Establishment and Application of Engineered NIH 3T3 Cell Line with Stable Human RAGE Expression

Xinyi Xiao; Hui Chen; Pameila Paerhati; Meiqi Zhou; Zhuoyi Yang; Siyi Bai; Yunsheng Yuan

doi:10.1055/s-0040-1708511

Pharmaceutical Fronts, Inhaltsverzeichnis

CC BY 4.0 · Pharmaceutical Fronts 2020; 02(01): e23-e27
DOI: 10.1055/s-0040-1708511

Original Article

Georg Thieme Verlag KG Stuttgart · New York

Establishment and Application of Engineered NIH 3T3 Cell Line with Stable Human RAGE Expression

Authors

Xinyi Xiao

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China
Hui Chen

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China
Pameila Paerhati

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China
Meiqi Zhou

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China
Zhuoyi Yang

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China
Siyi Bai

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China
Yunsheng Yuan

¹Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Jiao Tong University College of Pharmacy, Ministry of Education, Shanghai, People's Republic of China

Abstract

Aim NIH3T3 cell line with expression of human receptor for advanced glycation end-products (hRAGE) transduced with lentivirus vectors was used to analyze affinity, biological activity, and/or molecular mechanisms of molecules targeting the hRAGE pathway.

Method The DNA fragment coding for hRAGE gene was integrated into the genome of NIH3T3 cells using lentivirus transduction. Cells expressing hRAGE were selected with puromycin, and the level of hRAGE expression was analyzed by Western blot. To establish a stable cell line, colonies of hRAGE-expressing cells were generated, and the level of RAGE expression in each engineered cell line was analyzed within 20 generations. Flow cytometry assay was used to verify affinity of anti-hRAGE antibody binding to hRAGE on the surface of engineered cells. The engineered NIH3T3 cell line was applied to assess effects of anti-hRAGE blocking antibody on amyloid β-induced cells apoptosis by CCK-8 assay.

Results The engineered NIH3T3 cell line (hRAGE-NIH3T3) could stably express human RAGE. Commercial anti-RAGE polyclonal antibody could recognize and bind to human RAGE on the surface of hRAGE-NIH3T3 but not original NIH3T3 cells. In addition, hRAGE-NIH3T3 was more sensitive to RAGE pathway-dependent stimulation. Our data show that the hRAGE-NIH3T3 cell line established is an excellent tool in the study of RAGE-targeting molecules based on the cellular level, biological function, and RAGE-mediated molecular mechanisms.

Keywords

RAGE - engineered cell line - lentiviral - transduction - flow cytometry

Volltext

Referenzen

Reference
1 Rouhiainen A, Kuja-Panula J, Tumova S, Rauvala H. RAGE-mediated cell signaling. Methods Mol Biol 2013; 963: 239-263
2 Fritz G. RAGE: a single receptor fits multiple ligands. Trends Biochem Sci 2011; 36 (12) 625-632
3 Yamagishi S, Matsui T. Soluble form of a receptor for advanced glycation end products (sRAGE) as a biomarker. Front Biosci (Elite Ed) 2010; 2 (04) 1184-1195
4 Han SH, Kim YH, Mook-Jung I. RAGE: the beneficial and deleterious effects by diverse mechanisms of actions. Mol Cells 2011; 31 (02) 91-97
5 Huttunen HJ, Fages C, Rauvala H. Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-kappaB require the cytoplasmic domain of the receptor but different downstream signaling pathways. J Biol Chem 1999; 274 (28) 19919-19924
6 Fages C, Nolo R, Huttunen HJ, Eskelinen E, Rauvala H. Regulation of cell migration by amphoterin. J Cell Sci 2000; 113 (Pt 4): 611-620
7 Yan SD, Chen X, Fu J. , et al. RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 1996; 382 (6593): 685-691
8 Hofmann MA, Drury S, Fu C. , et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 1999; 97 (07) 889-901
9 Ray R, Juranek JK, Rai V. RAGE axis in neuroinflammation, neurodegeneration and its emerging role in the pathogenesis of amyotrophic lateral sclerosis. Neurosci Biobehav Rev 2016; 62: 48-55
10 Deane R, Singh I, Sagare AP. , et al. A multimodal RAGE-specific inhibitor reduces amyloid β-mediated brain disorder in a mouse model of Alzheimer disease. J Clin Invest 2012; 122 (04) 1377-1392
11 Li Y, Wu R, Tian Y. , et al. RAGE/NF-κB signaling mediates lipopolysaccharide induced acute lung injury in neonate rat model. Int J Clin Exp Med 2015; 8 (08) 13371-13376
12 Liu Y, Liang C, Liu X. , et al. AGEs increased migration and inflammatory responses of adventitial fibroblasts via RAGE, MAPK and NF-kappaB pathways. Atherosclerosis 2010; 208 (01) 34-42
13 Yeh CH, Sturgis L, Haidacher J. , et al. Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion. Diabetes 2001; 50 (06) 1495-1504
14 Bianchi R, Giambanco I, Donato R. S100B/RAGE-dependent activation of microglia via NF-kappaB and AP-1 Co-regulation of COX-2 expression by S100B, IL-1beta and TNF-alpha. Neurobiol Aging 2010; 31 (04) 665-677
15 Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 2007; 8 (01) 49-62
16 Tanji N, Markowitz GS, Fu C. , et al. Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease. J Am Soc Nephrol 2000; 11 (09) 1656-1666
17 Wendt T, Harja E, Bucciarelli L. , et al. RAGE modulates vascular inflammation and atherosclerosis in a murine model of type 2 diabetes. Atherosclerosis 2006; 185 (01) 70-77
18 Onyeagucha BC, Mercado-Pimentel ME, Hutchison J, Flemington EK, Nelson MA. S100P/RAGE signaling regulates microRNA-155 expression via AP-1 activation in colon cancer. Exp Cell Res 2013; 319 (13) 2081-2090
19 Tesarova P, Cabinakova M, Mikulova V, Zima T, Kalousova M. RAGE and its ligands in cancer - culprits, biomarkers, or therapeutic targets?. Neoplasma 2015; 62 (03) 353-364
20 Xia P, He H, Kristine MS. , et al. Therapeutic effects of recombinant human S100A6 and soluble receptor for advanced glycation end products(sRAGE) on CCl₄-induced liver fibrosis in mice. Eur J Pharmacol 2018; 833: 86-93
21 Pontén F, Jirström K, Uhlen M. The Human Protein Atlas--a tool for pathology. J Pathol 2008; 216 (04) 387-393
22 Shafaghat F, Abbasi-Kenarsari H, Majidi J, Movassaghpour AA, Shanehbandi D, Kazemi T. preparation of proper immunogen by cloning and stable expression of cDNA coding for human hematopoietic stem cell marker CD34 in NIH-3T3 mouse fibroblast cell line. Adv Pharm Bull 2015; 5 (01) 69-75
23 Salehi-Lalemarzi H, Shanehbandi D, Shafaghat F. , et al. Cloning and stable expression of cDNA coding for platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in NIH-3T3 cell line. Adv Pharm Bull 2015; 5 (02) 247-253
24 Abbasi-Kenarsari H, Shafaghat F, Baradaran B, Movassaghpour AA, Shanehbandi D, Kazemi T. Cloning and expression of CD19, a human B-cell marker in NIH-3T3 cell line. Avicenna J Med Biotechnol 2015; 7 (01) 39-44
25 Weng S, Zhou L, Deng Q. , et al. Niclosamide induced cell apoptosis via upregulation of ATF3 and activation of PERK in Hepatocellular carcinoma cells. BMC Gastroenterol 2016; 16: 25
26 Hudson BI, Carter AM, Harja E. , et al. Identification, classification, and expression of RAGE gene splice variants. FASEB J 2008; 22 (05) 1572-1580
27 Liu Z, O'Rourke J. Expediting antibody discovery with a cell and bead multiplexed competition assay. SLAS Discov 2018; 23 (07) 667-675
28 Elgundi Z, Reslan M, Cruz E, Sifniotis V, Kayser V. The state-of-play and future of antibody therapeutics. Adv Drug Deliv Rev 2017; 122: 2-19
29 Kim Y, Mook-Jung I. PRAK mediates Aβ-RAGE driven autophagy pathway. Oncotarget 2017; 8 (04) 5648-5649
30 Oczypok EA, Perkins TN, Oury TD. All the “RAGE” in lung disease: the receptor for advanced glycation endproducts (RAGE) is a major mediator of pulmonary inflammatory responses. Paediatr Respir Rev 2017; 23: 40-49
31 Litwinoff E, Hurtado Del Pozo C, Ramasamy R, Schmidt AM. Emerging targets for therapeutic development in diabetes and its complications: the RAGE signaling pathway. Clin Pharmacol Ther 2015; 98 (02) 135-144
32 Sakuma T, Barry MA, Ikeda Y. Lentiviral vectors: basic to translational. Biochem J 2012; 443 (03) 603-618
33 Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia 2018; 32 (07) 1529-1541