Regulation of complement C3 gene in the human hepatoma cells HepG2 under oxidative stress

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Accumulation of reactive oxygen and nitrogen species in cells during oxidative stress leads to oxidative damage to various cellular components, including DNA, proteins, and lipids, and, as a consequence, to the development of a number of severe diseases, such as atherosclerosis. Protein C3 is a central component of the complement cascade and a key player in the immune system. Proinflammatory activity of C3 can also contribute to the development of metabolic syndrome. Although hepatocytes are the main source of C3 in blood, regulation of C3 gene expression in hepatocytes under oxidative stress remains unexplored. Here we observe the suppression of C3 transcription and secretion during hydrogen peroxide-induced oxidative stress in human hepatoma cells HepG2. Transcription factor FOXO1 promoted C3 expression, and C3 repression by oxidative stress was mediated through regulation of FOXO1/HNF4α complex binding to the C3 promoter. We identified a novel cluster of FOXO1 binding sites in the distal region of the C3 promoter that was essential for regulation of C3 expression by FOXO1/HNF4α complex. Further, activation of the main MAP kinase cascades (ERK1/2, p38, and JNK), AMP kinase and the transcription factor NF-κB were necessary for suppression of C3 during oxidative stress. Together, we identified molecular mechanisms and transcription factors that mediate suppression of C3 production in HepG2 cells during oxidative stress.

About the authors

A. V. Babina

Institute of Experimental Medicine

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia

V. S. Shavva

Institute of Experimental Medicine

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia

A. V. Lisunov

Institute of Experimental Medicine; St. Petersburg State University

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia; St. Petersburg, 199034 Russia

G. N. Oleinikova

Institute of Experimental Medicine

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia

E. E. Larionova

Institute of Experimental Medicine

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia

A. A. Dmitrieva

Institute of Experimental Medicine

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia

E. V. Nekrasova

Institute of Experimental Medicine

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia

S. V. Orlov

Institute of Experimental Medicine; St. Petersburg State University

Email: serge@iem.spb.ru
St. Petersburg, 197376 Russia; St. Petersburg, 199034 Russia

References

  1. Rani V., Deep G., Singh R.K., Palle K., Yadav U.C.S. (2016) Oxidative stress and metabolic disorders: pathogenesis and therapeutic strategies. Life Sci. 148, 183–193. https://doi.org/10.1016/j.lfs.2016.02.002
  2. Kyriakis J.M., Avruch J. (2012) Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol. Rev. 92, 689–737. https://doi.org/10.1152/physrev.00028.2011
  3. Schieber M., Chandel N.S. (2014) ROS function in redox signaling and oxidative stress. Curr. Biol. 24, R453–R462. https://doi.org/10.1016/j.cub.2014.03.034
  4. Bedard K., Krause K.-H. (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313. https://doi.org/10.1152/physrev.00044.2005
  5. Donath M.Y., Shoelson S.E. (2011) Type 2 diabetes as an inflammatory disease. Nat. Rev. Immunol. 11, 98–107. https://doi.org/10.1038/nri2925
  6. Walport M.J. (2001) Complement. First of two parts. N. Engl. J. Med. 344, 1058–1066. https://doi.org/10.1056/NEJM200104053441406
  7. Sahu A., Lambris J.D. (2001) Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunol. Rev. 180, 35–48. https://doi.org/10.1034/j.1600-065x.2001.1800103.x
  8. Ricklin D., Hajishengallis G., Yang K., Lambris J.D. (2010) Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785‒797. https://doi.org/10.1038/ni.1923
  9. Mogilenko D.A., Danko K., Larionova E.E., Shavva V.S., Kudriavtsev I.V., Nekrasova E.V., Burnusuz A.V., Gorbunov N.P., Trofimov A.V., Zhakhov A.V., Ivanov I.A., Orlov S.V. (2022) Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion. Immunol. Cell. Biol. 100, 186‒204. https://doi.org/10.1111/imcb.12534
  10. Barbu A., Hamad O.A., Lind L., Ekdahl K.N., Nilsson B. (2015) The role of complement factor C3 in lipid metabolism. Mol. Immunol. 67, 101–107. https://doi.org/10.1016/j.molimm.2015.02.027
  11. Muscari A., Massarelli G., Bastagli L., Poggiopollini G., Tomassetti V., Drago G., Martignani C., Pacilli P., Boni P., Puddu P. (2000) Relationship of serum C3 to fasting insulin, risk factors and previous ischaemic events in middle-aged men. Eur. Heart J. 21, 1081–1090. https://doi.org/10.1053/euhj.1999.2013
  12. Hertle E., Van Greevenbroek M.M.J., Stehouwer C.D.A. (2012) Complement C3: an emerging risk factor in cardiometabolic disease. Diabetologia 55, 881–884. https://doi.org/10.1007/s00125-012-2462-z
  13. Clarke H.G., Freeman T., Pryse-Phillips W. (1971) Serum protein changes after injury. Clin. Sci. 40, 337‒344. https://doi.org/10.1042/cs0400337
  14. Alper C.A., Johnson A.M., Birtch A.G., Moore F.D. (1969) Human C3: evidence for the liver as the primary site of synthesis. Science. 163, 286–288. https://doi.org/10.1126/science.163.3864.286
  15. Einstein L.P., Hansen P.J., Ballow M., Davis A.E. 3rd, Davis J.S. 4th, Alper C.A., Rosen F.S., Colten H.R. (1977) Biosynthesis of the third component of complement (C3) in vitro by monocytes from both normal and homozygous C3-deficient humans. J. Clin. Invest. 60, 963–969. https://doi.org/10.1172/JCI108876
  16. Warren H.B., Pantazis P., Davies P.F. (1987) The third component of complement is transcribed and secreted by cultured human endothelial cells. Am.J. Pathol. 129, 9–13.
  17. Lévi-Strauss M., Mallat M. (1987) Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation. J. Immunol. 139, 2361–2366.
  18. Choy L.N., Rosen B.S., Spiegelman B.M. (1992) Adipsin and an endogenous pathway of complement from adipose cells. J. Biol. Chem. 267, 12736–12741. https://doi.org/10.1016/S0021-9258(18)42338-1
  19. Volanakis J.E. (1995) Transcriptional regulation of complement genes. Annu. Rev. Immunol. 12, 277–305. https://doi.org/10.1146/annurev.iy.13.040195.001425
  20. Mogilenko D.A., Kudriavtsev I.V., Shavva V.S., Dizhe E.B., Vilenskaya G., Efremov A.M., Perevozchikov A.P., Orlov S.V. (2013) Peroxisome proliferator-activated receptor α positively regulates complement C3 expression but inhibits tumor necrosis factor α-mediated activation of C3 gene in mammalian hepatic-derived cells. J. Biol. Chem. 288, 1726–1738. https://doi.org/10.1074/jbc.M112.437525
  21. Shavva V.S., Mogilenko D.A., Dizhe E.B., Oleinikova G.N., Perevozchikov A.P., Orlov S.V. (2013) Hepatic nuclear factor 4a positively regulates complement C3 expression and does not interfere with TNFα-mediated stimulation of C3 expression in HepG2 cells. Gene. 524, 187–192. https://doi.org/10.1016/j.gene.2013.04.036
  22. Shavva V.S., Bogomolova A.M., Efremov A.M., Trofimov A.N., Nikitin A.A., Babina A.V., Nekrasova E.V., Dizhe E.B., Oleinikova G.N., Missyul B.V., Orlov S.V. (2018) Insulin downregulates C3 gene expression in human HepG2 cells through activation of PPARγ. Eur. J. Cell. Biol. 97, 204–215. https://doi.org/10.1016/j.ejcb.2018.03.001
  23. Mogilenko D.A., Kudriavtsev I.V., Trulioff A.S., Shavva V.S., Dizhe E.B., Missyul B.V., Zhakhov A.V., Ischenko A.M., Perevozchikov A.P., Orlov S.V. (2012) Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Тoll-like receptor 4 activation in human macrophages. J. Biol. Chem. 287, 5954–5968. https://doi.org/10.1074/jbc.M111.289322.
  24. Pascual G., Glass C.K. (2006) Nuclear receptors versus inflammation: mechanisms of transrepression. Trends Endocrinol. Metab. 17, 321–327. https://doi.org/10.1016/j.tem.2006.08.005
  25. Glass C.K., Saijo K. (2010) Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells. Nat. Rev. Immunol. 10, 365–376. https://doi.org/10.1038/nri2748
  26. Collard C.D., Väkevä A., Büküsoglu C., Zünd G., Sperati C.J., Colgan S.P., Stahl G.L. (1997) Reoxygenation of hypoxic human umbilical vein endothelial cells activates the classic complement pathway. Circulation. 96, 326‒333. https://doi.org/10.1161/01.cir.96.1.326
  27. Collard C.D., Agah A., Stahl G.L. (1998) Complement activation following reoxygenation of hypoxic human endothelial cells: role of intracellular reactive oxygen species, NF-kappaB and new protein synthesis. Immunopharmacology. 39, 39–50. https://doi.org/10.1016/s0162-3109(97)00096-9
  28. Pei Y., Zhang J., Qu J., Rao Y., Li D., Gai X., Chen Y., Liang Y., Sun Y. (2022) Complement component 3 protects human bronchial epithelial cells from cigarette smoke-induced oxidative stress and prevents incessant apoptosis. Front. Immunol. 13, 1035930. https://doi.org/10.3389/fimmu.2022.1035930
  29. Zhong F., Hu Z., Jiang K., Lei B., Wu Z., Yuan G., Luo H., Dong C., Tang B., Zheng C., Yang S., Zeng Y., Guo Z., Yu S., Su H., Zhang G., Qiu X., Tomlinson S., He S. (2019) Complement C3 activation regulates the production of tRNA-derived fragments Gly-tRFs and promotes alcohol-induced liver injury and steatosis. Cell. Res. 29, 548–561. https://doi.org/10.1038/s41422-019-0175-2
  30. Диже Э.Б., Игнатович И.А., Буров С.В., Похвощева А.В., Акифьев Б.Н., Ефремов А.М., Перевозчиков А.П., Орлов С.В. (2006) Комплексы ДНК с катионными пептидами: условия формирования и факторы, влияющие на их проникновение в клетки млекопитающих. Биохимия. 71, 1659–1667.
  31. Shavva V.S., Bogomolova A.M., Nikitin A.A., Dizhe E.B., Oleinikova G.N., Lapikov I.A., Tanyanskiy D.A., Perevozchikov A.P., Orlov S.V. (2017) FOXO1 and LXRβ downregulate the apolipoprotein A-I gene expression during hydrogen peroxide-induced oxidative stress in HepG2 cells. Cell Stress Chaperones. 22, 123–134. https://doi.org/10.1007/s12192-016-0749-6
  32. Tangeman L., Wyatt C.N., Brown T.L. (2012) Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits. J. RNAi Gene Silenc. 8, 470–478.
  33. Mogilenko D.A., Dizhe E.B., Shavva V.S., Lapikov I.A., Orlov S.V., Perevozchikov A.P. (2009) Role of the nuclear receptors HNF4α, PPARα, and LXRs in the TNFα-mediated inhibition of human apolipoprotein A-I gene expression in HepG2 cells. Biochemistry. 48, 11950–11960. https://doi.org/10.1021/bi9015742
  34. Shavva V.S., Bogomolova A.M., Nikitin A.A., Dizhe E.B., Tanyanskiy D.A., Efremov A.M., Oleinikova G.N., Perevozchikov A.P., Orlov S.V. (2017) Insulin-mediated downregulation of apolipoprotein A-I gene in human hepatoma cell line HepG2: the role of interaction between FOXO1 and LXRβ transcription factors. J. Cell. Biochem. 118, 382–396. https://doi.org/10.1002/jcb.25651
  35. Shavva V.S., Mogilenko D.A., Bogomolova A.M., Nikitin A.A., Dizhe E.B., Efremov A.M., Oleinikova G.N., Perevozchikov A.P., Orlov S.V. (2016) PPARγ represses apolipoprotein A-I gene but impedes TNFα-mediated ApoA-I downregulation in HepG2 cells. J. Cell. Biochem. 117, 2010–2022. https://doi.org/10.1002/jcb.25498
  36. Andrews N.C., Faller D.V. (1991) A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucl. Acids Res. 19, 2499. https://doi.org/10.1093/nar/19.9.2499
  37. Aikawa R., Komuro I., Yamazaki T., Zou Y., Kudoh S., Tanaka M., Shiojima I., Hiro Y., Yazaki Y. (1997) Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J. Clin. Invest. 100, 1813–1821. https://doi.org/10.1172/JCI119709
  38. Kim M.J., Byun J.Y., Yun C.H., Park I.C., Lee K.H., Lee S.J. (2008) c-Src-p38 mitogen-activated protein kinase signaling is required for Akt activation in response to ionizing radiation. Mol. Cancer Res. 6, 1872–1880. https://doi.org/10.1158/1541-7786.MCR-08-0084
  39. Yoshizumi M., Abe J., Haendeler J., Huang Q., Berk B.C. (2000) Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J. Biol. Chem. 275, 11706–11712. https://doi.org/10.1074/jbc.275.16.11706
  40. Giannoni E., Buricchi F., Raugei G., Ramponi G., Chiarugi P. (2005) Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth. Mol. Cell. Biol. 25, 6391–6403. https://doi.org/10.1128/MCB.25.15.6391-6403.2005
  41. Han Y., Wang Q., Song P., Zhu Y., Zou M.H. (2010) Redox regulation of the AMP-activated protein kinase. PLoS One. 5, e15420. https://doi.org/10.1371/journal.pone.0015420
  42. Awad H., Nolette N., Hinton M., Dakshinamurti S. (2014) AMPK and FoxO1 regulate catalase expression in hypoxic pulmonary arterial smooth muscle. Pediatr. Pulmonol. 49, 885–897. https://doi.org/10.1002/ppul.22919
  43. Cheng Z., Guo S., Copps K., Dong X., Kollipara R., Rodgers J.T., Depinho R.A., Puigserver P., White M.F. (2009) Foxo1 integrates insulin signaling with mitochondrial function in the liver. Nat. Med. 15, 1307–1311. https://doi.org/10.1038/nm.2049
  44. Liu X., Cui Y., Li M., Xu H., Zuo J., Fang F., Chang Y. (2013) Cobalt protoporphyrin induces HO-1 expression mediated partially by FOXO1 and reduces mitochondria-derived reactive oxygen species production. PLoS One. 8, 1–9. https://doi.org/10.1371/journal.pone.0080521
  45. Sengupta A., Molkentin J.D., Paik J.H., DePinho R.A., Yutzey K.E. (2011) FoxO transcription factors promote cardiomyocyte survival upon induction of oxidative stress. J. Biol. Chem. 286, 7468–7478. https://doi.org/10.1074/jbc.M110.179242
  46. Klotz L.O., Sánchez-Ramos C., Prieto-Arroyo I., Urbánek P., Steinbrenner H., Monsalve M. (2015) Redox regulation of FoxO transcription factors. Redox Biol. 6, 51–72. https://doi.org/10.1016/j.redox.2015.06.019
  47. Rauluseviciute I., Riudavets-Puig R., Blanc-Mathieu R., Castro-Mondragon J. A., Ferenc K., Kumar V., Lemma R.B., Lucas J., Chèneby J., Baranasic D., Khan A., Fornes O., Gundersen S., Johansen M., Hovig E., Lenhard B., Sandelin A., Wasserman W.W., Parcy F., Mathelier A. (2024) JASPAR2024: 20th anniversary of the open-access database of transcription factor binding profiles. Nucl. Acids Res. 52, D174–D182. https://doi.org/10.1093/nar/gkad1059
  48. Hirota K., Sakamaki J.I., Ishida J., Shimamoto Y., Nishihara S., Kodama N., Ohta K., Yamamoto M., Tanimoto K., Fukamizu A. (2008) A combination of HNF-4 and Foxo1 is required for reciprocal transcriptional regulation of glucokinase and glucose-6-phosphatase genes in response to fasting and feeding. J. Biol. Chem. 283, 32432–32441. https://doi.org/10.1074/jbc.M806179200
  49. Ganjam G.K., Dimova E.Y., Unterman T.G., Kietzmann T. (2009) FoxO1 and HNF-4 are involved in regulation of hepatic glucokinase gene expression by resveratrol. J. Biol. Chem. 284, 30783–30797. https://doi.org/10.1074/jbc.M109.045260
  50. Ghezzi P. (2011) Role of glutathione in immunity and inflammation in the lung. Int. J. Gen. Med. 4, 105–113. https://doi.org/10.2147/IJGM.S15618
  51. Nikolaidou-Neokosmidou V., Zannis V.I., Kardassis D. (2006) Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor kappaB pathway. Biochem. J. 398, 439–450. https://doi.org/10.1042/BJ20060169
  52. Ehle C., Iyer-Bierhoff A., Wu Y., Xing S., Kiehntopf M., Mosig A.S., Godmann M., Heinzel T. (2024) Downregulation of HNF4A enables transcriptomic reprogramming during the hepatic acute-phase response. Commun. Biol. 7, 589. https://doi.org/10.1038/s42003-024-06288-1
  53. Cianflone K., Xia Z., Chen L.Y. (2003) Critical review of acylation-stimulating protein physiology in humans and rodents. Biochim. Biophys. Acta. 1609, 127–143. https://doi.org/10.1016/s0005-2736(02)00686-7
  54. Lehtinen M.K., Yuan Z., Boag P.R., Yang Y., Villen J., Becker E.B., DiBacco S., de la Iglesia N., Gygi S., Blackwell T.K., Bonni A. (2006) A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell. 125, 987–1001. https://doi.org/10.1016/j.cell.2006.03.046
  55. Yamagata K., Daitoku H., Takahashi Y., Namiki K., Hisatake K., Kako K., Mukai H., Kasuya Y., Fukamizu A. (2008) Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol. Cell. 32, 221–231. https://doi.org/10.1016/j.molcel.2008.09.013
  56. Asada S., Daitoku H., Matsuzaki H., Saito T., Sudo T., Mukai H., Iwashita S., Kako K., Kishi T., Kasuya Y., Fukamizu A. (2007) Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1. Cell Signal. 19, 519–527. https://doi.org/10.1016/j.cellsig.2006.08.015
  57. Van Der Heide L.P., Hoekman M.F.M., Smidt M.P. (2004) The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J. 380, 297–309. https://doi.org/10.1042/BJ20040167
  58. Sunayama J., Tsuruta F., Masuyama N., Gotoh Y. (2005) JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3. J. Cell Biol. 170, 295–304. https://doi.org/10.1083/jcb.200409117
  59. Weng Q., Liu Z., Li B., Liu K., Wu W., Liu H. (2016) Oxidative stress induces mouse follicular granulosa cells apoptosis via JNK/FoxO1 pathway. PLoS One. 11, e0167869. https://doi.org/10.1371/journal.pone.0167869
  60. Saline M., Badertscher L., Wolter M., Lau R., Gunnarsson A., Jacso T., Norris T., Ottmann C., Snijder A. (2019) AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins. J. Biol. Chem. 294, 13106–13116. https://doi.org/10.1074/jbc.RA119.008649

Supplementary files

Supplementary Files
Action
1. JATS XML

Copyright (c) 2025 Russian Academy of Sciences

Согласие на обработку персональных данных с помощью сервиса «Яндекс.Метрика»

1. Я (далее – «Пользователь» или «Субъект персональных данных»), осуществляя использование сайта https://journals.rcsi.science/ (далее – «Сайт»), подтверждая свою полную дееспособность даю согласие на обработку персональных данных с использованием средств автоматизации Оператору - федеральному государственному бюджетному учреждению «Российский центр научной информации» (РЦНИ), далее – «Оператор», расположенному по адресу: 119991, г. Москва, Ленинский просп., д.32А, со следующими условиями.

2. Категории обрабатываемых данных: файлы «cookies» (куки-файлы). Файлы «cookie» – это небольшой текстовый файл, который веб-сервер может хранить в браузере Пользователя. Данные файлы веб-сервер загружает на устройство Пользователя при посещении им Сайта. При каждом следующем посещении Пользователем Сайта «cookie» файлы отправляются на Сайт Оператора. Данные файлы позволяют Сайту распознавать устройство Пользователя. Содержимое такого файла может как относиться, так и не относиться к персональным данным, в зависимости от того, содержит ли такой файл персональные данные или содержит обезличенные технические данные.

3. Цель обработки персональных данных: анализ пользовательской активности с помощью сервиса «Яндекс.Метрика».

4. Категории субъектов персональных данных: все Пользователи Сайта, которые дали согласие на обработку файлов «cookie».

5. Способы обработки: сбор, запись, систематизация, накопление, хранение, уточнение (обновление, изменение), извлечение, использование, передача (доступ, предоставление), блокирование, удаление, уничтожение персональных данных.

6. Срок обработки и хранения: до получения от Субъекта персональных данных требования о прекращении обработки/отзыва согласия.

7. Способ отзыва: заявление об отзыве в письменном виде путём его направления на адрес электронной почты Оператора: info@rcsi.science или путем письменного обращения по юридическому адресу: 119991, г. Москва, Ленинский просп., д.32А

8. Субъект персональных данных вправе запретить своему оборудованию прием этих данных или ограничить прием этих данных. При отказе от получения таких данных или при ограничении приема данных некоторые функции Сайта могут работать некорректно. Субъект персональных данных обязуется сам настроить свое оборудование таким способом, чтобы оно обеспечивало адекватный его желаниям режим работы и уровень защиты данных файлов «cookie», Оператор не предоставляет технологических и правовых консультаций на темы подобного характера.

9. Порядок уничтожения персональных данных при достижении цели их обработки или при наступлении иных законных оснований определяется Оператором в соответствии с законодательством Российской Федерации.

10. Я согласен/согласна квалифицировать в качестве своей простой электронной подписи под настоящим Согласием и под Политикой обработки персональных данных выполнение мною следующего действия на сайте: https://journals.rcsi.science/ нажатие мною на интерфейсе с текстом: «Сайт использует сервис «Яндекс.Метрика» (который использует файлы «cookie») на элемент с текстом «Принять и продолжить».