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Transforming Growth Factor-β1 Increases the Expression of Lectin-like Oxidized Low-Density Lipoprotein Receptor-1
Biochemical and Biophysical Research Communications 272, 357–361 (2000) doi:10.1006/bbrc.2000.2778, available online at http://www.idealibrary.com on
Transforming Growth Factor- 1 Increases the Expression of Lectin-like Oxidized Low-Density Lipoprotein Receptor-1 Manabu Minami, Noriaki Kume, 1 Hiroharu Kataoka, Masafumi Morimoto, Kazutaka Hayashida, Tatsuya Sawamura,* Tomoh Masaki,* and Toru Kita Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; and *National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan
Received April 27, 2000
Lectin-like oxidized low-density lipoprotein (OxLDL) receptor-1 (LOX-1) is a novel cell-surface receptor for Ox-LDL, which can be expressed by vascular endothelial cells, smooth muscle cells, and macrophages. On the other hand, transforming growth factor (TGF)- 1, which plays crucial roles in vascular remodeling and the pathogenesis of atherosclerosis, has been shown to inhibit expression of class A scavenger receptors and CD36 in macrophages. Here we provide the evidence that TGF- 1 (0.1–10 ng/mL) induces LOX-1 protein and mRNA expression in both bovine aortic endothelial cells and smooth muscle cells in a doseand time-dependent fashion, probably at the transcriptional level. TGF- 1 also upregulates LOX-1 mRNA expression in murine peritoneal macrophages. Thus TGF- 1 can highly induce LOX-1 expression in vascular endothelial cells, smooth muscle cells, and macrophages, suggesting that TGF- 1 appears one of the key regulators that modulates expression of scavenger receptors. © 2000 Academic Press Key Words: atherosclerosis; LOX-1; oxidized lowdensity lipoprotein; TGF- 1; vascular remodeling.
Oxidized low-density lipoprotein (Ox-LDL) has been shown to play key roles in the pathogenesis of atherosclerosis (1–3). Ox-LDL and its lipid constituents have been shown to transcriptionally induce endothelial genes relevant to atherogenesis (4, 5). Furthermore, uptake of Ox-LDL in macrophages and vascular smooth muscle cells by receptor-mediated endocytosis appears to be involved in cellular accumulation of cholesteryl ester, and subsequent foam cell transformation in these cell types. Several different molecules, so far, have been identified to support cellular uptake of 1
To whom correspondence should be addressed. Fax: 81-75-7513574. E-mail: [email protected].
Ox-LDL (6 –12). Lectin-like Ox-LDL receptor-1 (LOX-1) is a novel receptor for Ox-LDL, which was initially identified in cultured vascular endothelial cells (13). LOX-1 can bind, internalize and proteolytically degrade Ox-LDL but not significant amount of acetylated LDL (14). In addition to modified lipoproteins, LOX-1 can also bind and mediate phagocytosis of aged red blood cells and apoptotic cells (15). Recent studies have shown that LOX-1 is also expressed by macrophages (16, 17) and vascular smooth muscle cells (18, 19). With regard to the factors that affect the expression of LOX-1, inflammatory stimuli, such as tumor necrosis factor-␣ (TNF-␣) and phorbol ester (20), fluid shear stress (21) and angiotensin II (22, 23) can induce its expression in endothelial cells. In addition, we have recently demonstrated that LOX-1 expression is upregulated in macrophages and smooth muscle cells in advanced atherosclerotic plaques, as well as endothelial cells covering early atherosclerotic lesions, by immunohistochemistry using human carotid atherosclerotic specimens (24). These data suggest that LOX-1 may play important roles in both endothelial activation and foam cell transformation of macrophages and vascular smooth muscle cells in atherogenesis. TGF- 1 is a multifunctional cytokine which affects cell growth, differentiation and migration, as well as extracellular matrix production (25, 26). Recent studies have demonstrated that TGF- 1 also regulates vascular cell apoptosis, and thus remodels vascular walls (27, 28). In human atherosclerotic plaques as well as restenosis lesions after angioplasty, TGF- 1 is highly expressed by vascular cells within lesions (29). And its expression in the vessel wall is also upregulated in animal models of diabetes mellitus and hypertension (30, 31). These data suggest that TGF- 1 may play crucial roles in various pathophysiological settings of vascular diseases including atherosclerosis. Concern-
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ing the regulation of other scavenger receptors, TGF- 1 has been shown to inhibit expression of class A scavenger receptors (32) and CD36 (33) in macrophages. In the present study, therefore, we sought to define whether TGF- 1 can induce or downregulate expression of LOX-1 in vascular cells. Here, we provide evidence that TGF- 1 can highly induce LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells and macrophages. MATERIALS AND METHODS Reagents and cells. DMEM was obtained from Nissui and fetal bovine serum (FBS) was from Irvine Scientific. Recombinant human TGF- 1 was purchased from R&D systems. Bovine aortic endothelial cells (BAEC) were isolated by scraping the inner surface of bovine aortas with a razor blade and cultured in DMEM containing 10% (vol/vol) FBS. Bovine aortic smooth muscle cells (BASMC) were isolated by an explant method, cultured in DMEM containing 10% (v/v) FBS. Murine peritoneal macrophages were harvested from peritoneal lavage of female DDY mice, which had been injected intraperitoneally with 2 mL of 3% thioglycollate broth (Difco) in PBS. Cells were suspended in DMEM containing 10% (v/v) FBS at a density of 3 ⫻ 10 6 cells/mL, allowed to adhere by 2-h incubation, washed with PBS to remove nonadherent cells, and then incubated overnight. Immunoblot analysis. Cells were washed with PBS and lysed in a buffer containing 50 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.01% bromophenol blue. After heated at 98° for 5 min, equal protein concentrations of the cell lysates were subjected to SDS– polyacrylamide (10%) gel electrophoresis and transferred onto nitrocellulose membranes (Hybond-N ⫹, Amersham) by electroblotting. After preincubation with blocking reagent (PBS containing 0.1% Tween 20 and 5% [w/v] nonfat dry milk) for 2 h at room temperature, blotted membranes were incubated with an anti-bovine LOX-1 mouse monoclonal antibody for 2 h at room temperature, followed by washing twice with the blocking reagent. Membranes were then incubated with a horseradish peroxidase-conjugated anti-mouse IgG (Amersham) for 1 h at room temperature, washed twice in PBS containing 0.04% Tween 20, and visualized by ECL Western blotting detection reagents (Amersham). Densitometric analysis was performed to measure the amounts of LOX-1 protein by use of NIH Image. Northern blot analysis. Total cellular RNA was isolated by TRIZOL Reagent (GIBCO BRL). Equal amounts of total RNA were subjected to electrophoresis through 1% agarose gels containing formaldehyde, and transferred onto nitrocellulose membranes (PROTRAN, Schleicher & Schuell). Membranes were hybridized with an XhoI/PstI fragment of bovine LOX-1 cDNA or XhoI fragment of
FIG. 1. Immunoblot analyses of LOX-1 induced by TGF- 1 in BAEC (A) and BASMC (B). Cells were incubated for 8 h with the indicated concentrations of TGF- 1 and subjected to immunoblot analyses. One of 3 independent experiments is shown.
FIG. 2. Time course of TGF- 1-induced LOX-1 expression in BAEC (A) and BASMC (B). Cells were incubated with 1 ng/mL of TGF- 1 for the indicated time periods; subsequently, cells were lysed, and immunoblot analyses were performed. A representative result from 2 independent experiments is shown.
mouse LOX-1 cDNA which had been labeled with (␣- 32P) dCTP (DuPont-New England Nuclear) using random hexanucleotide primers (DNA labeling kit, Pharmacia). Densitometric analysis was performed to measure the amounts of LOX-1 mRNA by use of NIH Image.
RESULTS TGF- 1 Induces LOX-1 Expression in Cultured Vascular Endothelial Cells as Well as Smooth Muscle Cells To examine whether TGF- 1 induces LOX-1 expression at the protein level, immunoblot analyses were performed in BAEC and BASMC. Figure 1 demonstrates that treatment with TGF- 1 increased LOX-1 protein expression in both cell types. Expression of LOX-1 protein was peaked at 1 ng/mL of TGF- 1 in both BAEC and BASMC; 4.2-fold and 2.8-fold increases, respectively. Time-course of TGF- 1-induced LOX-1 protein expression showed that increased levels of LOX-1 were detectable within 4 h and remained elevated after 20 h in both BAEC and BASMC (Fig. 2). To determine whether enhanced expression of LOX-1 protein by TGF- 1 depends upon induced expression of LOX-1 mRNA, Northern blot analyses were performed. As shown in Fig. 3, TGF- 1 treat-
FIG. 3. Dose–response relationship of LOX-1 mRNA upregulation elicited by TGF- 1 in BAEC (A) and BASMC (B). Cells were treated with the indicated concentrations of TGF- 1 for 4 h, and Northern blot analyses were performed to evaluate the amounts of LOX-1 mRNA. One of 2 independent experiments is shown.
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FIG. 4. Time course of LOX-1 mRNA expression induced by TGF- 1 in BAEC (A) and BASMC (B). Cells were incubated with 1 ng/mL of TGF- 1 for the indicated time periods, and levels of LOX-1 mRNA were measured by Northern blot analyses. A representative result from 2 similar experiments is shown.
ment increased the amount of LOX-1 mRNA; treatment with 1 ng/mL of TGF- 1 resulted in 4.9-fold and 3.0-fold increases in LOX-1 mRNA in BAEC and BASMC, respectively. Time-course experiments showed that increased levels of LOX-1 mRNA were detectable within 2 h, peaked at 4 – 6 h and remained elevated after 12 h, in response to 1 ng/mL of TGF- 1 (Fig. 4). TGF- 1-Induced LOX-1 Expression Requires de Novo RNA Synthesis To examine whether TGF- 1-induced expression of LOX-1 depends upon enhanced transcription of the LOX-1 gene, actinomycin D, an inhibitor of de novo RNA synthesis, was added to BAEC and BASMC 30 min before the application of TGF- 1. Pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by TGF- 1 (Fig. 5). These results suggest that TGF- 1 may stimulate transcription of the LOX-1 gene.
FIG. 5. Dependence of TGF- 1-induced LOX-1 expression on de novo RNA synthesis. After pretreatment with or without actinomycin D (2.5 or 5 g/mL) for 30 min, BAEC (A) or BASMC (B) were treated with 1 ng/mL of TGF- 1 for 4 h, levels of LOX-1 mRNA were evaluated by Northern blot analyses. A representative result from 2 similar experiments is shown.
FIG. 6. Effect of TGF- 1 on LOX-1 mRNA induction in murine peritoneal macrophages. After treatment with the indicated concentrations of TGF- 1 for 4 h (A), or after treatment with 1 ng/mL of TGF- 1 for the indicated time periods (B), total RNA was isolated and Northern blot analyses were performed. A representative result from 3 independent experiments is shown.
TGF- 1 Increases LOX-1 mRNA Levels in Murine Peritoneal Macrophages To test whether TGF- 1 also induces LOX-1 expression in macrophages, Northern blot analyses were performed. As shown in Fig. 6, TGF- 1 elevated LOX-1 mRNA levels, in a dose- and time-dependent fashion, in murine peritoneal macrophages. Densitometric analyses showed that treatment with 10 ng/mL of TGF- 1 for 4 h resulted in a 4.7-fold increase in the LOX-1 mRNA level. These results demonstrate that LOX-1 expression in macrophages also can be induced by TGF- 1. DISCUSSION Receptor-mediated endocytosis of Ox-LDL appears to play key roles in the pathogenesis of atherosclerosis. Although multiple receptors have been identified for atherogenic Ox-LDL, LOX-1, type II membrane glycoprotein with C-type lectin-like structure at C-terminus, may also be involved in this process (13). LOX-1 expression has been documented not only in endothelial cells but also in macrophages (16, 17) and smooth muscle cells (18, 19). In addition, LOX-1 expression is upregulated in macrophages and smooth muscle cells as well as endothelial cells in human atherosclerotic plaques (24). These data suggest that LOX-1 may play important roles in the pathogenesis of atherosclerosis. The present study provides evidence, for the first time, that TGF- 1 can dramatically induce LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells, and macrophages. TGF- 1 is an important secretary product of endothelial cells, smooth muscle cells, macrophages and platelets (34 –36). Previous studies have shown that TGF- 1(32), as well as TNF-␣ (37, 38), inhibited expression of class A scavenger receptors in macrophages. And recently, TGF- 1 has been also demonstrated to decrease expression of CD36, the type B
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scavenger receptor in THP-1 macrophages (33). LOX-1 expression, in contrast, can be upregulated by TNF-␣ (20) and TGF- 1; therefore, LOX-1 expression may be upregulated in atherosclerosis and inflammation, when and where these proinflammatory cytokines are secreted and class A scavenger receptor and CD36 expression is suppressed. Thus, LOX-1 might be a major scavenger receptor in these pathological settings. TGF- 1 appears to stimulate transcription of the LOX-1 gene, since pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by TGF- 1. It has been recently demonstrated that Smad 3 and Smad 4, which play key roles in intracellular signaling of TGF-, can interact directly with 12-O-tetradecanoyl-13-acetate-responsive elements (TREs) as well as AP-1, and activate TGF--dependent gene transcription in the presence or absence of AP-1 (39). Because consensus nucleotide sequences corresponding to TRE are found in the 5⬘-flanking region of the LOX-1 gene (40), the Smad-TRE pathway might be involved in LOX-1 gene transcription elicited by TGF 1. Further studies, however, would be necessary to elucidate this point. In summary, the present study demonstrates that TGF- 1 can highly induce LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells and macrophages. Further studies related to the pathophysiological relevance of TGF- 1-induced LOX-1 expression may provide new insights into the pathogenesis of atherosclerosis and other vascular diseases.
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ACKNOWLEDGMENTS This work has been supported, in part, by research grants from the Minister of Education, Science and Culture of Japan (No. 118338008 to N.K., and Nos. 08407026, 09281103, and 09281104 to T.K.). We thank Kyoto Red Cross Blood Center for gifts of unused human plasma. We also thank Ms. Kumiko Kanai for her assistance with the cell cultures.
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Transforming Growth Factor- 1 Increases the Expression of Lectin-like Oxidized Low-Density Lipoprotein Receptor-1 Manabu Minami, Noriaki Kume, 1 Hiroharu Kataoka, Masafumi Morimoto, Kazutaka Hayashida, Tatsuya Sawamura,* Tomoh Masaki,* and Toru Kita Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; and *National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan
Received April 27, 2000
Lectin-like oxidized low-density lipoprotein (OxLDL) receptor-1 (LOX-1) is a novel cell-surface receptor for Ox-LDL, which can be expressed by vascular endothelial cells, smooth muscle cells, and macrophages. On the other hand, transforming growth factor (TGF)- 1, which plays crucial roles in vascular remodeling and the pathogenesis of atherosclerosis, has been shown to inhibit expression of class A scavenger receptors and CD36 in macrophages. Here we provide the evidence that TGF- 1 (0.1–10 ng/mL) induces LOX-1 protein and mRNA expression in both bovine aortic endothelial cells and smooth muscle cells in a doseand time-dependent fashion, probably at the transcriptional level. TGF- 1 also upregulates LOX-1 mRNA expression in murine peritoneal macrophages. Thus TGF- 1 can highly induce LOX-1 expression in vascular endothelial cells, smooth muscle cells, and macrophages, suggesting that TGF- 1 appears one of the key regulators that modulates expression of scavenger receptors. © 2000 Academic Press Key Words: atherosclerosis; LOX-1; oxidized lowdensity lipoprotein; TGF- 1; vascular remodeling.
Oxidized low-density lipoprotein (Ox-LDL) has been shown to play key roles in the pathogenesis of atherosclerosis (1–3). Ox-LDL and its lipid constituents have been shown to transcriptionally induce endothelial genes relevant to atherogenesis (4, 5). Furthermore, uptake of Ox-LDL in macrophages and vascular smooth muscle cells by receptor-mediated endocytosis appears to be involved in cellular accumulation of cholesteryl ester, and subsequent foam cell transformation in these cell types. Several different molecules, so far, have been identified to support cellular uptake of 1
To whom correspondence should be addressed. Fax: 81-75-7513574. E-mail: [email protected].
Ox-LDL (6 –12). Lectin-like Ox-LDL receptor-1 (LOX-1) is a novel receptor for Ox-LDL, which was initially identified in cultured vascular endothelial cells (13). LOX-1 can bind, internalize and proteolytically degrade Ox-LDL but not significant amount of acetylated LDL (14). In addition to modified lipoproteins, LOX-1 can also bind and mediate phagocytosis of aged red blood cells and apoptotic cells (15). Recent studies have shown that LOX-1 is also expressed by macrophages (16, 17) and vascular smooth muscle cells (18, 19). With regard to the factors that affect the expression of LOX-1, inflammatory stimuli, such as tumor necrosis factor-␣ (TNF-␣) and phorbol ester (20), fluid shear stress (21) and angiotensin II (22, 23) can induce its expression in endothelial cells. In addition, we have recently demonstrated that LOX-1 expression is upregulated in macrophages and smooth muscle cells in advanced atherosclerotic plaques, as well as endothelial cells covering early atherosclerotic lesions, by immunohistochemistry using human carotid atherosclerotic specimens (24). These data suggest that LOX-1 may play important roles in both endothelial activation and foam cell transformation of macrophages and vascular smooth muscle cells in atherogenesis. TGF- 1 is a multifunctional cytokine which affects cell growth, differentiation and migration, as well as extracellular matrix production (25, 26). Recent studies have demonstrated that TGF- 1 also regulates vascular cell apoptosis, and thus remodels vascular walls (27, 28). In human atherosclerotic plaques as well as restenosis lesions after angioplasty, TGF- 1 is highly expressed by vascular cells within lesions (29). And its expression in the vessel wall is also upregulated in animal models of diabetes mellitus and hypertension (30, 31). These data suggest that TGF- 1 may play crucial roles in various pathophysiological settings of vascular diseases including atherosclerosis. Concern-
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ing the regulation of other scavenger receptors, TGF- 1 has been shown to inhibit expression of class A scavenger receptors (32) and CD36 (33) in macrophages. In the present study, therefore, we sought to define whether TGF- 1 can induce or downregulate expression of LOX-1 in vascular cells. Here, we provide evidence that TGF- 1 can highly induce LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells and macrophages. MATERIALS AND METHODS Reagents and cells. DMEM was obtained from Nissui and fetal bovine serum (FBS) was from Irvine Scientific. Recombinant human TGF- 1 was purchased from R&D systems. Bovine aortic endothelial cells (BAEC) were isolated by scraping the inner surface of bovine aortas with a razor blade and cultured in DMEM containing 10% (vol/vol) FBS. Bovine aortic smooth muscle cells (BASMC) were isolated by an explant method, cultured in DMEM containing 10% (v/v) FBS. Murine peritoneal macrophages were harvested from peritoneal lavage of female DDY mice, which had been injected intraperitoneally with 2 mL of 3% thioglycollate broth (Difco) in PBS. Cells were suspended in DMEM containing 10% (v/v) FBS at a density of 3 ⫻ 10 6 cells/mL, allowed to adhere by 2-h incubation, washed with PBS to remove nonadherent cells, and then incubated overnight. Immunoblot analysis. Cells were washed with PBS and lysed in a buffer containing 50 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, and 0.01% bromophenol blue. After heated at 98° for 5 min, equal protein concentrations of the cell lysates were subjected to SDS– polyacrylamide (10%) gel electrophoresis and transferred onto nitrocellulose membranes (Hybond-N ⫹, Amersham) by electroblotting. After preincubation with blocking reagent (PBS containing 0.1% Tween 20 and 5% [w/v] nonfat dry milk) for 2 h at room temperature, blotted membranes were incubated with an anti-bovine LOX-1 mouse monoclonal antibody for 2 h at room temperature, followed by washing twice with the blocking reagent. Membranes were then incubated with a horseradish peroxidase-conjugated anti-mouse IgG (Amersham) for 1 h at room temperature, washed twice in PBS containing 0.04% Tween 20, and visualized by ECL Western blotting detection reagents (Amersham). Densitometric analysis was performed to measure the amounts of LOX-1 protein by use of NIH Image. Northern blot analysis. Total cellular RNA was isolated by TRIZOL Reagent (GIBCO BRL). Equal amounts of total RNA were subjected to electrophoresis through 1% agarose gels containing formaldehyde, and transferred onto nitrocellulose membranes (PROTRAN, Schleicher & Schuell). Membranes were hybridized with an XhoI/PstI fragment of bovine LOX-1 cDNA or XhoI fragment of
FIG. 1. Immunoblot analyses of LOX-1 induced by TGF- 1 in BAEC (A) and BASMC (B). Cells were incubated for 8 h with the indicated concentrations of TGF- 1 and subjected to immunoblot analyses. One of 3 independent experiments is shown.
FIG. 2. Time course of TGF- 1-induced LOX-1 expression in BAEC (A) and BASMC (B). Cells were incubated with 1 ng/mL of TGF- 1 for the indicated time periods; subsequently, cells were lysed, and immunoblot analyses were performed. A representative result from 2 independent experiments is shown.
mouse LOX-1 cDNA which had been labeled with (␣- 32P) dCTP (DuPont-New England Nuclear) using random hexanucleotide primers (DNA labeling kit, Pharmacia). Densitometric analysis was performed to measure the amounts of LOX-1 mRNA by use of NIH Image.
RESULTS TGF- 1 Induces LOX-1 Expression in Cultured Vascular Endothelial Cells as Well as Smooth Muscle Cells To examine whether TGF- 1 induces LOX-1 expression at the protein level, immunoblot analyses were performed in BAEC and BASMC. Figure 1 demonstrates that treatment with TGF- 1 increased LOX-1 protein expression in both cell types. Expression of LOX-1 protein was peaked at 1 ng/mL of TGF- 1 in both BAEC and BASMC; 4.2-fold and 2.8-fold increases, respectively. Time-course of TGF- 1-induced LOX-1 protein expression showed that increased levels of LOX-1 were detectable within 4 h and remained elevated after 20 h in both BAEC and BASMC (Fig. 2). To determine whether enhanced expression of LOX-1 protein by TGF- 1 depends upon induced expression of LOX-1 mRNA, Northern blot analyses were performed. As shown in Fig. 3, TGF- 1 treat-
FIG. 3. Dose–response relationship of LOX-1 mRNA upregulation elicited by TGF- 1 in BAEC (A) and BASMC (B). Cells were treated with the indicated concentrations of TGF- 1 for 4 h, and Northern blot analyses were performed to evaluate the amounts of LOX-1 mRNA. One of 2 independent experiments is shown.
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FIG. 4. Time course of LOX-1 mRNA expression induced by TGF- 1 in BAEC (A) and BASMC (B). Cells were incubated with 1 ng/mL of TGF- 1 for the indicated time periods, and levels of LOX-1 mRNA were measured by Northern blot analyses. A representative result from 2 similar experiments is shown.
ment increased the amount of LOX-1 mRNA; treatment with 1 ng/mL of TGF- 1 resulted in 4.9-fold and 3.0-fold increases in LOX-1 mRNA in BAEC and BASMC, respectively. Time-course experiments showed that increased levels of LOX-1 mRNA were detectable within 2 h, peaked at 4 – 6 h and remained elevated after 12 h, in response to 1 ng/mL of TGF- 1 (Fig. 4). TGF- 1-Induced LOX-1 Expression Requires de Novo RNA Synthesis To examine whether TGF- 1-induced expression of LOX-1 depends upon enhanced transcription of the LOX-1 gene, actinomycin D, an inhibitor of de novo RNA synthesis, was added to BAEC and BASMC 30 min before the application of TGF- 1. Pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by TGF- 1 (Fig. 5). These results suggest that TGF- 1 may stimulate transcription of the LOX-1 gene.
FIG. 5. Dependence of TGF- 1-induced LOX-1 expression on de novo RNA synthesis. After pretreatment with or without actinomycin D (2.5 or 5 g/mL) for 30 min, BAEC (A) or BASMC (B) were treated with 1 ng/mL of TGF- 1 for 4 h, levels of LOX-1 mRNA were evaluated by Northern blot analyses. A representative result from 2 similar experiments is shown.
FIG. 6. Effect of TGF- 1 on LOX-1 mRNA induction in murine peritoneal macrophages. After treatment with the indicated concentrations of TGF- 1 for 4 h (A), or after treatment with 1 ng/mL of TGF- 1 for the indicated time periods (B), total RNA was isolated and Northern blot analyses were performed. A representative result from 3 independent experiments is shown.
TGF- 1 Increases LOX-1 mRNA Levels in Murine Peritoneal Macrophages To test whether TGF- 1 also induces LOX-1 expression in macrophages, Northern blot analyses were performed. As shown in Fig. 6, TGF- 1 elevated LOX-1 mRNA levels, in a dose- and time-dependent fashion, in murine peritoneal macrophages. Densitometric analyses showed that treatment with 10 ng/mL of TGF- 1 for 4 h resulted in a 4.7-fold increase in the LOX-1 mRNA level. These results demonstrate that LOX-1 expression in macrophages also can be induced by TGF- 1. DISCUSSION Receptor-mediated endocytosis of Ox-LDL appears to play key roles in the pathogenesis of atherosclerosis. Although multiple receptors have been identified for atherogenic Ox-LDL, LOX-1, type II membrane glycoprotein with C-type lectin-like structure at C-terminus, may also be involved in this process (13). LOX-1 expression has been documented not only in endothelial cells but also in macrophages (16, 17) and smooth muscle cells (18, 19). In addition, LOX-1 expression is upregulated in macrophages and smooth muscle cells as well as endothelial cells in human atherosclerotic plaques (24). These data suggest that LOX-1 may play important roles in the pathogenesis of atherosclerosis. The present study provides evidence, for the first time, that TGF- 1 can dramatically induce LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells, and macrophages. TGF- 1 is an important secretary product of endothelial cells, smooth muscle cells, macrophages and platelets (34 –36). Previous studies have shown that TGF- 1(32), as well as TNF-␣ (37, 38), inhibited expression of class A scavenger receptors in macrophages. And recently, TGF- 1 has been also demonstrated to decrease expression of CD36, the type B
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scavenger receptor in THP-1 macrophages (33). LOX-1 expression, in contrast, can be upregulated by TNF-␣ (20) and TGF- 1; therefore, LOX-1 expression may be upregulated in atherosclerosis and inflammation, when and where these proinflammatory cytokines are secreted and class A scavenger receptor and CD36 expression is suppressed. Thus, LOX-1 might be a major scavenger receptor in these pathological settings. TGF- 1 appears to stimulate transcription of the LOX-1 gene, since pretreatment with actinomycin D completely abolished LOX-1 mRNA induction elicited by TGF- 1. It has been recently demonstrated that Smad 3 and Smad 4, which play key roles in intracellular signaling of TGF-, can interact directly with 12-O-tetradecanoyl-13-acetate-responsive elements (TREs) as well as AP-1, and activate TGF--dependent gene transcription in the presence or absence of AP-1 (39). Because consensus nucleotide sequences corresponding to TRE are found in the 5⬘-flanking region of the LOX-1 gene (40), the Smad-TRE pathway might be involved in LOX-1 gene transcription elicited by TGF 1. Further studies, however, would be necessary to elucidate this point. In summary, the present study demonstrates that TGF- 1 can highly induce LOX-1 expression in cultured vascular endothelial cells, smooth muscle cells and macrophages. Further studies related to the pathophysiological relevance of TGF- 1-induced LOX-1 expression may provide new insights into the pathogenesis of atherosclerosis and other vascular diseases.
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ACKNOWLEDGMENTS This work has been supported, in part, by research grants from the Minister of Education, Science and Culture of Japan (No. 118338008 to N.K., and Nos. 08407026, 09281103, and 09281104 to T.K.). We thank Kyoto Red Cross Blood Center for gifts of unused human plasma. We also thank Ms. Kumiko Kanai for her assistance with the cell cultures.
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