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Am J Physiol Endocrinol Metab 292: E648 –E652, 2007. First published September 19, 2006; doi:10.1152/ajpendo.00084.2006.

Translational physiology

TRANSLATIONAL PHYSIOLOGY

Endothelin-1 decreases CD36 protein expression in vascular smooth muscle cells Ching Fai Kwok,1,3 Chi-Chang Juan,2,4 and Low-Tone Ho1,2,3,4 1 Division of Endocrinology and Metabolism, Department of Medicine, and 2Department of Medical Research and Education, Taipei Veterans General Hospital; and 3Faculty of Medicine and 4Institutes of Physiology and Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan

Submitted 19 February 2006; accepted in final form 13 September 2006

Kwok CF, Juan CC, Ho LT. Endothelin-1 decreases CD36 protein expression in vascular smooth muscle cells. Am J Physiol Endocrinol Metab 292: E648 –E652, 2007. First published September 19, 2006; doi:10.1152/ajpendo.00084.2006.—Recent studies have shown that CD36 plays important roles as a major scavenger receptor for oxidized low-density lipoproteins and as a crucial transporter for long-chain fatty acids. CD36 deficiency might be associated with insulin resistance and abnormal dynamics of long-chain fatty acids. Endothelin-1 (ET-1), which is synthesized and secreted by vascular endothelial cells, is the most potent endogenous vasoconstrictor known and also stimulates the proliferation of vascular smooth muscle cells (VSMCs) and thus is believed to play an important role in the development of various circulatory disorders, including hypertension and atherosclerosis. The aim of the present study was to investigate the regulatory effect of ET-1 on CD36 expression in cultured VSMCs. VSMCs were treated for different times (0 –24 h) with a fixed concentration (100 nM) of ET-1 or with different concentrations (0 –100 nM) for a fixed time (24 h); then CD36 expression was determined using Western blots. CD36 expression was significantly decreased by ET in a time- and dose-dependent manner. This inhibitory effect was prevented by the ETA receptor antagonist BQ-610 (10 ␮M) but not the ETB receptor antagonist BQ-788 (10 ␮M). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the tyrosine kinase-mediated and MAPK-mediated pathways. The inhibitory effect of ET-1 on CD36 protein expression was blocked by inhibition of tyrosine kinase activation by use of genistein (100 ␮M) and by the ERK inhibitor PD-98059 (75 ␮M) but not by the p38 MAPK inhibitor SB-203580 (20 ␮M). In conclusion, we have demonstrated that ET-1, acting via the ETA receptor, suppresses CD36 protein expression in VSMCs by activation of the tyrosine kinase and ERK pathways.

ATHEROSCLEROSIS AND CORONARY HEART DISEASE are the leading causes of mortality and morbidity in the developed world. During the atherosclerotic process, lipoproteins, such as lowdensity lipoprotein (LDL), are converted to oxidized-LDL (ox-LDL), which contains modified proteins and lipids (9). A family of membrane proteins, the scavenger receptors, recognizes and internalizes modified lipoproteins, making them susceptible to degradation. However, uncontrolled expression of scavenger receptors can lead to foam cell formation, one of

the major histopathological features of atherosclerosis (30). Although each scavenger receptor shows a broad specificity, clear preferences for certain ligands have been described (37). The CD36 scavenger receptor is specific for nitrated LDL and ox-LDL, the most atherogenic forms of modified LDL (31). CD36, originally identified as glycoprotein IV on platelets, is an 88-kDa integral membrane protein that has multiple ligands and is widely expressed in human tissues, including heart, skeletal muscles, adipose tissues, blood vessel walls, and intestines (2, 26, 42). CD36 expression is increased in macrophages in human atherosclerotic lesions (17, 30). Moreover, CD36 knockout mice exhibit a reduced uptake of modified LDL and a reduction in the occurrence of atherosclerosis (14). These results indicate that macrophage CD36 plays an important role in the development of atherosclerosis. However, the role of CD36 in vascular smooth muscle cells (VSMCs) in the pathogenesis of atherosclerosis is not as clear as that of macrophage. Finally, CD36 deficiency has been shown to be associated with insulin resistance (28). Recent studies have shown that CD36 is also a crucial transporter for long-chain fatty acids (LCFAs). Unlike sugars, amino acids, and nucleotides, LCFAs are apolar compounds and readily partition into the membrane, which adds complexity to the transport of LCFAs across the cellular membrane. However, cell studies have provided evidence for the involvement of high-affinity protein components in LCFA transport in adipose tissue (3, 4), liver (36, 40), skeletal muscle, and heart (27). This led to the identification of three major potential fatty acid (FA) transporting proteins, plasma membrane FA-binding protein (FABPpm), CD36 (FA translocase), and FA transport protein (FATP) (1, 18, 34, 38). Although evidence supporting a role for FATP and for FABPpm in FA metabolism has been obtained, CD36 plays the pivotal role. CD36 is a tightly regulated protein. Known regulatory mechanisms are at the level of mRNA, mediated by peroxisome proliferator-activated receptors (41), and at the translational level, mediated by glucose (16). Moreover, acute regulation of FA transport by insulin is accomplished at the protein level by translocation of CD36 from an intracellular store to the plasma membrane (25). All three regulatory mechanisms appear to be relevant to the development of diseases and their associated complications.

Address for reprint requests and other correspondence: Address for reprint requests and other correspondence: C. F. Kwok, Div. of Endocrinology and Metabolism, Taipei Veterans General Hospital, Taipei, Taiwan.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

smooth muscle cell; atherosclerosis

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Translational physiology ET-1 DECREASES CD36 EXPRESSION IN VSMCS

Endothelin-1 (ET-1), synthesized and secreted by vascular endothelial cells, is by far the most potent endogenous vasoconstrictor known (43). It also stimulates DNA synthesis in VSMCs and thus is suspected to play an important role in the development of various circulatory disorders, including hypertension and atherosclerosis (29). After being secreted from endothelial cells, ET-1 binds to specific receptors on nearby VSMCs and perhaps on VSMCs at other distal sites (35). Two distinct types of ET receptors, ETA and ETB, have been cloned and sequenced from bovine and rat tissues (8, 33). ETA receptors predominate in VSMCs and mediate vasoconstriction and cellular proliferation (13, 19). Recently, we (22) demonstrated overexpression of vascular ETA receptors in a fructoseinduced hypertensive rat model, which further supports the role of ET-1 and its receptors in hypertension. The aim of the present study was to investigate the effect of ET-1 on CD36 expression and its underlying regulatory mechanisms. Since both CD36 and ET-1 are involved in the pathology of atherosclerosis, this was expected to provide valuable information about the regulation of CD36 expression and to contribute to a better understanding of the development of atherosclerosis.

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Western blots. After treatment, the cells were washed twice with PBS and then lysed with 0.5 ml of lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 0.5% Nonidet P-40). Protein concentrations of lysates were measured using a Bio-Rad Protein Assay Kit (Hercules, CA), and then 100 ␮g of lysate proteins were separated by SDS-PAGE using a 7.5% polyacrylamide gel and electroblotted onto a PVDF membrane. After blocking of nonspecific binding by incubation for 1 h with 5% nonfat milk in PBS containing 0.05% Tween 20 (PBST), the membrane was incubated for 1 h at room temperature with polyclonal antibodies against CD36 or ␣-tubulin (both from Santa Cruz Biotechnology, Santa Cruz, CA), washed four times with PBST, incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat antirabbit IgG antibody (Santa Cruz Biotechnology), and then washed four times with PBST. To measure ERK phosphorylation, antibodies against phosphorylated or total ERK1/2 (Santa Cruz Biotechnology) were used. Bound antibody was visualized using a Western Blot Chemiluminescence Reagent Plus Kit and the intensity of the bands quantified using a densitometer (Molecular Dynamics, Sunnyvale, CA). Statistical analysis. Experiments were repeated at least three times. The results are expressed as means ⫾ SD. Statistical significance was assessed by one-way analysis of variance or Student’s t-test, a value of P ⬍ 0.05 being considered statistically significant. RESULTS

MATERIALS AND METHODS

Materials. ET-1 was purchased from the Peptide Institute (Osaka, Japan), BQ-610 and BQ-788 from Phoenix Pharmaceuticals (Belmont, CA), and genistein, PD-098059, and SB-203580 from BIOMOL International (Plymouth Meeting, PA). Experimental design. To explore the regulatory effect of ET-1 on CD36 protein expression, VSMCs were treated for different times (0 –24 h) with a fixed concentration (100 nM) of ET-1 or with different concentrations (0 –100 nM) for a fixed time (24 h), and then CD36 protein was measured using Western blots. To identify the receptor subtype(s) involved in the ET-1-mediated inhibition of CD36 protein expression, VSMCs were incubated for 1 h in the presence or absence of 10 ␮M BQ-610 (an ETA receptor antagonist) or 10 ␮M BQ-788 (an ETB receptor antagonist); then 100 nM ET-1 was added, and the cells were incubated for a further 24 h. To explore the underlying mechanisms of ET-1 action, VSMCs were preincubated for 1 h in the presence or absence of a tyrosine kinase inhibitor (genistein, 100 ␮M), an ERK inhibitor (PD-98059, 75 ␮M), or a p38 MAP kinase inhibitor (SB-203580, 20 ␮M) and then with 100 nM ET-1 in the continued presence or absence of these inhibitors for a further 24 h. The cell content of CD36 was then determined. Culture of VSMCs. This was performed as described previously (24). Briefly, male Sprague-Dawley rats (150 –200 g body wt) were decapitated, the thoracic aortas sterilely dissected out, and the adventitia and intima completely removed. The medial layers were then sliced into 2 ⫻ 2-mm squares, which were transferred to gelatincoated 60-mm diameter tissue culture dishes and incubated in DMEM low-glucose medium containing 100 U/ml penicillin and 100 ␮g/ml streptomycin (all from GIBCO-BRL, Gaithersburg, MD) and 10% fetal bovine serum (Biowest, Nuaille´, France) in a 37°C humidified incubator with an atmosphere of 95% air-5% CO2. Between days 5 and 7 after explant isolation, VSMCs started to migrate out of the explants and proliferate. At day 14, the explants were removed using fine forceps and the cells trypsinized and subcultured at a ratio of 1:3 into other dishes. Confluent VSMC cultures showed the characteristic “hill and valley” growth pattern and were able to form multilayers in culture. The cells were then harvested by treatment with 0.05% trypsin in 0.2% EDTA in serum-free medium and seeded in gelatincoated tissue culture dishes. Cells from passages 5–15 were used in these studies. Before each experiment, cells were incubated for 8 h in the absence of serum, using DMEM low-glucose medium. AJP-Endocrinol Metab • VOL

ET-1 decreases CD36 protein expression in VSMCs. As shown in Fig. 1, a significant decrease (⬃20%) in CD36 expression was observed after 6 h of incubation with ET-1, maximal inhibition (⬃50%) being seen at 12 h, and maintained for ⱖ24 h. To examine whether the effect was dose dependent, VSMCs were incubated for 24 h with various concentrations of ET-1 (0 –100 nM), and the results showed that 1 nM ET-1

Fig. 1. Time-dependent effect of endothelin-1 (ET-1) on CD36 protein expression. vascular smooth muscle cells (VSMCs) were incubated in serum-free medium for 8 h and then in the presence or absence of 100 nM ET-1 for various times (0 –24 h), and CD36 protein content was measured by Western blotting. Results are means ⫾ SD of 3 separate experiments. *P ⬍ 0.05 vs. time 0 control.

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ET-1 DECREASES CD36 EXPRESSION IN VSMCS

induced significant inhibition of CD36 protein expression (⬃30% inhibition vs. control group), maximal inhibition of ⬃50% being seen at 10 nM (Fig. 2). Effect of ET receptor antagonists on ET-1-mediated inhibition of CD36 protein expression. To examine the contribution of the two ET receptors to the ET-1-mediated inhibition of CD36 expression, VSMCs were pretreated for 1 h with the ETA receptor antagonist BQ-610 (10 ␮M) or the ETB receptor antagonist BQ-788 (10 ␮M); then 100 nM ET-1 was added, and the cells were incubated for 24 h. As shown in Fig. 3, BQ-610 or BQ-788 alone had no effect on CD36 protein expression, but BQ-610 completely prevented the inhibitory effect of ET-1 on CD36 expression, whereas BQ-788 had no effect. These results demonstrate that the inhibition of CD36 expression by ET-1 in VSMCs is mediated through the ETA receptor. Effects of a tyrosine kinase inhibitor and MAP kinase inhibitors on ET-1-mediated inhibition of CD36 protein expression. To further explore the signaling mechanism by which ET-1 suppressed CD36 protein expression, VSMCs were pretreated with inhibitors of tyrosine kinase, ERK, or p38 MAP kinase, and their effect on ET-1-mediated inhibition of CD36 expression was evaluated. As shown in Fig. 4, pretreatment with a tyrosine kinase inhibitor (genistein, 100 ␮M) or an ERK inhibitor (PD-98059, 75 ␮M), but not a p38 MAP kinase inhibitor (SB-203580, 20 ␮M), completely blocked the effect. These findings indicate that activation of tyrosine kinase and ERK, but not p38 MAP kinase, is necessary for the suppressive effect of ET-1 on CD36 protein expression in VSMCs. Effects of ET-1 on ERK phosphorylation. Western blots were used to determine whether ET-1 induced ERK phosphorylation. VSMCs were treated without (0 min) or with 100 nM

Fig. 2. Dose-dependent effect of ET-1 on CD36 protein expression. VSMCs were incubated in serum-free medium for 8 h and then with various concentrations of ET-1 (0 –100 nM) for 24 h, and CD36 protein content was measured. Results are means ⫾ SD of 3 separate experiments. *P ⬍ 0.05 vs. vehicle control. AJP-Endocrinol Metab • VOL

Fig. 3. Effect of ETA receptor (ETAR) and ETBR antagonists on ET-1mediated inhibition of CD36 expression. VSMCs were incubated in serum-free medium for 8 h, preincubated for 1 h in the presence or absence of the ETAR antagonist BQ-610 (10 ␮M) or the ETBR antagonist BQ-788 (10 ␮M) and then incubated in the presence or absence of ET-1 (100 nM) in the continued presence or absence of the antagonist for a further 24 h before CD36 protein content was measured. Results are means ⫾ SD of 3 separate experiments. *P ⬍ 0.05 vs. vehicle control.

Fig. 4. Effect of tyrosine kinase and MAP kinase inhibitors on ET-1-mediated inhibition of CD36 expression. VSMCs were incubated in serum-free medium for 8 h, preincubated for 1 h in the presence or absence of the tyrosine kinase inhibitor genistein (G; 100 ␮M), the ERK inhibitor PD-98059 (PD; 75 ␮M), or the p38 MAPK inhibitor SB-203580 (SB; 20 ␮M) and then incubated in the presence or absence of ET-1 (100 nM) in the continued presence of the inhibitor for a further 24 h before CD36 protein content was measured. Results are means ⫾ SD of 3 separate experiments. *P ⬍ 0.05 vs. vehicle control.

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Translational physiology ET-1 DECREASES CD36 EXPRESSION IN VSMCS

ET-1 for 5– 60 min, and then ERK phosphorylation was measured by immunostaining with antibodies against phosphorylated or total ERK1/2 (Santa Cruz Biotechnology), which detect the phosphorylation status of ERK1/2 (p42 and p44). The results showed that ET-1 caused a time-dependent increase in ERK phosphorylation, which peaked at 5 min, decreased at 15 min, and was no longer seen at 30 min (data not shown). DISCUSSION

In this study, we used primary rat aortic VSMCs as an in vitro model to examine the role of ET-1 in the regulation of CD36 protein expression. We discovered that, as in human aortic SMCs (32), CD36 scavenger receptor is also expressed in rat VSMCs. Our results also demonstrated that ET-1 caused time- and dose-dependent downregulation of CD36 protein expression in VSMCs via the ETA receptor. This suppressive effect of ET-1 involved the activation of tyrosine kinase and ERK. To the best of our knowledge, this is the first indication of regulation of CD36 by ET-1 in VSMCs. Recently, Amiri et al. (7) established a new murine model with endotheliumrestricted overexpression of human ET-1. In this model, human ET-1 induced vascular remodeling and endothelial dysfunction in the absence of significant increases in blood pressure. It will be very interesting to investigate the expression of VSMC CD36 in this animal model and to explore the role of VSMC CD36 in vascular remodeling and endothelial dysfunction. Studies on a possible role of ET-1 in lipid metabolism are very limited. Our previous study (21) demonstrated that ET-1 affects lipid metabolism by stimulating adipocyte lipolysis. In the present study, we showed that ET-1 might also manipulate homeostasis of lipid metabolism by downregulating CD36 expression in VSMCs. Since VSMCs play a pivotal role in the maintenance of vascular function, ET-1-induced CD36 downregulation might alter the properties of VSMCs and thus contribute to the development of several vascular diseases, such as hypertension and atherosclerosis. CD36 has been identified as an FA receptor/transporter (1). CD36 deficiency might result in defective clearance of FAs from the circulation and elevated blood FA levels and secondary hypertriglyceridemia (6). On the other hand, as CD36 functions as a receptor for ox-LDL and other lipoproteins (31), CD36 deficiency may reduce lipoprotein clearance. This notion is supported by the study of Aitman et al. (5), which demonstrated that CD36deficient spontaneously hypertensive rats have defective FA and glucose metabolism, leading to dysregulation of cardiovascular functions, insulin resistance, and diabetes. Our study also demonstrated that the ET-1-inhibited CD36 expression was mediated through ERK-dependent but not p38 MAPK-dependent pathway (Fig. 4). This signal transduction mechanism is compatible with Bisotto and Fixman’s finding (10) that ET-1 is able to stimulate Src family tyrosine kinases, which may mediate ERK activation. In addition, Chen et al. (12) demonstrated that ET-1 stimulated VSMC proliferation through two complementary signal transduction cascades including ERK and p38 MAPK. Furthermore, the ETA receptormediated growth-promoting effect of ET-1 requires activation of ERK via transactivation of epidermal growth factor (EGF) in rat VSMCs (20). Further studies are needed to clarify the role of EGF transactivation in the ET-1-mediated downregulation of CD36 expression in VSMCs. AJP-Endocrinol Metab • VOL

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The relevance of CD36 downregulation to pathobiology with specific respect to VSMCs is not clear. Deficiency of CD36 caused a significant increase in fasting levels of plasma triglycerides, cholesterol, and free FAs (5, 14). Patients with CD36 deficiency had increased plasma triglyceride and glucose, lower plasma HDL-cholesterol, and hypertension than did controls (28). Furthermore, the postprandial hyperlipidemia also occurred in patients with this monogenic disorder (23). These observations suggested that CD36 deficiency may provide a more atherogenic environment to accelerate the development of atherosclerosis. Whether this hypothesis is correct and operative in human beings must await the findings of further studies. In the present study, we used genistein to prove that ET-1 decreased CD36 via the tyrosine kinase-dependent pathway. However, we need to be concerned about the divergent effects of genistein. For example, it has been reported that genistein has estrogenic effects (15) and is able to inhibit cAMP-specific phosphodiesterases (11, 39). These actions of genistein may confound interpretation of our results. More specific inhibitors are needed to exactly clarify the role of the tyrosine kinasedependent cascade in ET-1-decreased CD36 expression in VSMCs. In summary, our findings indicate that ET-1, acting through the ETA receptor, decreases CD36 protein expression in VSMCs. The underlying mechanism involves the activation of the tyrosine kinase and ERK pathways. Further characterization of CD36 regulation in response to proatherogenic and antiatherogenic stimuli could lead to the development of therapeutic strategies to prevent or reverse the progression of atherosclerosis. ACKNOWLEDGMENTS We thank Ren Yeu Kwok for excellent editorial assistance. GRANTS This work was supported by grants from Taipei Veterans General Hospital (VGHUST93-P1-03, VGHUST 94-P1-11, and VGH94-219). REFERENCES 1. Abumrad N, Harmon C, Ibrahimi A. Membrane transport of long-chain fatty acids: evidence for a facilitated process. J Lipid Res 39: 2309 –2018, 1998. 2. Abumrad NA, el-Maghrabi MR, Amri EZ, Lopez E, Grimaldi PA. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during differentiation. Homology with human CD36. J Biol Chem 268: 17665–17668, 1993. 3. Abumrad NA, Park JH, Park CR. Permeation of long-chain fatty acid into adipocytes. Kinetics, specificity, and evidence for involvement of a membrane protein. J Biol Chem 259: 8945– 8953, 1984. 4. Abumrad NA, Perkins RC, Park JH, Park CR. Mechanism of long chain fatty acid permeation in the isolated adipocyte. J Biol Chem 256: 9183–9191, 1981. 5. Aitman TJ, Glazier AM, Wallace CA, Cooper LD, Norsworthy PJ, Wahid FN, Al-Majali KM, Trembling PM, Mann CJ, Shoulders CC, Graf D, St Lezin E, Kurtz TW, Kren V, Pravenec M, Ibrahimi A, Abumrad NA, Stanton LW, Scott J. Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats. Nat Genet 21: 76 – 83, 1999. 6. Aitman TJ, Godsland IF, Farren B, Crook D, Wong HJ, Scott J. Defects of insulin action on fatty acid and carbohydrate metabolism in familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol 17: 748 –754, 1997. 7. Amiri F, Virdis A, Neves MF, Iglarz M, Seidah NG, Touyz RM, Reudelhuber TL, Schiffrin EL. Endothelium-restricted overexpression

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