Skip to main content Accessibility help

Molecular characteristics and induction profiles of hypoxia-inducible factor-1α and other basic helix–loop–helix and Per–Arnt–Sim domain-containing proteins identified in a carcinogenic liver fluke Clonorchis sinensis

  • Seon-Hee Kim (a1), Gyu-Seok Oh (a1), Woon-Mok Sohn (a2), Kihyun Lee (a3), Hyun-Jong Yang (a4) and Young-An Bae (a1)...


Clonorchis sinensis (C. sinensis), a trematode parasite that invades the hypoxic hepatobiliary tract of vertebrate hosts requires a considerable amount of oxygen for its sexual reproduction and energy metabolism. However, little is known regarding the molecular mechanism of C. sinensis involved in the adaptation to the hypoxic environments. In this study, we investigated the molecular structures and induction patterns of hypoxia-inducible factor-1α (HIF-1α) and other basic helix–loop–helix and Per–Arnt–Sim (bHLH–PAS) domain-containing proteins such as HIF-1β, single-minded protein and aryl hydrocarbon receptor, which might prompt adaptive response to hypoxia, in C. sinensis. These proteins possessed various bHLH–PAS family-specific domains. Expression of C. sinensis HIF-1α (CsHIF-1α) was highly induced in worms which were either exposed to a hypoxic condition or co-incubated with human cholangiocytes. In addition to oxygen, nitric oxide and nitrite affected the CsHIF-1α expression depending on the surrounding oxygen concentration. Treatment using a prolyl hydroxylase-domain protein inhibitor under 20%-oxygen condition resulted in an increase in the CsHIF-1α level. Conversely, the other bHLH–PAS genes were less responsive to these exogenous stimuli. We suggest that nitrite and nitric oxide, as well as oxygen, coordinately involve in the regulation of HIF-1α expression to adapt to the hypoxic host environments in C. sinensis.


Corresponding author

Author for correspondence: Young-An Bae, E-mail:


Hide All
Adachi, J, Mori, Y, Matsui, S, Takigami, H, Fujino, J, Kitagawa, H, Miller, CA III, Kato, T, Saeki, K and Matsuda, T (2001) Indirubin and indigo are potent aryl hydrocarbon receptor ligands present in human urine. The Journal of Biological Chemistry 276, 3147531478.
Bae, YA, Ahn, DW, Lee, EG, Kim, SH, Cai, GB, Kang, I, Sohn, WM and Kong, Y (2013 a) Differential activation of diverse glutathione transferases of Clonorchis sinensis in response to the host bile and oxidative stressors. PLoS Neglected Tropical Diseases 7, e2211.
Bae, YA, Cai, GB, Kim, SH, Sohn, WM and Kong, Y (2013 b) Expression pattern and substrate specificity of Clonorchis sinensis tyrosinases. International Journal for Parasitology 43, 891900.
Bailey-Serres, J, Fukao, T, Gibbs, DJ, Holdsworth, MJ, Lee, SC, Licausi, F, Perata, P, Voesenek, ACJ and van Dongen, JT (2012) Making sense of low oxygen sensing. Trends in Plant Science 17, 129138.
Bartels, K, Grenz, A and Eltzschig, HK (2013) Hypoxia and inflammation are two sides of the same coin. Proceedings of the National Academy of Sciences of the United States of America 110, 1835118352.
Bertout, JA, Patel, SA and Simon, MC (2008) The impact of O2 availability on human cancer. Nature Reviews. Cancer 8, 967975.
Biddlestone, J, Bandarra, D and Rocha, S (2015) The role of hypoxia in inflammatory disease (review). International Journal of Molecular Medicine 35, 859869.
Bouvard, V, Baan, R, Straif, K, Grosse, Y, Secretan, B, El Ghissassi, F, Benbrahim-Tallaa, L, Guha, N, Freeman, C, Galichet, L, Cogliano, V and WHO International Agency for Research on Cancer Monograph Working Group (2009) A review of human carcinogens – part B: biological agents. The Lancet Oncology 10, 321322.
Brook, I (1989) Aerobic and anaerobic microbiology of biliary tract disease. Journal of Clinical Microbiology 27, 23732375.
Chandel, NS and Budinger, GR (2007) The cellular basis for diverse responses to oxygen. Free Radical Biology and Medicine 42, 165174.
Chandrasekharan, JA and Sharma-Walia, N (2015) Lipoxins: nature's way to resolve inflammation. Journal of Inflammation Research 30, 181192.
Cordingley, JS (1987) Trematode eggshells: novel protein biopolymers. Parasitology Today 3, 341344.
Cossins, A and Berenbrink, M (2008) Physiology: myoglobin's new clothes. Nature 454, 416417.
Crews, ST (1998) Control of cell lineage-specific development and transcription by bHLH-PAS proteins. Genes & Development 12, 607620.
Dennis, EA and Norris, PC (2015) Eicosanoid storm in infection and inflammation. Nature Reviews. Immunology 15, 511523.
Dyson, A and Singer, M (2011) Tissue oxygen tension monitoring: will it fill the void? Current Opinion in Critical Care 17, 281289.
Edgar, RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 17921797.
Fritsch, P, de Saint Blanquat, G and Klein, D (1985) Excretion of nitrates and nitrites in saliva and bile in the dog. Food and Chemical Toxicology 23, 655659.
Goto, M, Amino, H, Nakajima, M, Tsuji, N, Sakamoto, K and Kita, K (2013) Cloning and characterization of hypoxia-inducible factor-1 subunits from Ascaris suum – a parasitic nematode highly adapted to change of oxygen conditions during its life cycle. Gene 516, 3947.
Gunda, VG and Janapala, VR (2009) Effects of dissolved oxygen levels on survival and growth of Haliclona pigmentifera (Demospongiae). Cell and Tissue Research 337, 527535.
Huang, J, Zhao, Q, Mooney, SM and Lee, FS (2002) Sequence determinants in hypoxia-inducible factor-1 alpha for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3. The Journal of Biological Chemistry 277, 3979239800.
Huang, X and Madan, A (1999) CAP3: a DNA sequence assembly program. Genome Research 9, 868877.
Huang, Y, Chen, W, Wang, X, Liu, H, Chen, Y, Guo, L, Luo, F, Sun, J, Mao, Q, Liang, P, Xie, Z, Zhou, C, Tian, Y, Lv, X, Huang, L, Zhou, J, Hu, Y, Li, R, Zhang, F, Lei, H, Li, W, Hu, X, Liang, C, Xu, J, Li, X and Yu, X (2013) The carcinogenic liver fluke, Clonorchis sinensis: new assembly, reannotation and analysis of the genome and characterization of tissue transcriptomes. PLoS ONE 8, e54732.
Jiang, H, Guo, R and Powell-Coffman, J (2001) The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia. Proceedings of the National Academy of Sciences of the United States of America 98, 79167921.
Jürgens, KD, Papadopoulos, S, Peters, T and Gros, G (2000) Myoglobin: just an oxygen store or also an oxygen transporter? News in Physiological Sciences 15, 269274.
Keiser, J and Utzinger, J (2009) Food-borne trematodiases. Clinical Microbiology Reviews 22, 466483.
Kiger, L, Rashid, AK, Griffon, N, Haque, M, Moens, L, Gibson, QH, Poyart, C and Marden, MC (1998) Trematode hemoglobins show exceptionally high oxygen affinity. Biophysical Journal 75, 990998.
Kim, TI, Yoo, YG, Kwak, BK, Seok, JW and Hong, SJ (2011) Tracing of the bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT. PLoS Neglected Tropical Diseases 5, e1414.
Kiriakidis, S, Esteban, MA and Maxwell, PH (2007) Genetic insights into the hypoxia-inducible factor (HIF) pathway. Advances in Enzyme Regulation 47, 288306.
Li, KL, Lu, TM and Yu, JK (2014) Genome-wide survey and expression analysis of the bHLH-PAS genes in the amphioxus Branchiostoma floridae reveal both conserved and diverged expression patterns between cephalochordates and vertebrates. EvoDevo 5, 20.
Livak, KJ and Schmittgen, TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25, 402408.
Loenarz, C, Coleman, ML, Boleininger, A, Schierwater, B, Holland, PWH, Ratcliffe, PJ and Schofield, CJ (2011) The hypoxia-inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adherens. EMBO Reports 12, 6370.
Lun, ZR, Gasser, RB, Lai, DH, Li, AX, Zhu, XQ, Yu, XB and Fang, YY (2005) Clonorchiasis: a key foodborne zoonosis in China. The Lancet. Infectious Diseases 5, 3141.
Majmundar, AJ, Wong, WJ and Simon, MC (2010) Hypoxia-inducible factors and the response to hypoxic stress. Molecular Cell 40, 294309.
Mao, Q, Xie, Z, Wang, X, Chen, W, Ren, M, Shang, M, Lei, H, Tian, Y, Li, S, Liang, P, Chen, T, Liang, C, Xu, J, Li, X, Huang, Y and Yu, X (2015) Clonorchis sinensis ferritin heavy chain triggers free radicals and mediates inflammation signaling in human hepatic stellate cells. Parasitology Research 114, 659670.
Mei, Q and Dvornyk, V (2014) Evolution of PAS domains and PAS-containing genes in eukaryotes. Chromosoma 123, 385405.
Olson, N and van der Vliet, A (2011) Interactions between nitric oxide and hypoxia-inducible factor signaling pathways in inflammatory disease. Nitric Oxide 25, 125137.
Page, RD (1996) Tree view: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357358.
Powell-Coffman, J, Bradfield, CA and Wood, WB (1998) Caenorhabditis elegans orthologs of the aryl hydrocarbon receptor and its heterodimerization partner the aryl hydrocarbon nuclear translocator. Proceedings of the National Academy of Sciences of the United States of America 95, 28442849.
Qian, MB, Utzinger, J, Keiser, J and Zhou, XN (2016) Clonorchiasis. Lancet 387, 800810.
Rytkönen, KT, Williams, TA, Renshaw, GM, Primmer, CR and Nikinmaa, M (2011) Molecular evolution of the metazoan PHD-HIF oxygen-sensing system. Molecular Biology and Evolution 28, 19131926.
Sardiello, M, Palmieri, M, di Ronza, A, Medina, DL, Valenza, M, Gennarino, VA, Di Malta, C, Donaudy, F, Embrione, V, Polishchuk, RS, Banfi, S, Parenti, G, Cattaneo, E and Ballabio, A (2009) A gene network regulating lysosomal biogenesis and function. Science 325, 473477.
Schultz, J, Milpetz, F, Bork, P and Ponting, CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proceedings of the National Academy of Sciences of the United States of America 95, 58575864.
Seidel, SD, Winters, GM, Rogers, WJ, Ziccardi, MH, Li, V, Keser, B and Denison, MS (2001) Activation of the Ah receptor signaling pathway by prostaglandins. Journal of Biochemical and Molecular Toxicology 15, 187196.
Semenza, GL (2009) Regulation of oxygen homeostasis by hypoxia-inducible factor 1. Physiology 24, 97106.
Shin, HR, Oh, JK, Masuyer, E, Curado, MP, Bouvard, V, Fang, YY, Wianqnon, S, Sripa, B and Hong, ST (2010) Epidemiology of cholangiocarcinoma: an update focusing on risk factors. Cancer Science 101, 579585.
Sinal, CJ and Bend, JR (1997) Aryl hydrocarbon receptor-dependent induction of Cyp1a1 by bilirubin in mouse hepatoma hepa 1c1c7 cells. Molecular Pharmacology 52, 590599.
Smyth, JD and Halton, DW (1983) The Physiology of Trematodes, 2nd Edn. New York, NK: Cambridge University Press.
Sorci, G and Faivre, B (2009) Inflammation and oxidative stress in vertebrate host-parasite systems. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 364, 7183.
Tamura, K, Stecher, G, Peterson, D, Filipski, A and Kumar, S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 27252729.
Tielens, AG, van den Heuvel, JM and van den Bergh, SG (1984) The energy metabolism of Fasciola hepatica during its development in the final host. Molecular and Biochemical Parasitology 13, 301307.
Wang, G, Yu, Z, Zhen, Y, Mi, T, Shi, Y, Wang, J, Wang, M and Sun, S (2014) Molecular characterization, evolution and expression of hypoxia-inducible factor in Aurelia sp.1. PLoS ONE 9, e100057.
Wang, GL and Semenza, GL (1993) General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proceedings of the National Academy of Sciences of the United States of America 90, 43044308.
Wang, GL, Jiang, BH, Rue, EA and Semenza, GL (1995) Hypoxia-inducible factor 1 is a basic helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proceedings of the National Academy of Sciences of the United States of America 92, 55105514.
Wittenberg, JB (1959) Oxygen transport: a new function proposed for myoglobin. The Biological Bulletin 117, 402403.
Yang, QL, Shen, JQ, Xue, Y, Cheng, XB, Jiang, ZH, Yang, YC, Chen, YD and Zhou, XN (2015) Pathological lesions and inducible nitric oxide synthase expressions in the liver of mice experimentally infected with Clonorchis sinensis. Korean Journal of Parasitology 53, 777783.
Young, ND, Campbell, BE, Hall, RS, Jex, AR, Cantacessi, C, Laha, T, Sohn, WM, Sripa, B, Loukas, A, Brindley, PJ and Gasser, RB (2010) Unlocking the transcriptomes of two carcinogenic parasites, Clonorchis sinensis and Opisthorchis viverrini. PLoS Neglected Tropical Diseases 4, e719.
Young, ND, Nagarajan, N, Lin, SJ, Korhonen, PK, Jex, AR, Hall, RS, Safavi-Hemami, H, Kaewkong, W, Bertrand, D, Gao, S, Seet, Q, Wongkham, S, The, BT, Wongkham, C, Intapan, PM, Maleewong, W, Yang, X, Hu, M, Wang, Z, Hofmann, A, Sternberg, PW, Tan, P, Wang, J and Gasser, RB (2014) The Opisthorchis viverrini genome provides insights into life in the bile duct. Nature Communications 5, 4378.
Zhang, N, Fu, Z, Linke, S, Chicher, J, Gorman, JJ, Visk, D, Haddad, GG, Poellinger, L, Peet, DJ, Powell, F and Johnson, RS (2010) The asparaginyl hydroxylase factor inhibiting HIF-1α is an essential regulator of metabolism. Cell Metabolism 11, 364378.
Zhang, Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40.
Zhdanov, AV, Okkelman, IA, Collins, FW, Melgar, S and Papkovsky, DB (2015) A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF-target gene expression. Biochimica et Biophysica Acta 1847, 12541266.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 0031-1820
  • EISSN: 1469-8161
  • URL: /core/journals/parasitology
Please enter your name
Please enter a valid email address
Who would you like to send this to? *


Type Description Title
Supplementary materials

Kim et al. supplementary material
Tables S1-S2 and Figures S1-S2

 Word (663 KB)
663 KB


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed