Abstract
Hybridoma cells display an increase in antibody productivity following exposure to hypertonic conditions. However, the underlying mechanism is not well understood. In the present study, we hypothesize that the nuclear factor of activated T cells 5 (NFAT5)/tonicity enhancer binding protein (TonEBP) functions to increase the antibody productivity of hybridoma cells. NFAT5 is an osmosensitive mammalian transcription factor. However, its ubiquitous expression in various organs that are not bathed in hypertonic milieu suggests that NFAT5 may also regulate cell growth and function under isotonic conditions. In this study, we examined the expression of NFAT5 in hybridoma cells by Western blot analysis, and found that it increased significantly in hypertonic medium. To further define the function of NFAT5 in hybridoma cells, RNA interference technique was used to downregulate the expression of NFAT5 in SGB-8 cells (a hybridoma cell line). In isotonic medium, antibody productivity of hybridoma cells was reduced by downregulation of NFAT5 while cell proliferation was not influenced. The results presented here demonstrate that NFAT5 not only plays an important role in osmotic stress response pathway in hybridoma cells but also is essential for optimal antibody productivity.
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References
Kohler G, Milstein C . Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495–497.
deZengotita VM, Schmelzer AE, Miller WM . Characterization of hybridoma cell responses to elevated pCO(2) and osmolality: intracellular pH, cell size, apoptosis, and metabolism. Biotechnol Bioeng 2002; 77:369–380.
Balcarcel RR, Stephanopoulos G . Rapamycin reduces hybridoma cell death and enhances monoclonal antibody production. Biotechnol Bioeng 2001; 76:1–10.
Heilmann K, Groth T, Behrsing O, et al. The influence of the chemical composition of cell culture material on the growth and antibody production of hybridoma cells. J Biotechnol 2005; 115:291–301.
Hencsey Z, Fizil A, Inzelt-Kovacs M, Veszely G, Bankuti L . Effect of medium composition on hybridoma growth and antibody production. Acta Microbiol Immunol Hung 1996; 43:359–370.
Lee MS, Lee GM . Hyperosmotic pressure enhances immunoglobulin transcription rates and secretion rates of KR12H-2 transfectoma. Biotechnol Bioeng 2000; 68:260–268.
Sun Z, Zhou R, Liang S, McNeeley KM, Sharfstein ST . Hyperosmotic stress in murine hybridoma cells: effects on antibody transcription, translation, posttranslational processing, and the cell cycle. Biotechnol Prog 2004; 20:576–589.
Shen D, Sharfstein ST . Genome-wide analysis of the transcriptional response of murine hybridomas to osmotic shock. Biotechnol Bioeng 2006; 93:132–145.
Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM . Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity. Proc Natl Acad Sci USA 1999; 96:2538–2542.
Burg MB, Kwon ED, Kultz D . Regulation of gene expression by hypertonicity. Annu Rev Physiol 1997; 59:437–455.
Ho SN . Intracellular water homeostasis and the mammalian cellular osmotic stress response. J Cell Physiol 2006; 206:9–15.
Maouyo D, Kim JY, Lee SD, Wu YH, Woo SK, Kwon HM . Mouse TonEBP-NFAT5: expression in early development and alternative splicing. Am J Physiol-Renal Physiol 2002; 282:F802–F809.
Cai Q, Ferraris JD, Burg MB . High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA. Am J Physiol Renal Physiol 2005; 289:F803–F807.
Loyher ML, Mutin M, Woo SK, Kwon HM, Tappaz ML . Transcription factor tonicity-responsive enhancer-binding protein (TonEBP) which transactivates osmoprotective genes is expressed and upregulated following acute systemic hypertonicity in neurons in brain. Neuroscience 2004; 124:89–104.
Zhang Z, Ferraris JD, Brooks HL, Brisc I, Burg MB . Expression of osmotic stress-related genes in tissues of normal and hyposmotic rats. Am J Physiol Renal Physiol 2003; 285:F688–F693.
Phepherd P, Dean C . Monoclonal Antibodies. Oxford: Oxford University Press, 2000.
Lopez-Rodriguez C, Aramburu J, Rakeman AS, Rao A . NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun. Proc Natl Acad Sci USA 1999; 96:7214–7219.
Riechmann JL, Heard J, Martin G, et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 2000; 290:2105–2110.
Woo SK, Dahl SC, Handler JS, Kwon HM . Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity. Am J Physiol Renal Physiol 2000; 278:F1006–F1012.
Huang AM, Rubin GM . A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster. Genetics 2000; 156:1219–1230.
Go WY, Liu XB, Roti MA, Liu F, Ho SN . NFAT5/TonEBP mutant mice define osmotic stress as a critical feature of the lymphoid microenvironment. Proc Natl Acad Sci USA 2004; 101:10673–10678.
Junger WG, Liu FC, Loomis WH, Hoyt DB . Hypertonic saline enhances cellular immune function. Circ Shock 1994; 42:190–196.
Shapiro L, Dinarello CA . Osmotic regulation of cytokine synthesis in vitro. Proc Natl Acad Sci USA 1995; 92:12230–12234.
Macian F . NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol 2005; 5:472–484.
Lopez-Rodriguez C, Aramburu J, Jin L, Rakeman AS, Michino M, Rao A . Bridging the NFAT and NF-kappaB families: NFAT5 dimerization regulates cytokine gene transcription in response to osmotic stress. Immunity 2001; 15:47–58.
Loomis WH, Namiki S, Ostrom RS, Insel PA, Junger WG . Hypertonic stress increases T cell interleukin-2 expression through a mechanism that involves ATP release, P2 receptor, and p38 MAPK activation. J Biol Chem 2003; 278:4590–4596.
Trama J, Go WY, Ho SN . The osmoprotective function of the NFAT5 transcription factor in T cell development and activation. J Immunol 2002; 169:5477–5488.
Bird L . Lymphocyte responses – NFAT5: helping out in stressful times. Nat Rev Immunol 2004; 4:581.
Acknowledgements
This project was supported by the Major State Basic Research Program of China (2002AA2Z3329). We thank Drs LX Guo and YB Chen for thoughtful discussion during the course of study, and Z Tan for technical assistance with flow cytometry.
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Ju, J., Zou, K. & Xie, H. Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells. Cell Res 17, 264–270 (2007). https://doi.org/10.1038/cr.2007.3
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DOI: https://doi.org/10.1038/cr.2007.3
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