3 cells utilize physiological carriers of arachidonic acid such as plasma lipoproteins for PG synthesis. Our approach was to maintain Swiss 3T3 cells in culture medium deficient in both PDGF and lipoproteins and then study the synthesis ofPGE2 as a function of added PDGF and plasma lipoproteins. In the study described below we addressed the following questions: Do plasma lipoproteins affect PG synthesis in Swiss 3T3 cells? Does PDGF affect lipoprotein-dependent PG synthesis? Does the effect of lipoproteins require action of the low density lipoprotein (LDL) receptor? MATERIALS AND METHODS Materials. [3H]PGE2 (165 Ci/mmol; 1 Ci = 37 GBq) was obtained from New England Nuclear; Dulbecco's modified Eagle's medium and calf serum, from GIBCO; standard PGs, from Seragen (Boston, MA); antibodies, from the Institute Pasteur (Paris, France); and all other materials, from Sigma Chemie (Munich, Federal Republic of Germany). Plasma Proteins and PDGF. Calf plasma-derived lipoprotein-deficient serum (PDLDS) was prepared as described (4). PDGF was prepared as reported (5), plasma lipoproteins were prepared according to Havel et al. (6), and acetylated LDL was prepared according to Basu et al. (7). LDL was labeled with the fluorophore 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate (DiI) as described by Pitas et al. (8). Cell Culture. Cells were plated at a density of 1.25 x 10W cells in 35-mm Costar plates (Costar, Cambridge, MA) containing 1.5 ml of culture medium supplemented with PDLDS protein at 1.25 mg/ml. On days 2 and 4 the cells were fed with the same medium. On day 4, experimental solutions were added as described in the figure texts. PDGF was dissolved in 50 a.l of 10 mM acetic acid (carrier) before addition to quiescent cells (1-3). LDL was dissolved in 50 A.l of Dulbecco's phosphate-buffered saline (carrier). Assays, Flow Cytometry, and Ligand Blotting of LDL Receptor. The concentration of PGE2 was determined by radioimmunoassay as described (3). Protein was determined by the method of Lowry et al. (9). Flow cytometry analyses were performed on a cytofluorograph 30 L (Ortho Instruments) equipped with a low-power argon laser (10). Samples containing 106 cells per ml were analyzed at a flow rate of200 cells per sec. The results are displayed as two-parameter frequency distributions. To visualize the LDL receptor, Swiss 3T3 cells were maintained in PDLDS at a density of 1 x 106 cells per 100-mm dish as described in Cell Culture. On day 5, PDGF was added. Twenty-two hours later the cell monolayer was washed with phosphate-buffered saline; 21.6 x 106 cells of each group were disrupted by sonification in 1.2 ml of buffer (50 mM Tris/150 mM NaCl/1 mM CaCl2, pH 6.8) and centrifuged at 107 gamin. The pellet was suspended in buffer containing 40 mM n-octyl 6-D-glucopyranoside to solubilize the LDL receptor (11). After separation of 125 ;kg protein by 0.1% sodium dodecyl sulfate/9o polyacrylamide gel electrophoresis, the proteins were electroblotted onto nitrocellulose Abbreviations: PDGF, platelet-derived growth factor; PG, prostaglandin; VLDL, very low density lipoprotein(s); LDL, low density lipoprotein(s); HDL, high density lipoprotein(s); PDLDS, plasma-derived lipoprotein-deficient serum; DiI, 1,1'-dioctadecyl3,3,3',3'-tetramethylindocarbocyanine perchlorate. 1344 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 1345 paper, incubated with LDL at 40 ,.g of protein per ml, and then processed, using a horseradish peroxidase-linked antibody to LDL to identify the receptor-bound lipoprotein (12). |
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| Alternate Webpage(s) | http://www.pnas.org/content/83/5/1344.full.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
