The expression of extracellular-superoxide dismutase is increased by lysophosphatidylcholine in human monocytic U937 cells
Introduction
The occurrences of highly reactive oxygen species and their destruction by antioxidants are in equilibrium in healthy mammalian organisms, and disturbing this homeostasis causes numerous diseases. Highly reactive oxygen species, superoxide and other secondary generated species, can be produced by vascular cells and are involved in the initiation and progression of atherosclerosis. Low-density lipoprotein (LDL) is thought to become oxidatively modified within the arterial wall, where LDL particles are sequestered in a pro-oxidant environment, and oxidized LDL (oxLDL) plays an important role in atherogenesis [1], [2]. Several studies have demonstrated that oxLDL leads to the conversion of phosphatidylcholine to lysophosphatidylcholine (lysoPC) [3], which is known to induce the transcription of a variety of cellular genes [4], [5].
Superoxide dismutase (SOD) protects arteries and other tissues from the deleterious effects of reactive oxygen species by scavenging superoxide [6], [7]. There are three SOD isozymes in mammalian cells; copper, zinc-containing SOD (CuZn-SOD), manganese-containing SOD (Mn-SOD) and extracellular-SOD (EC-SOD). CuZn-SOD and Mn-SOD are intracellular SOD isozymes and are found predominantly in the cytoplasm and mitochondria, respectively. EC-SOD is a secretory, tetrameric glycoprotein with an affinity for heparin and glycosaminoglycans [8], and is the major SOD isozyme in the arterial wall, whereas CuZn-SOD and Mn-SOD are the major SOD isozymes in the extravascular tissues [7]. Due to the arterial wall is rich in connective tissue and EC-SOD is bound to the extracellular matrix [7], EC-SOD constitutes an important defense mechanism against superoxide in the arterial microenvironment. There has been substantial interest in the level of expression of EC-SOD in atherosclerotic vessels. EC-SOD is expressed in several human cell types such as fibroblasts [9], [10], [11], [12], smooth muscle cells (SMC) [7], [13], [14], and macrophages [15], but not in endothelial cells, epithelial cells, myelomas or lymphomas [9]. The synthesis of EC-SOD in human cells was reported to be highly responsible for various inflammatory cytokines, cyclic AMP, angiotensin II and heparin [10], [11], [12], [13], [14]. It has been shown that EC-SOD activity was higher in atherosclerotic lesions of WHHL rabbits than in normal aortas [15], and apo E-deficient mice [16]. EC-SOD was shown to be expressed by lipid-laden macrophages by immunohistochemical study in the above report [16]. Therefore, we investigated the change of EC-SOD expression in U937 human monocytic cells by lysoPC.
Section snippets
Reagents
L-α-lysoPC and RPMI 1640 medium were obtained from Sigma (St. Louis, MO, USA). Heparin-Sepharose CL-4B was purchased from Pharmacia Biotech (Uppsala, Sweden). TRIzol regent, M-MLV reverse transcriptase (M-MLV RT) and Taq DNA polymerase were obtained from GIBCO (Grand Island, NY, USA). LA Taq DNA polymerase was purchased from Takara Shuzo Co., Ltd. (Kyoto, Japan). U937 human myelomonocytic cells were purchased from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan).
Cell culture
U937 cells were maintained in
Upregulation of EC-SOD mRNA expression by lysoPC
The mRNA levels of EC-SOD in U937 cells were significantly increased by the incubation with more than 10 μg/ml lysoPC for 24 h (Fig. 1A). Incubation of the cells with 30 μg/ml lysoPC for 24 h did not affect on the proliferation and the total protein content of the cells (data not shown). The expression of EC-SOD mRNA was significantly increased by the incubation with lysoPC for 12 and 24 h at a concentration of 30 μg/ml (Fig. 1B).
mRNA expression of the other SOD isozymes
Next, we studied the changes in the mRNA expression of the SOD
Discussion
This study provides new information that the expression of EC-SOD mRNA and protein were increased by lysoPC, which is known to be an atherogenic substance, in human monocytic U937 cells. One of the earliest events in LDL oxidation is the hydrolysis of oxidatively modified phosphatidylcholine, which is mediated by phospholipase A2 and generates lysoPC [20]. LysoPC is a potent biological effector molecule able to induce adhesion molecules [21] and various growth factors that promote SMC migration
Acknowledgments
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports Science and Technology of Japan to T. Adachi.
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