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Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells
Biochemical and Biophysical Research Communications xxx (2018) 1e6
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells Li-Jun Zhao a, *, Shi-Fang Zhang b a b
Department of Internal Medicine, West China School of Medicine, Sichuan University, Chengdu 610041, Sichuan Province, China Department of Pulmonary Disease, Institute of Respiratory Disease, Chengdu Second People's Hospital, Chengdu 610041, Sichuan Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2018 Accepted 26 April 2018 Available online xxx
Impairment of mitochondrial biogenesis has been associated with vascular pathophysiology. The Gproteinecoupled receptor (TGR5) is an important mediator of bile acid signaling and glucose metabolism. However, the effects of TGR5 on mitochondrial biogenesis in endothelial cells remain elusive. In this study, we found that activation of TGR5 using its specific agonist taurolithocholic acid (TLCA) promoted the expression of PGC-1a, a master regulator of mitochondrial biogenesis in human aortic endothelial cells (HAECs). Additionally, activation of TGR5 increased the expression of PGC-1a target genes, such as NRF1 and TFAM. Indeed, we found that TLCA treatment promoted mitochondrial biogenesis by increasing mitochondrial mass, mitochondrial-to-nuclear DNA (mtDNA/nDNA), COX-Ⅰ expression, and cytochrome c oxidase activity in HAECs. Notably, our results displayed that activation of TGR5 resulted in a functional gain in mitochondria by increasing the rate of respiration and ATP production. Mechanistically, we found that TLCA treatment activated the transcriptional factor CREB by inducing the phosphorylation of CREB at Ser133. Using the PKA/CREB inhibitor H89 abolished the effects of TLCA on PGC-1a, NRF1 and TFAM expression as well as the increase in mtDNA/nDNA and ATP production. These findings suggest that activation of TGR5 promoted mitochondrial biogenesis in endothelial cells, which is mediated by the CREB/PGC-1a signaling pathway. © 2018 Elsevier Inc. All rights reserved.
Keywords: TGR5 Endothelial cells Mitochondrial biogenesis CREB PGC-1a
1. Introduction Mitochondria are essential organelles found in most eukaryotic organisms. Mitochondria possess numerous physiological functions and act as the main source of adenosine triphosphate (ATP) within the cell environment [1]. Mitochondrial biogenesis plays a critical role in maintaining normal mitochondrial homeostasis. Endothelial mitochondria have contributed a lot to vascular pathophysiology [2]. Dysregulation of mitochondrial biogenesis has been found in various vascular diseases, including atherosclerosis, heart failure, and cardiac ischemia/reperfusion injury [3]. Peroxisome proliferator-activated receptor g-coactivator-a (PGC-1a) has been considered as the master regulator of mitochondrial biogenesis [4]. In endothelial cells, PGC-1a regulates the expression of nuclear respiratory factor 1 (NRF1) and mitochondrial transcription
* Corresponding author. Department of internal Medicine, West China School of Medicine, Sichuan Universiy, No. 37 Guoxue Ally, Chengdu 610041, Sichuan Province, China. E-mail address: [email protected] (L.-J. Zhao).
factor A (TFAM). NRF1 is responsible for governing nuclear gene to encode mitochondrial proteins. TFAM plays a key role in initiating transcription and replication of mitochondrial DNA (mtDNA) [5]. Lower levels of PGC-1a are associated with the pathological development of atherosclerosis [6]. Stimulation of mitochondrial biogenesis by promoting the PGC-1a/NRF1/TFAM pathway has been considered as an important strategy for the treatment of endothelial dysfunction in vascular diseases. The transmembrane G-protein coupled bile acid receptor (TGR5) is an important member of the G-protein-coupled receptor (GPCR) family. TGR5 is expressed in diverse tissues and organelles and mediates the intracellular signaling pathways of bile acids [7]. TGR5 has been reported to possess a variety of biological functions. For example, activation of TGR5 was found to promote energy expenditure in brown adipose tissue [8]. Also, TGR5 contributes a great deal to glucose homeostasis, potentially by increasing the secretion of GLP-1 [9]. TGR5 exerts important protective actions against gastric and liver inflammation by suppressing the NF-kB signaling pathway [10]. Interestingly, activation of TGR5 increases intracellular levels of the second messenger cyclic adenosine
https://doi.org/10.1016/j.bbrc.2018.04.210 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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monophosphate (cAMP) and activates the cAMP response elementbinding protein (CREB) transcription factor [11]. CREB plays a critical role in mediating the transcription and expression of PGC-1a [12]. However, the effects of TGR5 in mitochondrial function in endothelial cells remain unclear. The stimulatory effects of TGR5 on CREB activation lead us to speculate that activation of TGR5 might promote mitochondrial biogenesis through activation of the PGC1a pathway in endothelial cells. We aim to verify this hypothesis in this study.
random individual cells were selected to calculate average integrated optical density (IOD) of fluorescence with Image-Pro Plus software (Version 5.0) to index mitochondrial mass. 2.5. Determination of cytochrome c oxidase activity
2. Materials and methods
Cytochrome c oxidase activity in HAECs was measured by a colorimetric assay kit (Sigma-Aldrich, USA) in 96-well plates. Briefly, 10 ml cell lysate was added to assay buffer. A kinetic program for 30e45 min at 30 s intervals was run to measure cytochrome c oxidase activity.
2.1. Cell culture and treatment
2.6. Measurement of intracellular ATP
Human aortic endothelial cells (HAECs), obtained from Lonza, USA, were maintained in EBM-2 media supplemented with growth factors. Cells were treated with TLCA (10, 30, and 100 mM) to determine the expressions of PGC-1a, NRF-1, and TFAM. Cells were treated with 30 mM TLCA with or without 10 mM PKA inhibitor H89 (Sigma-Aldrich, USA) to evaluate mitochondrial biogenesis.
Intracellular levels of ATP were determined by a commercial ATP detection kit (Life Technologies, USA). Briefly, HAECs were lysed, followed by centrifugation at 12,000 g for 15 min at 4 C. Supernatant was collected to be mixed with an equal volume (50 ml) of the luciferin/luciferase reagent. Chemiluminescence signals were evaluated with a microplate luminometer.
2.2. Real-time PCR analysis and the ratio of mtDNA to nDNA
2.7. Measurement of mitochondrial respiration rate
Total intracellular RNA was isolated from HAECs using the Trizol reagent (Life Technologies, USA). Complementary DNA (cDNA) was produced with an iScript-RT kit (Bio-Rad, USA) and 2 mg RNA in a reverse transcription PCR system. Expressions of target genes were determined by real-time PCR analysis on the step one ABI 7500 real-time PCR system. Primers used in this study are listed in Table 1. The ratio of mtDNA to nDNA (mtDNA/nDNA) was indexed by mtDNA to nDNA 18S [13].
O2 consumption of HAECs was evaluated using a commercial respirometer equipped with a Peltier thermostat and electromagnetic stirrer. After the indicated treatment, 5 106 cells were collected and put in a glass chamber equilibrated in ambient room air with continuous stirring (800 r.p.m.) for 10 min. The oxygen consumption was detected at 2 S intervals and the recording was stopped after stabilization of the O2 consumption. 2.8. Statistical analysis
2.3. Western blot analysis HAECs were lysed to extract proteins. Protein concentration was measured by the BCA method (Thermo Fisher Scientific, USA). Protein samples were separated by 12% SDS-PAGE. Blots were then transferred to PVDF membranes, followed by blocking with 5% nonfat milk at RT for 2 h. Then membranes were sequentially incubated with PGC-1a, NRF-1, TFAM, COX-1, p-CREB, total CREB, and b-actin primary antibodies at 4 C overnight and HRP-conjugated secondary antibodies (1:5000) for 1 h at RT. Blots were developed by enhanced chemiluminescence (Thermo Fisher Scientific, USA). 2.4. Mitochondria mass determination Mitochondria mass in HAECs was determined by staining with the mitochondria-specific dye Mitotracker red. Briefly, HAECs (5 105) were seeded onto cover-slips in 6-well plates and incubated for 12 h. Then cells were treated with 30 mM TLCA for 48 h. After washing 3 times with HBST, HAECs were loaded with 20 nM Mitotracker red (Life Technologies, USA) for 15 min. Nuclei of HAECs were stained with DAPI. Fluorescence signals were visualized and recorded at 100 oil immersion using a Zeiss fluorescence microscope. One hundred
Experimental data are presented as means ± S.E.M. The statistical significance of differences was determined by one-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant. 3. Results 3.1. Activation of TGR5 increased the expression of PGC-1a and its target genes NRF1 and TFAM PGC-1a is a master modulator of mitochondrial biogenesis. TLCA is a well-known agonist of TGR5. Our findings showed that TLCA treatment significantly increased the expression of PGC-1a at both the mRNA level (Fig. 1A) and the protein level (Fig. 1B) in a dosedependent manner (10, 30, and 100 mM) in HAECs. NRF1 and TFAM are two important target genes of PGC-1a and the executors of mitochondrial biogenesis. Indeed, real-time PCR analysis revealed that the gene expressions of NRF1 and TFAM were markedly increased in response to treatment with 30 mM TLCA (Fig. 1C). Accordingly, western blot results displayed that 30 mM TLCA treatment significantly increased the protein expressions of
Table 1 Real-time PCR primers. Gene name
Forward
Reverse
PGC-1a NRF-1 TFAM GAPDH mtDNA nDNA (18S)
50 -CAATGAATGCAGCGGTCTTA-30 50 -CTAGTG TGGGACAGCAA-30 50 -GGCACAGGAAACCAGTTAGG-30 50 -CCACGCTCAGACACCAT-30 50 -CAAACCTACGCCAAAATCCA-30 50 -ACGGACCAGAGCGAAAGCA-30
50 -ACGTCTTTGTGGCTTTTGCT-30 50 -AATTCCGTCGATGGTGAGA-30 50 -CAGAACACCGTGGCTTCTAC-30 50 -CCAGGCGCCCAATACG-30 50 -GAAATGAATGAGCCTACAGA-30 50 -GACATCTAAGGGCATCACAGAC-30
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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Fig. 1. TGR5 agonism induced expression of PGC-1a and its target proteins NRF1 and TFAM in HAECs. (AeB). HAECs were stimulated with various concentrations of the specific TGR5 agonist taurolithocholic acid (TLCA: 10, 30, and 100 mM) for 48 h. mRNA and protein levels of PGC-1a were determined by real-time PCR and western blot analysis, respectively (*, P < 0.01 vs. non-treatment group, n ¼ 5); (CeD). HAECs were stimulated with TLCA (30 mM) for 48 h mRNA and protein levels of NRF1 and TFAM were determined by real-time PCR and western blot analysis, respectively (*, #, $ P < 0.01 vs. previous column group, n ¼ 5e6).
NRF 1 and TFAM (Fig. 1D). 3.2. Activation of TGR5 promoted mitochondrial biogenesis We then examined the biological markers of mitochondrial biogenesis. The Mitotracker red staining assay revealed that activation of TGR5 by treatment with TLCA (30 mM) increased mitochondrial mass (Fig. 2A). Increased expression of TFAM has been associated with upregulated expression of mtDNA. Importantly, TLCA (30 mM) treatment led to a significant increase in mtDNA/ nDNA (Fig. 2B). TFAM and other nuclear-encoded mitochondrial proteins enter mitochondria where they promote expression of the 13 mtDNA-encoded proteins, including respiratory chain complex I (COX-I). Here, we found that the protein expression of COX-I was significantly increased in response to TLCA treatment (Fig. 2C). Cytochrome c oxidase, an important member of the respiratory complexes, catalyzes the transfer of electrons from reduced
cytochrome c to the final acceptor of electrons. Here, we found that activation of TGR5 by treatment with TLCA (30 mM) significantly increased the activity of cytochrome c oxidase (Fig. 2D). 3.3. Activation of TGR5 led to functional gain in mitochondria Increased mitochondrial biogenesis has been associated with functional gain in mitochondria. To evaluate a possible functional gain in mitochondria induced by activation of TGR5, we assessed mitochondrial oxygen consumption and mitochondrial respiratory rate in response to TLCA (30 mM) treatment. As expected, we found that TLCA treatment significantly increased oxygen consumption by HAECs (Fig. 3A). Importantly, TLCA treatment led to a significant increase in respiratory rate from 16.3 ± 2.1 to 24.6 ± 3.0 [pmol/ (s*Mill)] (p < 0.01) (Fig. 4B). Mitochondria are the main source of intracellular ATP. Here, we found that activation of TGR5 caused by TLCA treatment significantly elevated intracellular ATP levels.
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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Fig. 2. TGR5 agonism promoted mitochondrial biogenesis in HAECs. HAECs were stimulated with TLCA (30 mM) for 48 h (A). Mitochondrial mass was determined by staining with MitoTracker red dye. The cells were visualized by fluorescence microscopy. Scale bar, 20 mm; (B). Mitochondrial-to-nuclear DNA (mtDNA/nDNA) was determined by quantitative real-time PCR and was expressed relative to control cells; (C). Representative immunoblot and quantification analysis revealed that the level of COX-Ⅰwas significantly increased by TLCA treatment; (D). Cytochrome c oxidase activity assay (*, P < 0.01 vs. vehicle group, n ¼ 5).
Fig. 3. TGR5 agonism induced a gain of mitochondrial function in HAECs. HAECs were stimulated with TLCA (30 mM) for 48 h (A). Representative recording of oxygen content reduction in non-treated (green curve) and TLCA-treated (red curve) cells; (B). Summarized mitochondrial respiratory rate obtained from 5 independent experiments in non-treated (green curve) and TLCA-treated (red curve) cells; (C). ATP production (*, P < 0.01 vs. vehicle group, n ¼ 5). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
These results suggest that activation of TGR5 by TLCA led to a functional gain in mitochondria. 3.4. The effect of TGR5 on mitochondrial biogenesis was mediated by the transcriptional factor CREB Intracellular cAMP, an important PKA activator, could activate
the transcriptional factor CREB through the PKA pathway. Activation of TGR5 has been reported to induce CREB phosphorylation by increasing intracellular cAMP. PGC1-a expression is regulated by the transcriptional factor CREB [14]. Here, we found that stimulation with TLCA (30 mM) obviously increased the phosphorylation of CREB at Ser 133 (Fig. 4A). Importantly, blockage of the PKA/CREB signaling pathway using the specific inhibitor H89 significantly
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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Fig. 4. The effects of TGR5 agonism on mitochondrial biogenesis are mediated by the CREB pathway. (A). HAECs were stimulated with TLCA (TLCA: 10, 30, and 100 mM) for 6 h. Phosphorylated levels of CREB at Ser133 was determined by western blot analysis (*, P < 0.01 vs. vehicle group, n ¼ 5); (B). HAECs were stimulated with TLCA (30 mM) in the presence or absence of CREB inhibitor H89 for 48 h. Expressions of PGC-1a, NRF1 and TFAM were determined by western blot analysis; (C). HAECs were stimulated with TLCA (30 mM) in the presence or absence of CREB inhibitor H89 for 48 h. Mitochondrial-to-nuclear DNA (mtDNA/nDNA) was determined; (D). HAECs were stimulated with TLCA (30 mM) in the presence or absence of CREB inhibitor H89 for 48 h. ATP production was determined (ANOVA, *, P < 0.01 vs. vehicle group; #, P < 0.01 vs. TLCA treatment group, n ¼ 5).
suppressed the expressions of PGC1-a, NRF-1, and TFAM (Fig. 4B). Also, the presence of H89 abolished the TLCA (30 mM)-induced increase in mtDNA/nDNA (Fig. 4C) and ATP production (Fig. 4D). These results suggest that the effects of TGR5 on mitochondrial biogenesis are mediated by the transcriptional factor CREB. 4. Discussion TGR5 is an important member of the GPCR family. It is wellknown that TGR5 acts as a receptor of bile acid and modulates its intracellular signaling pathway [15]. TGR5 is expressed in various tissues and organs. Notably, TGR5 is important for the maintenance of glucose homeostasis [16]. The effects of TGR5 in the cardiovascular system have been reported before. For example, activation of TGR5 using the semisynthetic bile acid 6a-ethyl-23(S)-methylcholic acid (6-EMCA) protected against the development of atherosclerosis by inhibiting the production of pro-inflammatory
cytokines [17]. Importantly, activation of TGR5 using TLCA reduced the expression of vascular cell adhesion molecule-1 as well as the adhesion of monocytes to endothelial cells by increasing NO production and inhibiting nuclear factor-kB (NF-kB) activation [18]. Mitochondrial health in the vascular endothelium plays a pivotal role in maintaining normal vascular physiology. However, the biological function of TGR5 on mitochondrial function, and especially mitochondrial biogenesis, remains unknown. In the current study, for the first time, we report that activation of TGR5 using TLCA could promote mitochondrial biogenesis and increase mitochondrial contents in HAECs. Firstly, treatment with TLCA increased the expression of PGC-1a, which acts as a “master switch” of mitochondrial biogenesis by regulating its downstream genes, such as NRF1 and TFAM. Secondly, TLCA treatment caused an increase in mitochondrial mass, mtDNA/nDNA, cytochrome c oxidase activity, and the expression of COX-I. Thirdly, activation of TGR5 resulted in a functional gain in mitochondria by increasing ATP generation and
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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mitochondrial respiratory rate. Finally, we found that blockage of CREB using its specific inhibitor H89 abolished the effects of TGR5 on the activation of the PGC-1a/NRF1/TFAM pathway and mitochondrial biogenesis, suggesting the potential involvement of CREB. Mitochondrial damage and dysfunction have been associated with the pathological progressions of several diseases, including vascular diseases such as atherosclerosis [19]. Particularly, impairment of mitochondrial biogenesis signaling has been associated with disruption of endothelial homeostasis. Reduced expression of PGC-1a has been found in the aortic endothelial cells of patients with atherosclerosis [20]. PGC-1a also mitigated TNF-aeinduced generation of mitochondrial ROS and expression of adhesion molecules in endothelial cells [21]. Transcriptional regulation of PGC1a is complex. CREB has been reported to be an important regulator of PGC-1a transcription. In this study, we found that the effects of TGR5 on the PGC-1a signaling pathway are mediated by CREB. Consistently, TGR5 can transduce the chemical signal through the Gs subunit to promote cAMP synthesis and CREB phosphorylation [22]. It should be noted that PGC-1a plays a pivotal role in the regulation of glucose homeostasis and energy expenditure [23]. Combining the essential roles of TGR5 in glucose homeostasis and energy balance with our findings showing that TGR5 activation promoted PGC-1a expression, we speculate that PGC-1a might play an important role in mediating the physiological function of TGR5. Taken together, results in this study suggested that TGR5 agonism might be a therapeutic approach for mitochondrial disorders in vascular diseases. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.04.210. References [1] M.A. Kluge, J.L. Fetterman, J.A. Vita, Mitochondria and endothelial function, Circ. Res. 112 (2013) 1171e1188. [2] A.O. Kadlec, A.M. Beyer, K. Ait-Aissa, D.D. Gutterman, Mitochondrial signaling in the vascular endothelium: beyond reactive oxygen species, Basic Res. Cardiol. 111 (2016) 26. [3] S. Xing, X. Yang, W. Li, F. Bian, D. Wu, J. Chi, G. Xu, Y. Zhang, S. Jin, Salidroside stimulates mitochondrial biogenesis and protects against H₂O₂-induced endothelial dysfunction, Oxid. Med. Cell Longev. 2014 (2014), 904834. [4] J.A. Villena, New insights into PGC-1 coactivators: redefining their role in the regulation of mitochondrial function and beyond, FEBS J. 282 (2015) 647e672. [5] H. Islam, B.A. Edgett, B.J. Gurd, Coordination of mitochondrial biogenesis by PGC-1a in human skeletal muscle: a re-evaluation, Metabolism 79 (2018)
42e51. [6] A.O. Kadlec, D.S. Chabowski, K. Ait-Aissa, D.D. Gutterman, Role of PGC-1a in vascular regulation: implications for atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 36 (2016) 1467e1474. €ge, L. Spomer, D. Ha €ussinger, [7] K. Deutschmann, M. Reich, C. Klindt, C. Dro V. Keitel, Bile acid receptors in the biliary tree: TGR5 in physiology and disease, Biochim. Biophys. Acta 1864 (2018) 1319e1325. [8] M. Watanabe, S.M. Houten, C. Mataki, et al., Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation, Nature 439 (2006) 484e489. [9] C. Thomas, A. Gioiello, L. Noriega, et al., TGR5-mediated bile acid sensing controls glucose homeostasis, Cell Metabol. 10 (2009) 167e177. [10] C. Guo, H. Qi, Y. Yu, Q. Zhang, J. Su, D. Yu, W. Huang, W.D. Chen, Y.D. Wang, The G-protein-coupled bile acid receptor Gpbar1 (TGR5) inhibits gastric infammation through antagonizing NF-kappaB signaling pathway, Front. Pharmacol. 6 (2015) 287. [11] I. Casaburi, P. Avena, M. Lanzino, D. Sisci, F. Giordano, P. Maris, S. Catalano, , Chenodeoxycholic acid through a TGR5-dependent CREB C. Morelli, S. Ando signaling activation enhances cyclin D1 expression and promotes human endometrial cancer cell proliferation, Cell Cycle 11 (2012) 2699e2710. [12] D.M. Lonard, B.W. O'Malley, The expanding cosmos of nuclear receptor coactivators, Cell 125 (2006) 411e414. [13] B. Sheng, X. Wang, B. Su, H.G. Lee, G. Casadesus, G. Perry, X. Zhu, Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in Alzheimer's disease, J. Neurochem. 120 (2012) 419e429. [14] S. Herzig, F. Long, U.S. Jhala, S. Hedrick, R. Quinn, A. Bauer, et al., CREB regulates hepatic gluconeogenesis through the coactivator PGC-1, Nature 413 (2001) 179e183. € ge, L. Spomer, D. Ha €ussinger, [15] K. Deutschmann, M. Reich, C. Klindt, C. Dro V. Keitel, Bile acid receptors in the biliary tree: TGR5 in physiology and disease, Biochim. Biophys. Acta 1864 (2018) 1319e1325. [16] C. Thomas, R. Pellicciari, M. Pruzanski, et al., Targeting bile-acid signalling for metabolic diseases, Nat. Rev. Drug Discov. 7 (2008) 678e693. [17] T.W. Pols, M. Nomura, T. Harach, G. Lo Sasso, M.H. Oosterveer, C. Thomas, G. Rizzo, A. Gioiello, L. Adorini, R. Pellicciari, J. Auwerx, K. Schoonjans, TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading, Cell Metabol. 14 (2011) 747e757. [18] T. Kida, Y. Tsubosaka, M. Hori, H. Ozaki, T. Murata, Bile acid receptor TGR5 agonism induces NO production and reduces monocyte adhesion in vascular endothelial cells, Arterioscler. Thromb. Vasc. Biol. 33 (2013) 1663e1669. [19] E. Yu, J. Mercer, M. Bennett, Mitochondria in vascular disease, Cardiovasc. Res. 95 (2012) 173e182. [20] C. McCarthy, N.T. Lieggi, D. Barry, D. Mooney, M. de Gaetano, W.G. James, S. McClelland, M.C. Barry, L. Escoubet-Lozach, A.C. Li, C.K. Glass, D.J. Fitzgerald, O. Belton, Macrophage PPAR gamma Co-activator-1 alpha participates in repressing foam cell formation and atherosclerosis in response to conjugated linoleic acid, EMBO Mol. Med. 5 (2013) 1443e1457. [21] H.J. Kim, K.G. Park, E.K. Yoo, Y.H. Kim, Y.N. Kim, H.S. Kim, H.T. Kim, J.Y. Park, K.U. Lee, W.G. Jang, J.G. Kim, B.W. Kim, I.K. Lee, Effects of PGC-1alpha on TNFalpha-induced MCP-1 and VCAM-1 expression and NF-kappaB activation in human aortic smooth muscle and endothelial cells, Antioxidants Redox Signal.Sig. 9 (2007) 301e307. [22] I. Casaburi, P. Avena, M. Lanzino, D. Sisci, F. Giordano, P. Maris, S. Catalano, , Chenodeoxycholic acid through a TGR5-dependent CREB C. Morelli, S. Ando signaling activation enhances cyclin D1 expression and promotes human endometrial cancer cell proliferation, Cell Cycle 11 (2012) 2699e2710. [23] G.C. Rowe, Z. Arany, Genetic models of PGC-1 and glucose metabolism and homeostasis, Rev. Endocr. Metab. Disord. 15 (2014) 21e29.
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells Li-Jun Zhao a, *, Shi-Fang Zhang b a b
Department of Internal Medicine, West China School of Medicine, Sichuan University, Chengdu 610041, Sichuan Province, China Department of Pulmonary Disease, Institute of Respiratory Disease, Chengdu Second People's Hospital, Chengdu 610041, Sichuan Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2018 Accepted 26 April 2018 Available online xxx
Impairment of mitochondrial biogenesis has been associated with vascular pathophysiology. The Gproteinecoupled receptor (TGR5) is an important mediator of bile acid signaling and glucose metabolism. However, the effects of TGR5 on mitochondrial biogenesis in endothelial cells remain elusive. In this study, we found that activation of TGR5 using its specific agonist taurolithocholic acid (TLCA) promoted the expression of PGC-1a, a master regulator of mitochondrial biogenesis in human aortic endothelial cells (HAECs). Additionally, activation of TGR5 increased the expression of PGC-1a target genes, such as NRF1 and TFAM. Indeed, we found that TLCA treatment promoted mitochondrial biogenesis by increasing mitochondrial mass, mitochondrial-to-nuclear DNA (mtDNA/nDNA), COX-Ⅰ expression, and cytochrome c oxidase activity in HAECs. Notably, our results displayed that activation of TGR5 resulted in a functional gain in mitochondria by increasing the rate of respiration and ATP production. Mechanistically, we found that TLCA treatment activated the transcriptional factor CREB by inducing the phosphorylation of CREB at Ser133. Using the PKA/CREB inhibitor H89 abolished the effects of TLCA on PGC-1a, NRF1 and TFAM expression as well as the increase in mtDNA/nDNA and ATP production. These findings suggest that activation of TGR5 promoted mitochondrial biogenesis in endothelial cells, which is mediated by the CREB/PGC-1a signaling pathway. © 2018 Elsevier Inc. All rights reserved.
Keywords: TGR5 Endothelial cells Mitochondrial biogenesis CREB PGC-1a
1. Introduction Mitochondria are essential organelles found in most eukaryotic organisms. Mitochondria possess numerous physiological functions and act as the main source of adenosine triphosphate (ATP) within the cell environment [1]. Mitochondrial biogenesis plays a critical role in maintaining normal mitochondrial homeostasis. Endothelial mitochondria have contributed a lot to vascular pathophysiology [2]. Dysregulation of mitochondrial biogenesis has been found in various vascular diseases, including atherosclerosis, heart failure, and cardiac ischemia/reperfusion injury [3]. Peroxisome proliferator-activated receptor g-coactivator-a (PGC-1a) has been considered as the master regulator of mitochondrial biogenesis [4]. In endothelial cells, PGC-1a regulates the expression of nuclear respiratory factor 1 (NRF1) and mitochondrial transcription
* Corresponding author. Department of internal Medicine, West China School of Medicine, Sichuan Universiy, No. 37 Guoxue Ally, Chengdu 610041, Sichuan Province, China. E-mail address: [email protected] (L.-J. Zhao).
factor A (TFAM). NRF1 is responsible for governing nuclear gene to encode mitochondrial proteins. TFAM plays a key role in initiating transcription and replication of mitochondrial DNA (mtDNA) [5]. Lower levels of PGC-1a are associated with the pathological development of atherosclerosis [6]. Stimulation of mitochondrial biogenesis by promoting the PGC-1a/NRF1/TFAM pathway has been considered as an important strategy for the treatment of endothelial dysfunction in vascular diseases. The transmembrane G-protein coupled bile acid receptor (TGR5) is an important member of the G-protein-coupled receptor (GPCR) family. TGR5 is expressed in diverse tissues and organelles and mediates the intracellular signaling pathways of bile acids [7]. TGR5 has been reported to possess a variety of biological functions. For example, activation of TGR5 was found to promote energy expenditure in brown adipose tissue [8]. Also, TGR5 contributes a great deal to glucose homeostasis, potentially by increasing the secretion of GLP-1 [9]. TGR5 exerts important protective actions against gastric and liver inflammation by suppressing the NF-kB signaling pathway [10]. Interestingly, activation of TGR5 increases intracellular levels of the second messenger cyclic adenosine
https://doi.org/10.1016/j.bbrc.2018.04.210 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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monophosphate (cAMP) and activates the cAMP response elementbinding protein (CREB) transcription factor [11]. CREB plays a critical role in mediating the transcription and expression of PGC-1a [12]. However, the effects of TGR5 in mitochondrial function in endothelial cells remain unclear. The stimulatory effects of TGR5 on CREB activation lead us to speculate that activation of TGR5 might promote mitochondrial biogenesis through activation of the PGC1a pathway in endothelial cells. We aim to verify this hypothesis in this study.
random individual cells were selected to calculate average integrated optical density (IOD) of fluorescence with Image-Pro Plus software (Version 5.0) to index mitochondrial mass. 2.5. Determination of cytochrome c oxidase activity
2. Materials and methods
Cytochrome c oxidase activity in HAECs was measured by a colorimetric assay kit (Sigma-Aldrich, USA) in 96-well plates. Briefly, 10 ml cell lysate was added to assay buffer. A kinetic program for 30e45 min at 30 s intervals was run to measure cytochrome c oxidase activity.
2.1. Cell culture and treatment
2.6. Measurement of intracellular ATP
Human aortic endothelial cells (HAECs), obtained from Lonza, USA, were maintained in EBM-2 media supplemented with growth factors. Cells were treated with TLCA (10, 30, and 100 mM) to determine the expressions of PGC-1a, NRF-1, and TFAM. Cells were treated with 30 mM TLCA with or without 10 mM PKA inhibitor H89 (Sigma-Aldrich, USA) to evaluate mitochondrial biogenesis.
Intracellular levels of ATP were determined by a commercial ATP detection kit (Life Technologies, USA). Briefly, HAECs were lysed, followed by centrifugation at 12,000 g for 15 min at 4 C. Supernatant was collected to be mixed with an equal volume (50 ml) of the luciferin/luciferase reagent. Chemiluminescence signals were evaluated with a microplate luminometer.
2.2. Real-time PCR analysis and the ratio of mtDNA to nDNA
2.7. Measurement of mitochondrial respiration rate
Total intracellular RNA was isolated from HAECs using the Trizol reagent (Life Technologies, USA). Complementary DNA (cDNA) was produced with an iScript-RT kit (Bio-Rad, USA) and 2 mg RNA in a reverse transcription PCR system. Expressions of target genes were determined by real-time PCR analysis on the step one ABI 7500 real-time PCR system. Primers used in this study are listed in Table 1. The ratio of mtDNA to nDNA (mtDNA/nDNA) was indexed by mtDNA to nDNA 18S [13].
O2 consumption of HAECs was evaluated using a commercial respirometer equipped with a Peltier thermostat and electromagnetic stirrer. After the indicated treatment, 5 106 cells were collected and put in a glass chamber equilibrated in ambient room air with continuous stirring (800 r.p.m.) for 10 min. The oxygen consumption was detected at 2 S intervals and the recording was stopped after stabilization of the O2 consumption. 2.8. Statistical analysis
2.3. Western blot analysis HAECs were lysed to extract proteins. Protein concentration was measured by the BCA method (Thermo Fisher Scientific, USA). Protein samples were separated by 12% SDS-PAGE. Blots were then transferred to PVDF membranes, followed by blocking with 5% nonfat milk at RT for 2 h. Then membranes were sequentially incubated with PGC-1a, NRF-1, TFAM, COX-1, p-CREB, total CREB, and b-actin primary antibodies at 4 C overnight and HRP-conjugated secondary antibodies (1:5000) for 1 h at RT. Blots were developed by enhanced chemiluminescence (Thermo Fisher Scientific, USA). 2.4. Mitochondria mass determination Mitochondria mass in HAECs was determined by staining with the mitochondria-specific dye Mitotracker red. Briefly, HAECs (5 105) were seeded onto cover-slips in 6-well plates and incubated for 12 h. Then cells were treated with 30 mM TLCA for 48 h. After washing 3 times with HBST, HAECs were loaded with 20 nM Mitotracker red (Life Technologies, USA) for 15 min. Nuclei of HAECs were stained with DAPI. Fluorescence signals were visualized and recorded at 100 oil immersion using a Zeiss fluorescence microscope. One hundred
Experimental data are presented as means ± S.E.M. The statistical significance of differences was determined by one-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant. 3. Results 3.1. Activation of TGR5 increased the expression of PGC-1a and its target genes NRF1 and TFAM PGC-1a is a master modulator of mitochondrial biogenesis. TLCA is a well-known agonist of TGR5. Our findings showed that TLCA treatment significantly increased the expression of PGC-1a at both the mRNA level (Fig. 1A) and the protein level (Fig. 1B) in a dosedependent manner (10, 30, and 100 mM) in HAECs. NRF1 and TFAM are two important target genes of PGC-1a and the executors of mitochondrial biogenesis. Indeed, real-time PCR analysis revealed that the gene expressions of NRF1 and TFAM were markedly increased in response to treatment with 30 mM TLCA (Fig. 1C). Accordingly, western blot results displayed that 30 mM TLCA treatment significantly increased the protein expressions of
Table 1 Real-time PCR primers. Gene name
Forward
Reverse
PGC-1a NRF-1 TFAM GAPDH mtDNA nDNA (18S)
50 -CAATGAATGCAGCGGTCTTA-30 50 -CTAGTG TGGGACAGCAA-30 50 -GGCACAGGAAACCAGTTAGG-30 50 -CCACGCTCAGACACCAT-30 50 -CAAACCTACGCCAAAATCCA-30 50 -ACGGACCAGAGCGAAAGCA-30
50 -ACGTCTTTGTGGCTTTTGCT-30 50 -AATTCCGTCGATGGTGAGA-30 50 -CAGAACACCGTGGCTTCTAC-30 50 -CCAGGCGCCCAATACG-30 50 -GAAATGAATGAGCCTACAGA-30 50 -GACATCTAAGGGCATCACAGAC-30
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Fig. 1. TGR5 agonism induced expression of PGC-1a and its target proteins NRF1 and TFAM in HAECs. (AeB). HAECs were stimulated with various concentrations of the specific TGR5 agonist taurolithocholic acid (TLCA: 10, 30, and 100 mM) for 48 h. mRNA and protein levels of PGC-1a were determined by real-time PCR and western blot analysis, respectively (*, P < 0.01 vs. non-treatment group, n ¼ 5); (CeD). HAECs were stimulated with TLCA (30 mM) for 48 h mRNA and protein levels of NRF1 and TFAM were determined by real-time PCR and western blot analysis, respectively (*, #, $ P < 0.01 vs. previous column group, n ¼ 5e6).
NRF 1 and TFAM (Fig. 1D). 3.2. Activation of TGR5 promoted mitochondrial biogenesis We then examined the biological markers of mitochondrial biogenesis. The Mitotracker red staining assay revealed that activation of TGR5 by treatment with TLCA (30 mM) increased mitochondrial mass (Fig. 2A). Increased expression of TFAM has been associated with upregulated expression of mtDNA. Importantly, TLCA (30 mM) treatment led to a significant increase in mtDNA/ nDNA (Fig. 2B). TFAM and other nuclear-encoded mitochondrial proteins enter mitochondria where they promote expression of the 13 mtDNA-encoded proteins, including respiratory chain complex I (COX-I). Here, we found that the protein expression of COX-I was significantly increased in response to TLCA treatment (Fig. 2C). Cytochrome c oxidase, an important member of the respiratory complexes, catalyzes the transfer of electrons from reduced
cytochrome c to the final acceptor of electrons. Here, we found that activation of TGR5 by treatment with TLCA (30 mM) significantly increased the activity of cytochrome c oxidase (Fig. 2D). 3.3. Activation of TGR5 led to functional gain in mitochondria Increased mitochondrial biogenesis has been associated with functional gain in mitochondria. To evaluate a possible functional gain in mitochondria induced by activation of TGR5, we assessed mitochondrial oxygen consumption and mitochondrial respiratory rate in response to TLCA (30 mM) treatment. As expected, we found that TLCA treatment significantly increased oxygen consumption by HAECs (Fig. 3A). Importantly, TLCA treatment led to a significant increase in respiratory rate from 16.3 ± 2.1 to 24.6 ± 3.0 [pmol/ (s*Mill)] (p < 0.01) (Fig. 4B). Mitochondria are the main source of intracellular ATP. Here, we found that activation of TGR5 caused by TLCA treatment significantly elevated intracellular ATP levels.
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Fig. 2. TGR5 agonism promoted mitochondrial biogenesis in HAECs. HAECs were stimulated with TLCA (30 mM) for 48 h (A). Mitochondrial mass was determined by staining with MitoTracker red dye. The cells were visualized by fluorescence microscopy. Scale bar, 20 mm; (B). Mitochondrial-to-nuclear DNA (mtDNA/nDNA) was determined by quantitative real-time PCR and was expressed relative to control cells; (C). Representative immunoblot and quantification analysis revealed that the level of COX-Ⅰwas significantly increased by TLCA treatment; (D). Cytochrome c oxidase activity assay (*, P < 0.01 vs. vehicle group, n ¼ 5).
Fig. 3. TGR5 agonism induced a gain of mitochondrial function in HAECs. HAECs were stimulated with TLCA (30 mM) for 48 h (A). Representative recording of oxygen content reduction in non-treated (green curve) and TLCA-treated (red curve) cells; (B). Summarized mitochondrial respiratory rate obtained from 5 independent experiments in non-treated (green curve) and TLCA-treated (red curve) cells; (C). ATP production (*, P < 0.01 vs. vehicle group, n ¼ 5). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
These results suggest that activation of TGR5 by TLCA led to a functional gain in mitochondria. 3.4. The effect of TGR5 on mitochondrial biogenesis was mediated by the transcriptional factor CREB Intracellular cAMP, an important PKA activator, could activate
the transcriptional factor CREB through the PKA pathway. Activation of TGR5 has been reported to induce CREB phosphorylation by increasing intracellular cAMP. PGC1-a expression is regulated by the transcriptional factor CREB [14]. Here, we found that stimulation with TLCA (30 mM) obviously increased the phosphorylation of CREB at Ser 133 (Fig. 4A). Importantly, blockage of the PKA/CREB signaling pathway using the specific inhibitor H89 significantly
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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Fig. 4. The effects of TGR5 agonism on mitochondrial biogenesis are mediated by the CREB pathway. (A). HAECs were stimulated with TLCA (TLCA: 10, 30, and 100 mM) for 6 h. Phosphorylated levels of CREB at Ser133 was determined by western blot analysis (*, P < 0.01 vs. vehicle group, n ¼ 5); (B). HAECs were stimulated with TLCA (30 mM) in the presence or absence of CREB inhibitor H89 for 48 h. Expressions of PGC-1a, NRF1 and TFAM were determined by western blot analysis; (C). HAECs were stimulated with TLCA (30 mM) in the presence or absence of CREB inhibitor H89 for 48 h. Mitochondrial-to-nuclear DNA (mtDNA/nDNA) was determined; (D). HAECs were stimulated with TLCA (30 mM) in the presence or absence of CREB inhibitor H89 for 48 h. ATP production was determined (ANOVA, *, P < 0.01 vs. vehicle group; #, P < 0.01 vs. TLCA treatment group, n ¼ 5).
suppressed the expressions of PGC1-a, NRF-1, and TFAM (Fig. 4B). Also, the presence of H89 abolished the TLCA (30 mM)-induced increase in mtDNA/nDNA (Fig. 4C) and ATP production (Fig. 4D). These results suggest that the effects of TGR5 on mitochondrial biogenesis are mediated by the transcriptional factor CREB. 4. Discussion TGR5 is an important member of the GPCR family. It is wellknown that TGR5 acts as a receptor of bile acid and modulates its intracellular signaling pathway [15]. TGR5 is expressed in various tissues and organs. Notably, TGR5 is important for the maintenance of glucose homeostasis [16]. The effects of TGR5 in the cardiovascular system have been reported before. For example, activation of TGR5 using the semisynthetic bile acid 6a-ethyl-23(S)-methylcholic acid (6-EMCA) protected against the development of atherosclerosis by inhibiting the production of pro-inflammatory
cytokines [17]. Importantly, activation of TGR5 using TLCA reduced the expression of vascular cell adhesion molecule-1 as well as the adhesion of monocytes to endothelial cells by increasing NO production and inhibiting nuclear factor-kB (NF-kB) activation [18]. Mitochondrial health in the vascular endothelium plays a pivotal role in maintaining normal vascular physiology. However, the biological function of TGR5 on mitochondrial function, and especially mitochondrial biogenesis, remains unknown. In the current study, for the first time, we report that activation of TGR5 using TLCA could promote mitochondrial biogenesis and increase mitochondrial contents in HAECs. Firstly, treatment with TLCA increased the expression of PGC-1a, which acts as a “master switch” of mitochondrial biogenesis by regulating its downstream genes, such as NRF1 and TFAM. Secondly, TLCA treatment caused an increase in mitochondrial mass, mtDNA/nDNA, cytochrome c oxidase activity, and the expression of COX-I. Thirdly, activation of TGR5 resulted in a functional gain in mitochondria by increasing ATP generation and
Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210
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mitochondrial respiratory rate. Finally, we found that blockage of CREB using its specific inhibitor H89 abolished the effects of TGR5 on the activation of the PGC-1a/NRF1/TFAM pathway and mitochondrial biogenesis, suggesting the potential involvement of CREB. Mitochondrial damage and dysfunction have been associated with the pathological progressions of several diseases, including vascular diseases such as atherosclerosis [19]. Particularly, impairment of mitochondrial biogenesis signaling has been associated with disruption of endothelial homeostasis. Reduced expression of PGC-1a has been found in the aortic endothelial cells of patients with atherosclerosis [20]. PGC-1a also mitigated TNF-aeinduced generation of mitochondrial ROS and expression of adhesion molecules in endothelial cells [21]. Transcriptional regulation of PGC1a is complex. CREB has been reported to be an important regulator of PGC-1a transcription. In this study, we found that the effects of TGR5 on the PGC-1a signaling pathway are mediated by CREB. Consistently, TGR5 can transduce the chemical signal through the Gs subunit to promote cAMP synthesis and CREB phosphorylation [22]. It should be noted that PGC-1a plays a pivotal role in the regulation of glucose homeostasis and energy expenditure [23]. Combining the essential roles of TGR5 in glucose homeostasis and energy balance with our findings showing that TGR5 activation promoted PGC-1a expression, we speculate that PGC-1a might play an important role in mediating the physiological function of TGR5. Taken together, results in this study suggested that TGR5 agonism might be a therapeutic approach for mitochondrial disorders in vascular diseases. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.04.210. References [1] M.A. Kluge, J.L. Fetterman, J.A. Vita, Mitochondria and endothelial function, Circ. Res. 112 (2013) 1171e1188. [2] A.O. Kadlec, A.M. Beyer, K. Ait-Aissa, D.D. Gutterman, Mitochondrial signaling in the vascular endothelium: beyond reactive oxygen species, Basic Res. Cardiol. 111 (2016) 26. [3] S. Xing, X. Yang, W. Li, F. Bian, D. Wu, J. Chi, G. Xu, Y. Zhang, S. Jin, Salidroside stimulates mitochondrial biogenesis and protects against H₂O₂-induced endothelial dysfunction, Oxid. Med. Cell Longev. 2014 (2014), 904834. [4] J.A. Villena, New insights into PGC-1 coactivators: redefining their role in the regulation of mitochondrial function and beyond, FEBS J. 282 (2015) 647e672. [5] H. Islam, B.A. Edgett, B.J. Gurd, Coordination of mitochondrial biogenesis by PGC-1a in human skeletal muscle: a re-evaluation, Metabolism 79 (2018)
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Please cite this article in press as: L.-J. Zhao, S.-F. Zhang, Activation of TGR5 promotes mitochondrial biogenesis in human aortic endothelial cells, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.04.210