J Nutr Sci Vitaminol, 60, 450–454, 2014
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Deoxycholic Acid Is Involved in the Proliferation and Migration of Vascular Smooth Muscle Cells Hidehisa Shimizu1, Masahito Hagio2, Hitoshi Iwaya1, Ikuya Tsuneki1, Ja-Young Lee1, Satoru Fukiya1, Atsushi Yokota1, Hitoshi Miyazaki3, Hiroshi Hara1 and Satoshi Ishizuka1 1
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060–8589, Japan 2 Faculty of Life Sciences, Toyo University, Gunma 374–0193, Japan 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305–8572, Japan (Received April 15, 2014)
Summary Obesity is increasingly becoming associated with increased risk of atherosclerosis. Serum levels of the bile acid deoxycholic acid (DCA) are elevated in mice with obesity induced by a high-fat (HF) diet. Therefore, we investigated the influence of DCA on the functions of vascular smooth muscle cells (VSMCs) because the initiation and progression of atherosclerosis are associated with VSMC proliferation and migration. DCA induced c-jun N-terminal kinase (JNK) activation whereas a JNK inhibitor prevented DCA-induced VSMC proliferation and migration. Based on these findings, we examined whether DCA promotes the expression of platelet-derived growth factor b-receptor (PDGFRb) that has a c-Jun binding site in its promoter region. The mRNA and protein expression levels of PDGFRb were upregulated in VSMCs after a 24- and 48-h incubation with DCA, respectively. The effects of PDGF such as proliferation and migration of VSMCs were promoted after a 48-h incubation with DCA despite the absence of DCA during PDGF stimulation. These findings suggest that elevated serum concentrations of DCA are involved in the pathogenesis of atherosclerosis in HF-induced obesity. Key Words high-fat diet, enterobacterium, bile acid, atherosclerosis
Atherosclerosis remains a major health problem throughout the western world, particularly in moderately to severely obese individuals because current therapeutic regimens prevent only 25% of all cardiovascular events (1). Obesity is an important established risk factor for the development of atherosclerosis and subsequent cardiovascular disease (CVD) (2). One of the main factors that contribute to the development of obesity is the consumption of high-fat (HF) diets that enhance bile secretion to facilitate lipid digestion (3). The gut microbiota in humans modifies the steroid nucleus of bile acids during transit to the large intestine to yield secondary bile acids (4). The most prevalent of these is deoxycholic acid (DCA) that is generated from cholic acid (CA), which is the most abundant bile acid in biliary bile (5). High-fat diets alter the gut microbiota of obese experimental animals, thus increasing serum DCA levels (6). Plasma and serum concentrations of DCA are increased in patients with insulin resistance (IR) and chronic renal failure (CRF), respectively (7, 8) and the risk of developing atherosclerosis is greater for these patients than healthy individuals. Furthermore, CVD is the leading cause of mortality in such patients (9, 10). The initiation and progression of atherosclerosis are associated with the proliferation and migration of E-mail: [email protected]
vascular smooth muscle cells (VSMCs), and with dysfunctional endothelial cells (ECs) (11). Although DCA directly promotes the adhesion of THP-1 cells, a human monocytic cell line derived from a patient with acute monocytic leukemia, to human umbilical vein endothelial cells (HUVECs) by inducing the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (12), the functions of DCA in VSMCs remain unclear. Platelet-derived growth factor (PDGF)-dependent signaling plays a key role in the development of athero sclerosis (11). Among the five isoforms of PDGF, PDGFBB is involved in the proliferation and migration of VSMCs through binding its specific PDGF b-receptor (PDGFRb). Therefore, the present study aimed to determine the effects of DCA on VSMC proliferation and migration, and on the upregulation of PDGFRb expression in VSMCs. We also investigated whether DCA and PDGF-BB coordinately promote the VSMC functions. Materials and Methods Materials and reagents. Antibodies were obtained from the following suppliers: anti-PDGF-b receptor antibody, Santa Cruz Biotechnology (Santa Cruz, CA); anti-a-tubulin, Calbiochem (La Jolla, CA); anti-phosphoc-jun N-terminal kinase (JNK), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody, anti-mouse
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Fig. 1. Proliferation and migration of VSMCs is induced by DCA via JNK activation. (A) DCA induces JNK activation. Serum-starved VSMCs were incubated with or without DCA (5 mm) for 10 min and then cell lysates were immunoblotted using anti-phospho-JNK antibody. Each value represents the average of band intensities from three independent experiments. (B) After incubation with or without SP600125 (JNK inhibitor; 5 mm) for 30 min, serum-starved VSMCs were stimulated with or without DCA (5 mm). Cell proliferation was assessed after stimulation with DCA (5 mm) for 48 h. (C) Experimental conditions were as described in (B) except cell migration was assessed after DCA (5 mm) stimulation for 5 h. Data are expressed as means6SE of three independent experiments for (B) and (C).
IgG, HRP-linked antibodies and PDGF-BB, Cell Signaling Technology (Beverly, MA); the JNK inhibitor SP600125 and Dulbecco’s modified Eagle’s medium (DMEM), Wako Pure Chemical Insustries, Ltd. (Osaka, Japan); trypsinEDTA and fetal bovine serum (FBS), GIBCO (Grand Island, NY); DCA and penicillin/streptomycin, Nacalai Tesque, Inc. (Kyoto, Japan). DCA and SP600125 were dissolved in dimethyl sulfoxide. PDGF-BB was dissolved in distilled deionized water. Cell culture. We isolated VSMCs from the thoracic aorta of adult Sprague-Dawley rats as described (13). The Institutional Animal Care and Use Committees of the University of Tsukuba approved all experimental protocols (approval number: 12-088). We maintained VSMCs in DMEM supplemented with 10% FBS, 100 U/ mL penicillin, and 100 mg/mL streptomycin. VSMCs were analyzed between passages 8 and 10 after a 48-h incubation in DMEM containing 0.1% FBS. Immunoblotting. Serum-starved VSMCs (23105 cells/3.5 cm dish) were incubated with or without SP600125 (5 mm) for 30 min, then with or without DCA (5 mm) for 10 min in Fig. 1A and 48 h in Fig. 2C. Proteins from cell lysates were immunoblotted as described (13). In brief, cell lysates were fractionated by SDS-PAGE and proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, MA). Levels of phospho-JNK and PDGFRb detected using specific antibodies were normalized to those of a-tubulin. Protein bands were visualized using the enhanced ChemiLumi One Super system (Nacalai Tesque), and densito-
metrically analyzed and calculated using Lumi Vision PRO 400EX (Aisin, Aichi, Japan). Evaluation of cell proliferation. Serum-starved VSMCs (23105 cells/6 cm dish) were incubated with or without SP600125 (5 mm) for 30 min, then with or without DCA (5 mm) for 48 h in Fig. 1B. In Fig. 3A, serumstarved VSMCs (23105 cells/6 cm dish) were incubated with or without DCA (5 mm) for 48 h. After removing DCA, cells were stimulated with or without PDGF-BB (20 ng/mL) for another 48 h. After culture under various conditions, VSMCs were harvested by trypsin digestion, stained with Trypan blue and counted. Cell migration assay. Cell migration was assayed as described (13). In brief, it was determined using a modified Boyden chamber assay (Corning Costar, Acton, MA). Polyvinylpyrrolidone-free polycarbonate filters with an 8 mm-pore size were coated with fibronectin. In Fig. 1C, serum-starved VSMCs (23105 cells/well) were incubated with or without SP600125 (5 mm) for 30 min. In Fig. 3B, serum-starved VSMCs (23105 cells/ well) were incubated with or without DCA (5 mm) for 48 h. Reagent-treated cells added to the upper Boyden chambers. These were inserted into the bottom chamber containing medium and incubated with or without DCA (5 mm) or PDGF-BB (20 ng/mL) for 5 h. Cells that migrated to the bottom surface of the membrane were fixed in methanol, stained with hematoxylin and eosin, and counted in 10 representative fields. Quantitative real-time PCR. Serum-starved VSMCs (23105 cells/3.5 cm dish) were incubated with or with-
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Fig. 2. Deoxycholic acid upregulates PDGFRb expression through JNK activation. (A) Serum-starved VSMCs were incubated with DCA (5 mm) for indicated periods and then purified mRNA was analyzed by real-time PCR using PDGFRb primer. *Different from 0 h, p,0.05. (B) After incubation with or without SP600125 (JNK inhibitor; 5 mm) for 30 min, serum-starved VSMCs were incubated with or without DCA (5 mm) for 24 h and then purified mRNA was analyzed by real-time PCR using PDGFRb primer. (C) After incubation with or without SP600125 (5 mm) for 30 min, serum-starved VSMCs were incubated with or without DCA (5 mm) for 48 h and then cell lysates were immunoblotted using anti-PDGFRb antibody. Each value represents the average of band intensities from three independent experiments. Data are expressed as means6SE of four independent experiments for (A) and (B).
Fig. 3. Incubation with DCA enhances subsequent PDGF-BB-induced VSMC proliferation and migration. Serum-starved VSMCs were incubated with or without DCA (5 mm) for 48 h. After removal of DCA, cells were incubated with or without PDGF-BB (20 ng/mL). (A) Cell proliferation assessed after stimulation with PDGF-BB for 48 h. (B) Cells migration assessed after stimulation with PDGF-BB for 5 h. Data are expressed as means6SE of four independent experiments for (A) and (B).
out SP600125 (5 mm) for 30 min, then with or without DCA (5 mm) for indicated periods in Fig. 2A and 24 h in Fig. 2B. Quantitative real time PCR proceeded as described (14). In brief, total RNA was isolated SepasolRNA I Super G (Nacalai Tesque). First-strand cDNAs were synthesized from template RNA (2 mg) using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Quantitative real time PCR proceeded using Syber Premix Ex Taq II Green (Takara Bio, Shiga, Japan) and the Mx3000P real-time PCR system (Stratagene, La Jolla, CA, UA), according to the manufacturer’s protocol with the following oligonucleotide primers: rat PDGFRb, 5′-GCATGGAATCGTCGTCTCAG-3′ (forward) and 5′-CAACTCACTGGGGCCAGAG-3′ (reverse); rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-AGGTTGTCTCCTGTGACTTC-3′ (for-
ward) and 5′-CTGTTGCTGTAGCCATATTC-3′ (reverse). Levels of mRNA expression were measured as ratios of the amount of GAPDH mRNA. Statistical analysis. Statistical analyses were performed with JMP software (ver. 5.0; SAS Institute, Cary, NC). A two-way ANOVA was used for evaluation in Fig. 1B, 1C, 2B, 3A, and 3B. Differences in Figs. 1B and 1C were determined with Tukey’s test. Differences in Fig. 2A were determined using Dunnett’s test. Differences were considered significant at p,0.05. All data are shown as mean6SE. Results The JNK activation leads to neointimal formation through VSMC proliferation and migration (15, 16). Therefore, we initially investigated whether DCA induces
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JNK activation in VSMCs. Figure 1A shows that DCA stimulation activated JNK, and Figs. 1B and 1C show that the JNK inhibitor SP600125 suppressed the DCAinduced proliferation and migration of VSMCs. These results indicate that DCA triggered VSMC proliferation and migration through JNK activation. Because JNK activation induces PDGFRb upregulation in hepatic stellate cells (17), we postulated that DCA would also promote PDGFRb in these cells. Figure 2A shows that DCA time-dependently increased the amount of PDGFRb mRNA expression in VSMCs. Figures 2B and 2C show that SP600125 partially sup pressed the mRNA and protein expression of PDGFRb induced by DCA, respectively. Thus, DCA evokes PDGFRb expression via JNK activation in VSMCs. We investigated the relationship between the DCAinduced upregulation of PDGFRb, and the PDGF-BBelicited proliferation and migration of VSMCs. Figures 3A and 3B show augmented VSMC proliferation and migration after a 48-h incubation with DCA even when the cells were not exposed to DCA during the incubation with PDGF-BB. These results suggest that DCA promotes PDGF-induced VSMC proliferation and migration through the upregulation of PDGFRb expression promoted by JNK. Discussion The novel findings of the present study are that DCA elicited VSMC proliferation and migration through JNK activation and that DCA accelerates PDGF-BB-induced VSMC proliferation and migration via the upregulation of PDGFRb expression promoted by JNK. Together with a report indicating that DCA evokes EC dysfunction (12), the present findings support the notion that DCA is critically involved in the development of atherosclerosis and that DCA acts in concert with PDGFRb to stimulate the pathogenesis of atherosclerosis. We found that 5 mm DCA can induce VSMC functions, whereas most previous studies have examined cellular functions using $100 mm. Our results suggest that even a very low concentration of DCA can lead to VSMC dysfunction. The effects of low DCA concentrations should also be investigated in other types of cells. In healthy WKAH rats, the maximum plasma concentration of DCA is about 1 mm (18). However, we consider 5 mm DCA to be a physiological concentration because the plasma concentration of bile acids is about 20 mm in rats with CRF (19) and DCA comprises about 25% of serum bile acids in patients with CRF (8). Bile acids are ligands for farnesoid X receptors (FXR) that are expressed in VSMCs and prevent neointima formation (20, 21). However, our results suggested that DCA is involved in VSMC proliferation and migration. Although chenodeoxycholic acid (CDCA) is reportedly the best activator of FXR (22), DCA interferes with the ability of CDCA to cause the recruitment of steroid receptor coactivator-1 (SRC-1) to FXR and thus might result in a decline in the activation of some FXR pathways (23). Thus, we speculate that DCA does not activate FXR and that it induces the activation of an
unknown signaling pathway through JNK activation for VSMC functions. The present results showed that DCA promotes PDGFRb expression in VSMCs. The expression of PDGFRb might be upregulated in VSMCs during the process of atherosclerosis induced by diabetes or CRF because increased concentrations of serum glucose in diabetic rats and of indoxyl sulfate, a tryptophan metabolite, in CRF rats promote such expression (13, 24). Therefore, analysis of crosstalk between DCA and high glucose or indoxyl sulfate might identify novel upregulatory mechanisms of PDGFRb expression in atherosclerosis induced by diabetes and CRF. To study diet-induced atherosclerosis, atherogenic diets are used and CA is supplemented in the diets (25). Because a part of CA is converted to DCA by intestinal bacteria (5), on the basis of the present results, DCA might influence diet-induced atherosclerosis. We propose that the intestinal microbiota plays a role in these cardiometabolic diseases because DCA generated by enterobacteria induces the initiation and development of atherosclerosis through VSMC proliferation and migration. Older literature supports our notion that germ-free animals are less susceptible to atherosclerotic plaque (26). In addition, prebiotics administered to ApoE2/2 mice for 16 wk obviously altered the composition of the gut microbiota and reduced the size of atherosclerotic lesions by 35% (27). Therefore, controlling intestinal microbiota using prebiotics might attenuate the initiation and progression of CVD. Acknowledgments We thank Norma Foster for help with preparing the manuscript. This study was supported by the Regional Innovation Strategy Support Program of the MEXT (Ministry of Education, Culture, Sports, Science and Technology) from the Japanese government and a Grantin-Aid for Young Scientists (B) 25750356 (to H.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. REFERENCES 1) Murray CJ, Lopez AD. 1997. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 349: 1269–1276. 2) Yusuf S, Reddy S, Ounpuu S, Anand S. 2001. Global burden of cardiovascular diseases: part I: general considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation 104: 2746–2753. 3) Reddy BS. 1981. Diet and excretion of bile acids. Cancer Res 41: 3766–3768. 4) Ridlon JM, Kang DJ, Hylemon PB. 2006. Bile salt biotransformations by human intestinal bacteria. J Lipid Res 47: 241–259. 5) Islam KB, Fukiya S, Hagio M, Fujii N, Ishizuka S, Ooka T, Ogura Y, Hayashi T, Yokota A. 2011. Bile acid is a host factor that regulates the composition of the cecal microbiota in rats. Gastroenterology 141: 1773–1781. 6) Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, Iwakura Y, Oshima K, Morita H, Hattori M, Honda K, Ishikawa Y, Hara E, Ohtani N. 2013. Obesityinduced gut microbial metabolite promotes liver cancer
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through senescence secretome. Nature 499: 97–101. 7) Haeusler RA, Astiarraga B, Camastra S, Accili D, Ferrannini E. 2013. Human insulin resistance is associated with increased plasma levels of 12a-hydroxylated bile acids. Diabetes 62: 4184–4191. 8) Jimenez F, Monte MJ, El-Mir MY, Pascual MJ, Marin JJ. 2002. Chronic renal failure-induced changes in serum and urine bile acid profiles. Dig Dis Sci 47: 2398–2406. 9) Bornfeldt KE, Tabas I. 2011. Insulin resistance, hyperglycemia, and atherosclerosis. Cell Metab 14: 575–585. 10) London GM, Drueke TB. 1997. Atherosclerosis and arteriosclerosis in chronic renal failure. Kidney Int 51: 1678–1695. 11) Ross R. 1995. Cell biology of atherosclerosis. Annu Rev Physiol 57: 791–804. 12) Qin P, Tang X, Elloso MM, Harnish DC. 2006. Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NFkappaB, and p38. Am J Physiol Heart Circ Physiol 291: H741-H747. 13) Shimizu H, Hirose Y, Nishijima F, Tsubakihara Y, Miyazaki H. 2009. ROS and PDGF-b receptors are critically involved in indoxyl sulfate actions that promote vascular smooth muscle cell proliferation and migration. Am J Physiol Cell Physiol 297: C389–C396. 14) Shimizu H, Saito S, Higashiyama Y, Nishijima F, Niwa T. 2013. CREB, NF-kB, and NADPH oxidase coordinately upregulate indoxyl sulfate-induced angiotensinogen expression in proximal tubular cells. Am J Physiol Cell Physiol 304: C685–C692. 15) Izumi Y, Kim S, Namba M, Yasumoto H, Miyazaki H, Hoshiga M, Kaneda Y, Morishita R, Zhan Y, Iwao H. 2001. Gene transfer of dominant-negative mutants of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase prevents neointimal formation in ballooninjured rat artery. Circ Res 88: 1120–1126. 16) Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H. 2003. Role of JNK, p38, and ERK in plateletderived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 23: 795–801. 17) Chen A, Zhang L. 2003. The antioxidant (2)-epigallocatechin-3-gallate inhibits rat hepatic stellate cell
18)
19)
20)
21)
22)
23)
24)
25) 26)
27)
proliferation in vitro by blocking the tyrosine phosphorylation and reducing the gene expression of platelet-derived growth factor-b receptor. J Biol Chem 278: 23381–23389. Hagio M, Matsumoto M, Fukushima M, Hara H, Ishizuka S. 2009. Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS. J Lipid Res 50: 173–180. Yamakado M, Ise M. 1999. Mechanism of oral absorbent AST-120 in lipid abnormalities in experimental uremic rats. Kidney Int Suppl 71: S190–S192. Li YT, Swales KE, Thomas GJ, Warner TD, Bishop-Bailey D. 2007. Farnesoid x receptor ligands inhibit vascular smooth muscle cell inflammation and migration. Arterioscler Thromb Vasc Biol 27: 2606–2611. Zhang Q, He F, Kuruba R, Gao X, Wilson A, Li J, Billiar TR, Pitt BR, Xie W, Li S. 2008. FXR-mediated regulation of angiotensin type 2 receptor expression in vascular smooth muscle cells. Cardiovasc Res 77: 560–569. Lew JL, Zhao A, Yu J, Huang L, De Pedro N, Peláez F, Wright SD, Cui J. 2004. The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion. J Biol Chem 279: 8856–8861. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, Lehmann JM. 1999. Bile acids: natural ligands for an orphan nuclear receptor. Science 284: 1365–1368. Tamura K, Kanzaki T, Tashiro J, Yokote K, Mori S, Ueda S, Saito Y, Morisaki N. 2000. Increased atherogenesis in Otsuka Long-Evans Tokushima fatty rats before the onset of diabetes mellitus: association with overexpression of PDGF b-receptors in aortic smooth muscle cells. Atherosclerosis 149: 351–358. Getz GS, Reardon CA. 2006. Diet and murine atherosclerosis. Arterioscler Thromb Vasc Biol 26: 242–249. Nordin AA. 1968. The occurrence of plaque forming cells in normal and immunized conventional and germfree mice. Proc Soc Exp Biol Med 129: 57–62. Rault-Nania M, Gueux E, Demougeot C, Demigné C, Rock E, Mazur A. 2006. Inulin attenuates atherosclerosis in apolipoprotein E-deficient mice. Br J Nutr 96: 840–844.
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Deoxycholic Acid Is Involved in the Proliferation and Migration of Vascular Smooth Muscle Cells Hidehisa Shimizu1, Masahito Hagio2, Hitoshi Iwaya1, Ikuya Tsuneki1, Ja-Young Lee1, Satoru Fukiya1, Atsushi Yokota1, Hitoshi Miyazaki3, Hiroshi Hara1 and Satoshi Ishizuka1 1
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060–8589, Japan 2 Faculty of Life Sciences, Toyo University, Gunma 374–0193, Japan 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305–8572, Japan (Received April 15, 2014)
Summary Obesity is increasingly becoming associated with increased risk of atherosclerosis. Serum levels of the bile acid deoxycholic acid (DCA) are elevated in mice with obesity induced by a high-fat (HF) diet. Therefore, we investigated the influence of DCA on the functions of vascular smooth muscle cells (VSMCs) because the initiation and progression of atherosclerosis are associated with VSMC proliferation and migration. DCA induced c-jun N-terminal kinase (JNK) activation whereas a JNK inhibitor prevented DCA-induced VSMC proliferation and migration. Based on these findings, we examined whether DCA promotes the expression of platelet-derived growth factor b-receptor (PDGFRb) that has a c-Jun binding site in its promoter region. The mRNA and protein expression levels of PDGFRb were upregulated in VSMCs after a 24- and 48-h incubation with DCA, respectively. The effects of PDGF such as proliferation and migration of VSMCs were promoted after a 48-h incubation with DCA despite the absence of DCA during PDGF stimulation. These findings suggest that elevated serum concentrations of DCA are involved in the pathogenesis of atherosclerosis in HF-induced obesity. Key Words high-fat diet, enterobacterium, bile acid, atherosclerosis
Atherosclerosis remains a major health problem throughout the western world, particularly in moderately to severely obese individuals because current therapeutic regimens prevent only 25% of all cardiovascular events (1). Obesity is an important established risk factor for the development of atherosclerosis and subsequent cardiovascular disease (CVD) (2). One of the main factors that contribute to the development of obesity is the consumption of high-fat (HF) diets that enhance bile secretion to facilitate lipid digestion (3). The gut microbiota in humans modifies the steroid nucleus of bile acids during transit to the large intestine to yield secondary bile acids (4). The most prevalent of these is deoxycholic acid (DCA) that is generated from cholic acid (CA), which is the most abundant bile acid in biliary bile (5). High-fat diets alter the gut microbiota of obese experimental animals, thus increasing serum DCA levels (6). Plasma and serum concentrations of DCA are increased in patients with insulin resistance (IR) and chronic renal failure (CRF), respectively (7, 8) and the risk of developing atherosclerosis is greater for these patients than healthy individuals. Furthermore, CVD is the leading cause of mortality in such patients (9, 10). The initiation and progression of atherosclerosis are associated with the proliferation and migration of E-mail: [email protected]
vascular smooth muscle cells (VSMCs), and with dysfunctional endothelial cells (ECs) (11). Although DCA directly promotes the adhesion of THP-1 cells, a human monocytic cell line derived from a patient with acute monocytic leukemia, to human umbilical vein endothelial cells (HUVECs) by inducing the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (12), the functions of DCA in VSMCs remain unclear. Platelet-derived growth factor (PDGF)-dependent signaling plays a key role in the development of athero sclerosis (11). Among the five isoforms of PDGF, PDGFBB is involved in the proliferation and migration of VSMCs through binding its specific PDGF b-receptor (PDGFRb). Therefore, the present study aimed to determine the effects of DCA on VSMC proliferation and migration, and on the upregulation of PDGFRb expression in VSMCs. We also investigated whether DCA and PDGF-BB coordinately promote the VSMC functions. Materials and Methods Materials and reagents. Antibodies were obtained from the following suppliers: anti-PDGF-b receptor antibody, Santa Cruz Biotechnology (Santa Cruz, CA); anti-a-tubulin, Calbiochem (La Jolla, CA); anti-phosphoc-jun N-terminal kinase (JNK), anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody, anti-mouse
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Fig. 1. Proliferation and migration of VSMCs is induced by DCA via JNK activation. (A) DCA induces JNK activation. Serum-starved VSMCs were incubated with or without DCA (5 mm) for 10 min and then cell lysates were immunoblotted using anti-phospho-JNK antibody. Each value represents the average of band intensities from three independent experiments. (B) After incubation with or without SP600125 (JNK inhibitor; 5 mm) for 30 min, serum-starved VSMCs were stimulated with or without DCA (5 mm). Cell proliferation was assessed after stimulation with DCA (5 mm) for 48 h. (C) Experimental conditions were as described in (B) except cell migration was assessed after DCA (5 mm) stimulation for 5 h. Data are expressed as means6SE of three independent experiments for (B) and (C).
IgG, HRP-linked antibodies and PDGF-BB, Cell Signaling Technology (Beverly, MA); the JNK inhibitor SP600125 and Dulbecco’s modified Eagle’s medium (DMEM), Wako Pure Chemical Insustries, Ltd. (Osaka, Japan); trypsinEDTA and fetal bovine serum (FBS), GIBCO (Grand Island, NY); DCA and penicillin/streptomycin, Nacalai Tesque, Inc. (Kyoto, Japan). DCA and SP600125 were dissolved in dimethyl sulfoxide. PDGF-BB was dissolved in distilled deionized water. Cell culture. We isolated VSMCs from the thoracic aorta of adult Sprague-Dawley rats as described (13). The Institutional Animal Care and Use Committees of the University of Tsukuba approved all experimental protocols (approval number: 12-088). We maintained VSMCs in DMEM supplemented with 10% FBS, 100 U/ mL penicillin, and 100 mg/mL streptomycin. VSMCs were analyzed between passages 8 and 10 after a 48-h incubation in DMEM containing 0.1% FBS. Immunoblotting. Serum-starved VSMCs (23105 cells/3.5 cm dish) were incubated with or without SP600125 (5 mm) for 30 min, then with or without DCA (5 mm) for 10 min in Fig. 1A and 48 h in Fig. 2C. Proteins from cell lysates were immunoblotted as described (13). In brief, cell lysates were fractionated by SDS-PAGE and proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, MA). Levels of phospho-JNK and PDGFRb detected using specific antibodies were normalized to those of a-tubulin. Protein bands were visualized using the enhanced ChemiLumi One Super system (Nacalai Tesque), and densito-
metrically analyzed and calculated using Lumi Vision PRO 400EX (Aisin, Aichi, Japan). Evaluation of cell proliferation. Serum-starved VSMCs (23105 cells/6 cm dish) were incubated with or without SP600125 (5 mm) for 30 min, then with or without DCA (5 mm) for 48 h in Fig. 1B. In Fig. 3A, serumstarved VSMCs (23105 cells/6 cm dish) were incubated with or without DCA (5 mm) for 48 h. After removing DCA, cells were stimulated with or without PDGF-BB (20 ng/mL) for another 48 h. After culture under various conditions, VSMCs were harvested by trypsin digestion, stained with Trypan blue and counted. Cell migration assay. Cell migration was assayed as described (13). In brief, it was determined using a modified Boyden chamber assay (Corning Costar, Acton, MA). Polyvinylpyrrolidone-free polycarbonate filters with an 8 mm-pore size were coated with fibronectin. In Fig. 1C, serum-starved VSMCs (23105 cells/well) were incubated with or without SP600125 (5 mm) for 30 min. In Fig. 3B, serum-starved VSMCs (23105 cells/ well) were incubated with or without DCA (5 mm) for 48 h. Reagent-treated cells added to the upper Boyden chambers. These were inserted into the bottom chamber containing medium and incubated with or without DCA (5 mm) or PDGF-BB (20 ng/mL) for 5 h. Cells that migrated to the bottom surface of the membrane were fixed in methanol, stained with hematoxylin and eosin, and counted in 10 representative fields. Quantitative real-time PCR. Serum-starved VSMCs (23105 cells/3.5 cm dish) were incubated with or with-
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Fig. 2. Deoxycholic acid upregulates PDGFRb expression through JNK activation. (A) Serum-starved VSMCs were incubated with DCA (5 mm) for indicated periods and then purified mRNA was analyzed by real-time PCR using PDGFRb primer. *Different from 0 h, p,0.05. (B) After incubation with or without SP600125 (JNK inhibitor; 5 mm) for 30 min, serum-starved VSMCs were incubated with or without DCA (5 mm) for 24 h and then purified mRNA was analyzed by real-time PCR using PDGFRb primer. (C) After incubation with or without SP600125 (5 mm) for 30 min, serum-starved VSMCs were incubated with or without DCA (5 mm) for 48 h and then cell lysates were immunoblotted using anti-PDGFRb antibody. Each value represents the average of band intensities from three independent experiments. Data are expressed as means6SE of four independent experiments for (A) and (B).
Fig. 3. Incubation with DCA enhances subsequent PDGF-BB-induced VSMC proliferation and migration. Serum-starved VSMCs were incubated with or without DCA (5 mm) for 48 h. After removal of DCA, cells were incubated with or without PDGF-BB (20 ng/mL). (A) Cell proliferation assessed after stimulation with PDGF-BB for 48 h. (B) Cells migration assessed after stimulation with PDGF-BB for 5 h. Data are expressed as means6SE of four independent experiments for (A) and (B).
out SP600125 (5 mm) for 30 min, then with or without DCA (5 mm) for indicated periods in Fig. 2A and 24 h in Fig. 2B. Quantitative real time PCR proceeded as described (14). In brief, total RNA was isolated SepasolRNA I Super G (Nacalai Tesque). First-strand cDNAs were synthesized from template RNA (2 mg) using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Quantitative real time PCR proceeded using Syber Premix Ex Taq II Green (Takara Bio, Shiga, Japan) and the Mx3000P real-time PCR system (Stratagene, La Jolla, CA, UA), according to the manufacturer’s protocol with the following oligonucleotide primers: rat PDGFRb, 5′-GCATGGAATCGTCGTCTCAG-3′ (forward) and 5′-CAACTCACTGGGGCCAGAG-3′ (reverse); rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-AGGTTGTCTCCTGTGACTTC-3′ (for-
ward) and 5′-CTGTTGCTGTAGCCATATTC-3′ (reverse). Levels of mRNA expression were measured as ratios of the amount of GAPDH mRNA. Statistical analysis. Statistical analyses were performed with JMP software (ver. 5.0; SAS Institute, Cary, NC). A two-way ANOVA was used for evaluation in Fig. 1B, 1C, 2B, 3A, and 3B. Differences in Figs. 1B and 1C were determined with Tukey’s test. Differences in Fig. 2A were determined using Dunnett’s test. Differences were considered significant at p,0.05. All data are shown as mean6SE. Results The JNK activation leads to neointimal formation through VSMC proliferation and migration (15, 16). Therefore, we initially investigated whether DCA induces
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JNK activation in VSMCs. Figure 1A shows that DCA stimulation activated JNK, and Figs. 1B and 1C show that the JNK inhibitor SP600125 suppressed the DCAinduced proliferation and migration of VSMCs. These results indicate that DCA triggered VSMC proliferation and migration through JNK activation. Because JNK activation induces PDGFRb upregulation in hepatic stellate cells (17), we postulated that DCA would also promote PDGFRb in these cells. Figure 2A shows that DCA time-dependently increased the amount of PDGFRb mRNA expression in VSMCs. Figures 2B and 2C show that SP600125 partially sup pressed the mRNA and protein expression of PDGFRb induced by DCA, respectively. Thus, DCA evokes PDGFRb expression via JNK activation in VSMCs. We investigated the relationship between the DCAinduced upregulation of PDGFRb, and the PDGF-BBelicited proliferation and migration of VSMCs. Figures 3A and 3B show augmented VSMC proliferation and migration after a 48-h incubation with DCA even when the cells were not exposed to DCA during the incubation with PDGF-BB. These results suggest that DCA promotes PDGF-induced VSMC proliferation and migration through the upregulation of PDGFRb expression promoted by JNK. Discussion The novel findings of the present study are that DCA elicited VSMC proliferation and migration through JNK activation and that DCA accelerates PDGF-BB-induced VSMC proliferation and migration via the upregulation of PDGFRb expression promoted by JNK. Together with a report indicating that DCA evokes EC dysfunction (12), the present findings support the notion that DCA is critically involved in the development of atherosclerosis and that DCA acts in concert with PDGFRb to stimulate the pathogenesis of atherosclerosis. We found that 5 mm DCA can induce VSMC functions, whereas most previous studies have examined cellular functions using $100 mm. Our results suggest that even a very low concentration of DCA can lead to VSMC dysfunction. The effects of low DCA concentrations should also be investigated in other types of cells. In healthy WKAH rats, the maximum plasma concentration of DCA is about 1 mm (18). However, we consider 5 mm DCA to be a physiological concentration because the plasma concentration of bile acids is about 20 mm in rats with CRF (19) and DCA comprises about 25% of serum bile acids in patients with CRF (8). Bile acids are ligands for farnesoid X receptors (FXR) that are expressed in VSMCs and prevent neointima formation (20, 21). However, our results suggested that DCA is involved in VSMC proliferation and migration. Although chenodeoxycholic acid (CDCA) is reportedly the best activator of FXR (22), DCA interferes with the ability of CDCA to cause the recruitment of steroid receptor coactivator-1 (SRC-1) to FXR and thus might result in a decline in the activation of some FXR pathways (23). Thus, we speculate that DCA does not activate FXR and that it induces the activation of an
unknown signaling pathway through JNK activation for VSMC functions. The present results showed that DCA promotes PDGFRb expression in VSMCs. The expression of PDGFRb might be upregulated in VSMCs during the process of atherosclerosis induced by diabetes or CRF because increased concentrations of serum glucose in diabetic rats and of indoxyl sulfate, a tryptophan metabolite, in CRF rats promote such expression (13, 24). Therefore, analysis of crosstalk between DCA and high glucose or indoxyl sulfate might identify novel upregulatory mechanisms of PDGFRb expression in atherosclerosis induced by diabetes and CRF. To study diet-induced atherosclerosis, atherogenic diets are used and CA is supplemented in the diets (25). Because a part of CA is converted to DCA by intestinal bacteria (5), on the basis of the present results, DCA might influence diet-induced atherosclerosis. We propose that the intestinal microbiota plays a role in these cardiometabolic diseases because DCA generated by enterobacteria induces the initiation and development of atherosclerosis through VSMC proliferation and migration. Older literature supports our notion that germ-free animals are less susceptible to atherosclerotic plaque (26). In addition, prebiotics administered to ApoE2/2 mice for 16 wk obviously altered the composition of the gut microbiota and reduced the size of atherosclerotic lesions by 35% (27). Therefore, controlling intestinal microbiota using prebiotics might attenuate the initiation and progression of CVD. Acknowledgments We thank Norma Foster for help with preparing the manuscript. This study was supported by the Regional Innovation Strategy Support Program of the MEXT (Ministry of Education, Culture, Sports, Science and Technology) from the Japanese government and a Grantin-Aid for Young Scientists (B) 25750356 (to H.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. REFERENCES 1) Murray CJ, Lopez AD. 1997. Mortality by cause for eight regions of the world: Global Burden of Disease Study. Lancet 349: 1269–1276. 2) Yusuf S, Reddy S, Ounpuu S, Anand S. 2001. Global burden of cardiovascular diseases: part I: general considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation 104: 2746–2753. 3) Reddy BS. 1981. Diet and excretion of bile acids. Cancer Res 41: 3766–3768. 4) Ridlon JM, Kang DJ, Hylemon PB. 2006. Bile salt biotransformations by human intestinal bacteria. J Lipid Res 47: 241–259. 5) Islam KB, Fukiya S, Hagio M, Fujii N, Ishizuka S, Ooka T, Ogura Y, Hayashi T, Yokota A. 2011. Bile acid is a host factor that regulates the composition of the cecal microbiota in rats. Gastroenterology 141: 1773–1781. 6) Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, Iwakura Y, Oshima K, Morita H, Hattori M, Honda K, Ishikawa Y, Hara E, Ohtani N. 2013. Obesityinduced gut microbial metabolite promotes liver cancer
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Shimizu H et al.
through senescence secretome. Nature 499: 97–101. 7) Haeusler RA, Astiarraga B, Camastra S, Accili D, Ferrannini E. 2013. Human insulin resistance is associated with increased plasma levels of 12a-hydroxylated bile acids. Diabetes 62: 4184–4191. 8) Jimenez F, Monte MJ, El-Mir MY, Pascual MJ, Marin JJ. 2002. Chronic renal failure-induced changes in serum and urine bile acid profiles. Dig Dis Sci 47: 2398–2406. 9) Bornfeldt KE, Tabas I. 2011. Insulin resistance, hyperglycemia, and atherosclerosis. Cell Metab 14: 575–585. 10) London GM, Drueke TB. 1997. Atherosclerosis and arteriosclerosis in chronic renal failure. Kidney Int 51: 1678–1695. 11) Ross R. 1995. Cell biology of atherosclerosis. Annu Rev Physiol 57: 791–804. 12) Qin P, Tang X, Elloso MM, Harnish DC. 2006. Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NFkappaB, and p38. Am J Physiol Heart Circ Physiol 291: H741-H747. 13) Shimizu H, Hirose Y, Nishijima F, Tsubakihara Y, Miyazaki H. 2009. ROS and PDGF-b receptors are critically involved in indoxyl sulfate actions that promote vascular smooth muscle cell proliferation and migration. Am J Physiol Cell Physiol 297: C389–C396. 14) Shimizu H, Saito S, Higashiyama Y, Nishijima F, Niwa T. 2013. CREB, NF-kB, and NADPH oxidase coordinately upregulate indoxyl sulfate-induced angiotensinogen expression in proximal tubular cells. Am J Physiol Cell Physiol 304: C685–C692. 15) Izumi Y, Kim S, Namba M, Yasumoto H, Miyazaki H, Hoshiga M, Kaneda Y, Morishita R, Zhan Y, Iwao H. 2001. Gene transfer of dominant-negative mutants of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase prevents neointimal formation in ballooninjured rat artery. Circ Res 88: 1120–1126. 16) Zhan Y, Kim S, Izumi Y, Izumiya Y, Nakao T, Miyazaki H, Iwao H. 2003. Role of JNK, p38, and ERK in plateletderived growth factor-induced vascular proliferation, migration, and gene expression. Arterioscler Thromb Vasc Biol 23: 795–801. 17) Chen A, Zhang L. 2003. The antioxidant (2)-epigallocatechin-3-gallate inhibits rat hepatic stellate cell
18)
19)
20)
21)
22)
23)
24)
25) 26)
27)
proliferation in vitro by blocking the tyrosine phosphorylation and reducing the gene expression of platelet-derived growth factor-b receptor. J Biol Chem 278: 23381–23389. Hagio M, Matsumoto M, Fukushima M, Hara H, Ishizuka S. 2009. Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS. J Lipid Res 50: 173–180. Yamakado M, Ise M. 1999. Mechanism of oral absorbent AST-120 in lipid abnormalities in experimental uremic rats. Kidney Int Suppl 71: S190–S192. Li YT, Swales KE, Thomas GJ, Warner TD, Bishop-Bailey D. 2007. Farnesoid x receptor ligands inhibit vascular smooth muscle cell inflammation and migration. Arterioscler Thromb Vasc Biol 27: 2606–2611. Zhang Q, He F, Kuruba R, Gao X, Wilson A, Li J, Billiar TR, Pitt BR, Xie W, Li S. 2008. FXR-mediated regulation of angiotensin type 2 receptor expression in vascular smooth muscle cells. Cardiovasc Res 77: 560–569. Lew JL, Zhao A, Yu J, Huang L, De Pedro N, Peláez F, Wright SD, Cui J. 2004. The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion. J Biol Chem 279: 8856–8861. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, Lehmann JM. 1999. Bile acids: natural ligands for an orphan nuclear receptor. Science 284: 1365–1368. Tamura K, Kanzaki T, Tashiro J, Yokote K, Mori S, Ueda S, Saito Y, Morisaki N. 2000. Increased atherogenesis in Otsuka Long-Evans Tokushima fatty rats before the onset of diabetes mellitus: association with overexpression of PDGF b-receptors in aortic smooth muscle cells. Atherosclerosis 149: 351–358. Getz GS, Reardon CA. 2006. Diet and murine atherosclerosis. Arterioscler Thromb Vasc Biol 26: 242–249. Nordin AA. 1968. The occurrence of plaque forming cells in normal and immunized conventional and germfree mice. Proc Soc Exp Biol Med 129: 57–62. Rault-Nania M, Gueux E, Demougeot C, Demigné C, Rock E, Mazur A. 2006. Inulin attenuates atherosclerosis in apolipoprotein E-deficient mice. Br J Nutr 96: 840–844.
