IFN-? Induction of Apolipoprotein A-I Expression is Mediated by NF-?B Signalling in HepG2 Cells

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Lui Siang Tong
Hong Kin Wong
Choy Hoong Chew


Liver inflammation is associated with changes in lipoprotein and apolipoprotein expression. Interferon-? (IFN-?), the sole representative of type II IFN, plays a pivotal role in modulating and intensifying inflammatory responses. This study was designed to identify the effect of IFN-? on apolipoproteinA-I (APOA-I) and to identify the involvement of nuclear factor–kappa B (NF-?B) in its regulation. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analysis were performed to quantify the APOA-I expression in treated HepG2 cells. Here, we show that 50 ng/mL of IFN-? induced APOA-I mRNA and protein expression. Pretreatment of cells with NF-?B signalling pathway inhibitors, however, decreased the APOA-I expression levels. This study also demonstrated the direct involvement of NF-?B signalling in IFN-?-induced APOA-I expression, whereby IFN-? induced the levels of phosphorylated NF-?B p65 Ser468 and Ser536 expression to 2.59-fold and 1.63-fold, respectively. However, pretreatment of cells with NF-?B signalling pathway inhibitors attenuated their increment and subsequently reduced APOA-I expression in HepG2 cells. In summary, the present study successfully confirmed the role of NF-?B signalling and activation of p65 Ser468 and Ser536 in mediating IFN-? induction of APOA-I expression in HepG2 cells.


Keradangan hati berkait rapat dengan perubahan dalam ekspresi lipoprotein dan apolipoprotein. Interferon-? (IFN-?), wakil tunggal jenis kedua IFN, memainkan peranan yang penting dalam memodulasi dan mempergiatkan tindak balas keradangan. Justeru itu, kajian ini direka untuk mengenal pasti kesan IFN-? terhadap apolipoprotein A-I (APOA-I) dan penglibatan nuclear factor–kappa B (NF-?B) dalam laluan isyarat tersebut. Tindak balas rantai polymerase transkripsi berbalik kuantitatif (qRT-PCR) dan analisis blot western telah dilaksanakan untuk menguantifikasi ekspresi APOA-I dalam sel-sel HepG2 selepas dirawat dengan IFN-?. Kajian ini menunjukkan bahawa 50 ng/mL IFN-? merangsangkan ekspresi mRNA dan protein APOA-I. Walau bagaimanapun, pra-rawatan sel dengan inhibitor laluan isyarat NF-?B mengurangkan tahap ekspresi APOA-I. Kajian ini juga mendemonstrasikan penglibatan langsung isyarat NF-?B dalam ekspresi APOA-I akibat rangsangan IFN-?, di mana IFN-? meningkatkan tahap fosforilasi NF-?B p65 Ser468 dan Ser536 kepada 2.59-ganda and 1.63-ganda. Namun demikian, pra-rawatan sel dengan perencat laluan isyarat NF-?B melumpuhkan peningkatkan tersebut dan kemudian mengurangkan ekspresi APOA-I dalam sel HepG2. Sebagai rumusan, kajian ini berjaya mengenalpasti peranan isyarat NF-?B dan pengaktifan p65 Ser468 dan Ser536 sebagai pengantara IFN-? ke atas induksi APOA-I di dalam sel-sel HepG2.

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Lui Siang Tong, Hong Kin Wong, & Choy Hoong Chew. (2019). IFN-? Induction of Apolipoprotein A-I Expression is Mediated by NF-?B Signalling in HepG2 Cells. Tropical Life Sciences Research, 30(2), 39–50. https://doi.org/10.21315/tlsr2019.30.2.4
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Chew C H, Chew G S, Najimudin N and Tengku-Muhammad T S. (2007). Interleukin-6 inhibits human peroxisome proliferator activated receptor alpha gene expression via CCAAT/enhancer-binding proteins in hepatocytes. International Journal of Biochemistry & Cell 39(10): 1975–1986. https://doi.org/10.1016/j.biocel.2007.05.015

Delerive P, Bosscher K D, Besnard S, Berghe W V, Peters J M, Gonzalez F J, Frunchart J C, Tedgui A, Haegeman G and Staels B. (1999). Peroxisome proliferatoractivated receptor ? negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kB and AP-1. The Journal of Biological Chemistry 274(54): 32048–32054. https://doi.org/10.1074/jbc.274.45.32048

Deb A, Haque S J, Mogensen T, Silverman R H and Williams R G. (2001). RNA-dependent protein kinase PKR is required for activation of NF-?B by IFN-? in a STAT1-independent pathway. Journal of Immunology 166(10): 6170–6180. https://doi.org/10.4049/jimmunol.166.10.6170

Deng Y Q, Zhao H, Ma A L, Zhou J Y, Xie S B, Zhang X Q, Zhang D Z, Xie Q, Zhang G, Shang J and Cheng J. (2015). Selected cytokines serve as potential biomarkers for predicting liver inflammation and fibrosis in chronic hepatitis B patients with normal to mildly elevated aminotransferases. Medicine 94(45): e2003. https://doi.org/10.1097/MD.0000000000002003

Dominiczak M H and Caslake M J. (2011). Apolipoproteins: Metabolic role and clinical biochemistry applications. Annals of Clinical Biochemistry 48: 485–486. https://doi.org/10.1258/acb.2011.011111

El Jamal S M, Taylor E B, Elmageed Z Y A, Alamodi A A, Selimovic D, Alkhateeb A, Hannig M, Hassan S Y, Santourlidis S, Friedlander P L and Haikel Y. (2016). Interferon gamma-induced apoptosis of head and neck squamous cell carcinoma is connected to indoleamine-2, 3-dioxygenase via mitochondrial and ER stressassociated pathways. Cell Division 11(1): 11. https://doi.org/10.1186/s13008-016-0023-4

Ertunc M E and Hotamisligil G S. (2016). Lipid signaling and lipotoxicity in metaflammation: indications for metabolic disease pathogenesis and treatment. Journal of Lipid Research 57(12): 2099–2114. https://doi.org/10.1194/jlr.R066514

Esteve E, Ricart W and Ferna´ndez-Real J M. (2005). Dyslipidemia and inflammation: An evolutionary conserved mechanism. Clinical Nutrition 24: 16–31. https://doi.org/10.1016/j.clnu.2004.08.004

Hayden M S and Ghosh S. (2014). Regulation of NF-?B by TNF family cytokines. Seminars in Immunology 26: 253–206. https://doi.org/10.1016/j.smim.2014.05.004

Irshad M and Dubey R. (2005). Apolipoproteins and their role in different clinical conditions: An overview. Indian Journal of Biochemistry & Biophysics 42: 73–80. Ishii T, Kwon H, Hiscott J, Mosialos G and Koromilas A E. (2001). Activation of the I?B? kinase (IKK) complex by double-stranded RNA-binding defective and catalytic inactive mutants of the interferon-inducible protein kinase PKR. Oncogene 20: 1900–1912. https://doi.org/10.1038/sj.onc.1204267

Jaramillo M, Gowda D C, Radzioch D and Olivier M. (2003). Hemozoin increases IFN?-inducible macrophage nitric oxide generation through extracellular signalregulated kinase-and NF-?B-dependent pathways. The Journal of Immunology 171(8): 4243–4253. https://doi.org/10.4049/jimmunol.171.8.4243

Kindt T J, Goldsby R A and Osborne B A. (2004). Kuby immunology (6th ed.). New York: W.H. Freeman and Company.

Knight B, Lim R, Yeoh G C and Olynyk J K. (2007). Interferon-? exacerbates liver damage, the hepatic progenitor cell response and fibrosis in a mouse model of chronic liver injury. Journal of Hepatology 47(6): 826–833. https://doi.org/10.1016/j.jhep.2007.06.022

L´opez-Vel´azquez J A, Carrillo-C´ordova L D, Ch´avez-Tapia N C, Uribe M and M´endezS´anchez N. (2011). Nuclear receptors in nonalcoholic fatty liver disease. Journal of Lipids 2012: 1–10. https://doi.org/10.1155/2012/139875

Lim W S, Ng D L, Kor S B, Wong H K, Tengku-Muhammad T S, Choo Q C and Chew C H. (2013). Tumour necrosis factor alpha down-regulates the expression of peroxisome proliferator activated receptor alpha (PPAR?) in human hepatocarcinoma HepG2 cells by activation of NF-?B pathway. Cytokine 61(1): 266–274. https://doi.org/10.1016/j.cyto.2012.10.007

Masoodi M, Kuda O, Rossmeisl M, Flachs P and Kopecky J. (2015). Lipid signaling in adipose tissue: Connecting inflammation & metabolism. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids 1851(4): 503–518. https://doi.org/10.1016/j.bbalip.2014.09.023

Matsuura F, Oku H, Koseki M, Sandoval J C, Yuasa-Kawase M, Tsubakio-Yamamoto K, Masuda D, Maeda N, Tsujii K, Nishida M, Hirano K, Kihara S, Hori M, Shimomura I and Yamashita S. (2007). Adiponectin accelerates reverse cholesterol transport by increasing high density lipoprotein assembly in the liver. Biochemical and Biophysical Research Communication 358(4): 1091–1095. https://doi.org/10.1016/j.bbrc.2007.05.040

Mihm S, Hutschenreiter A, Fayyazi A, Pingel S and Ramadori G. (1996). High inflammatory activity is associated with an increased amount of IFN-gamma transcripts in peripheral blood cells of patients with chronic hepatitis C virus infection. Medical Microbiology and Immunology 185(2): 95–102.

Mizuhara H, Uno M, Seki N, Yamashita M, Yamaoka M, Ogawa T, Kaneda K, Fujii T, Senoh H and Fujiwara H. (1996). Critical involvement of interferon gamma in the pathogenesis of T-cell activation-associated hepatitis and regulatory mechanisms of interleukin-6 for the manifestations of hepatitis. Hepatology 23(6): 1608–1615. https://doi.org/10.1053/jhep.1996.v23.pm0008675184

Mogilenko D A, Dizhe E B, Shavva V S, Lapikov I A, Orlov, S V and Perevozchikov A P. (2009). Role of the nuclear receptors HNF4R, PPAR, and LXRs in the TNF?mediated inhibition of human apolipoprotein A-I gene expression in HepG2 cells. Biochemistry 48: 11950–11960. https://doi.org/10.1021/bi9015742

Panousis C G and Zuckerman S H. (2000). Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-?. Journal of Lipid Research 41(1):75–83.

Pérez-Baos S, Barrasa J I, Gratal P, Larrañaga-Vera A, Prieto-Potin I, Herrero-Beaumont G and Largo R. (2017). Tofacitinib restores the inhibition of reverse cholesterol transport induced by inflammation: Understanding the lipid paradox associated with rheumatoid arthritis. British Journal of Pharmacology 174(18): 3018–3031. https://doi.org/10.1111/bph.13932

Peters J M, Hennuyer N, Staels B, Frunchart J C, Fievet C, Gonzalez F J and Auwerx J. (1997). Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor ?-deficient mice. Journal of Biological Chemistry 272(43): 27307–27312. https://doi.org/10.1074/jbc.272.43.27307

Platanias L C. (2005). Mechanisms of type-I- and type-II-interferon-mediated signaling. Nature Review Immunology 5: 375–386. https://doi.org/10.1038/nri1604

Ramalingam T R, Gieseck R L, Acciani T H, M Hart K, Cheever A W, Mentink-Kane M M, Vannella K M and Wynn T A. (2016). Enhanced protection from fibrosis and inflammation in the combined absence of IL-13 and IFN-?. The Journal of Pathology 239(3): 344–354. https://doi.org/10.1002/path.4733

Ramana C V, Gil M P, Schreiber R D and Stark G R. (2002). Stat1-dependent and independent pathways in IFN-?-dependent signaling. Trends in Immunology 23(2): 96–101. https://doi.org/10.1016/S1471-4906(01)02118-4

Rani S M R, Shultz D B, Fuller J D, Ransohoff R M and Stark G R. (2009). Roles of IKK-?, IRF1, and p65 in the activation of chemokine genes by interferon-?. Journal of Interferon & Cytokine Research 29(12): 817–824. https://doi.org/10.1089/jir.2009.0034

Reiss A B, Patel C A, Rahman M M, Chan E S, Hasneen K, Montesinos M C, Trachman J D and Cronstein B N. (2004). Interferon-? impedes reverse cholesterol transport and promotes foam cell transformation in THP-1 human monocytes/macrophages. Medical Science Monitor 10(11): BR420–BR425.

Sen G C and Sarkar S N. (2007). The interferon-stimulated genes: targets of direct signaling by interferons, double-stranded RNA, and viruses. Current Topics in Microbiology and Immunology 316: 233–250.

Shultz D B, Fuller J D, Yang Y, Sizemore N, Rani M R and Stark G R. (2007). Activation of a subset of genes by IFN-gamma requires IKKbeta but not interferon-dependent activation of NF-kappaB. Journal of Interferon & Cytokine Research 27(10): 875–884. https://doi.org/10.1089/jir.2007.0031

Sizemore N, Agarwal A, Das K, Lerner N, Sulak M, Rani S, Ransohoff R, Shultz D and Stark G R. (2004). Inhibitor of ?B kinase is required to activate a subset of interferon ?-stimulated genes. Proceedings of the National Academy of Sciences of USA 101(21): 7994–7998. https://doi.org/10.1073/pnas.0401593101

Stark G R, Kerr IM, Williams B R G, Silverman R H and Schreiber R D. (1998). How cells respond to interferons. Annual Reviews of Biochemistry 67: 227–264. https://doi.org/10.1146/annurev.biochem.67.1.227

Terkeltaub R. (2014). Apolipoprotein A-I at the interface of vascular inflammation and arthritis. Arteriosclerosis, Thrombosis, and Vascular Biology 34: 474–476. https://doi.org/10.1161/ATVBAHA.114.303112

Thapa R J, Basagoudanavar S H, Nogusa S, Irrinki K, Mallilankaraman K, Slifker M J, Beg A A, Madesh M and Balachandran S. (2011). NF-?B protects cells from gamma interferon-induced RIP1-dependent necroptosis. Molecular and Cellular Biology 31(4): 2934–2946. https://doi.org/10.1128/MCB.05445-11

Thomsen M K, Bakiri L, Hasenfuss S C, Hamacher R, Martinez L and Wagner E F. (2013). JUNB/AP-1 controls IFN-? during inflammatory liver disease. The Journal of Clinical Investigation 123(12): 5258–5268. https://doi.org/10.1172/JCI70405

Yang F, Yin Y, Wang F, Zhang L, Wang Y and Sun S. (2010). An altered pattern of liver apolipoprotein A-I isoforms is implicated in male chronic Hepatitis B progression. Journal of Proteome Research 9(1): 134–143. https://doi.org/10.1021/pr900593r.l

Yoshida K, Okamura H, Hiroshima Y, Abe K, Kido J I, Shinohara Y and Ozaki K. (2017). PKR induces the expression of NLRP3 by regulating the NF-?B pathway in Porphyromonas gingivalis-infected osteoblasts. Experimental Cell Research 354(1): 57–64. https://doi.org/10.1016/j.yexcr.2017.03.028

Yu X H, Zhang J, Zheng X L, Yang Y H and Tang C K. (2015). Interferon-? in foam cell formation and progression of atherosclerosis. Clinica Chimica Acta 441: 33–43. https://doi.org/10.1016/j.cca.2014.12.007

Zamanian-Daryoush M, Mogensen T H, Didonato J A and Williams B R G. (1999). NF-?B activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-?B-inducing kinase and I?B kinase. Molecular and Cellular Biology 20(4): 1278–1290. https://doi.org/10.1128/MCB.20.4.1278-1290.2000

Zhang J. (2007). Yin and yang interplay of IFN-? in inflammation and autoimmune disease. The Journal of Clinical Investigation 117(4): 871–873. https://doi.org/10.1172/JCI31860

Zhang N, Chu E S, Zhang J, Li X, Liang Q, Chen J, Chen M, Teoh N, Farrell G, Sung J J and Yu J. (2014). Peroxisome proliferator activated receptor alpha inhibits hepatocarcinogenesis through mediating NF-?B signaling pathway. Oncotarget 5(18): 8330. https://doi.org/10.18632/oncotarget.2212