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Molecular Cloning and Transient Expression of Recombinant Human PPARγ in HEK293T Cells Under an Inducible Tet-on System

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Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 μg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.

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Abbreviations

hPPARγ:

Human peroxisome proliferator-activated receptor gamma

HEK293T:

Human embryonic kidney 293T

References

  1. Rosen, E. D., & Spiegelman, B. M. (2001). PPARgamma: a nuclear regulator of metabolism, differentiation, and cell growth. Journal of Biological Chemistry, 276, 37731–37734. https://doi.org/10.1074/jbc.R100034200.

    Article  CAS  PubMed  Google Scholar 

  2. Berger, J., & Moller, D. E. (2002). The mechanisms of action of PPARs. Annual Review of Medicine, 53, 409–435. https://doi.org/10.1146/annurev.med.53.082901.104018.

    Article  CAS  PubMed  Google Scholar 

  3. Rizzo, B., & Fiorucci, S. (2006). PPARs and other nuclear receptors in inflammation. Current Opinion in Pharmacology, 6, 421–427. https://doi.org/10.1016/j.coph.2006.03.012.

    Article  CAS  PubMed  Google Scholar 

  4. Lehrke, M., & Lazar, M. (2005). The many faces of PPAR gamma. Cell, 123, 993–999. https://doi.org/10.1016/j.cell.2005.11.026.

    Article  CAS  PubMed  Google Scholar 

  5. Ahmadian, M., Suh, S., Hah, N., Liddle, C., Atkins, A., Downes, M., et al. (2013). PPARγ signaling and metabolism: the good, the bad and the future. Nature Medicine, 19, 557–566. https://doi.org/10.1038/nm.3159.

    Article  CAS  PubMed  Google Scholar 

  6. Zieleniak, A., Wojcik, M., & Wozniak, L. A. (2008). Structure and physiological functions of the human peroxisome. Arch Immunol Ther Exp, 56, 331–345. https://doi.org/10.1007/s00005-008-0037-y.

    Article  CAS  Google Scholar 

  7. Glass, C. K., Rose, D. W., & Rosenfeld, M. G. (1997). Nuclear receptors coactivators. Current Opinion in Cell Biology, 9, 222–232. https://doi.org/10.1016/S0955-0674(97)80066-X.

    Article  CAS  PubMed  Google Scholar 

  8. Raspé, E., Schoonjans, K., Lefebvre, A. M., Saladin, R., Najib, J., Laville, M., et al. (1997). The organization, promoter analysis, and expression of the human PPARgamma gene. Journal of Biological Chemistry, 272, 18779–18789. https://doi.org/10.1074/jbc.272.30.18779.

    Article  PubMed  Google Scholar 

  9. Shim, J., Kim, B., Kim, B. H., Il, Y., & Kim, K. Y. (2010). The peroxisome proliferator-activated receptor gamma ligands, pioglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2), have antineoplastic effects against hepatitis B virus-associated hepatocellular carcinoma cells. International Journal of Oncology, 36, 223–231. https://doi.org/10.3892/ijo_00000493.

    Article  CAS  PubMed  Google Scholar 

  10. Chan, S. H., Wu, K. L., Kung, P. S., & Chan, J. Y. (2010). Oral intake of rosiglitazone promotes a central antihypertensive effect via upregulation of peroxisome proliferator-activated receptor-gamma and alleviation of oxidative stress in rostral ventrolateral medulla of spontaneously hypertensive rats. Hypertension, 55, 1444–1453. https://doi.org/10.1161/HYPERTENSIONAHA.109.149146.

    Article  CAS  PubMed  Google Scholar 

  11. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., & Evans, R. M. (1995). 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell, 83, 803–812. https://doi.org/10.1016/0092-8674(95)90193-0.

    Article  CAS  PubMed  Google Scholar 

  12. Nissen, S. E., & Wolski, T. (2007). Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. New England Journal of Medicine, 356, 2457–2471. https://doi.org/10.1056/NEJMoa072761.

    Article  CAS  PubMed  Google Scholar 

  13. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., & Kliewer, S. A. (1995). An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). Journal of Biological Chemistry, 270, 12953–12956. https://doi.org/10.1074/jbc.270.22.12953.

    Article  CAS  PubMed  Google Scholar 

  14. Zhu, Z., Zheng, T., Chun, G., Homer, R. J., & Elias, J. A. (2002). Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Seminars in Cell & Developmental Biology, 13, 121–128. https://doi.org/10.1016/S1084-9521(02)00018-6.

    Article  CAS  Google Scholar 

  15. Loew, R., Heinz, N., Hampf, M., Bujard, H., & Gossen, M. (2010). Improved Tet-responsive promoters with minimized background expression. BMC Biotechnology, 10, 81–93. https://doi.org/10.1186/1472-6750-10-81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sun, Y., Chen, X., & Xiao, D. (2007). Tetracycline-inducible expression systems: new strategies and practices in the transgenic mouse modeling. Acta Biochimica et Biophysica Sinica, 39, 235–246. https://doi.org/10.1111/j.1745-7270.2007.00258.x.

    Article  CAS  PubMed  Google Scholar 

  17. Elbrecht, A., Chen, Y., Cullinan, C. A., Hayes, N., Leibowitz, M., Moller, D. E., et al. (1996). Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma 1 and gamma 2. Biochemical and Biophysical Research Communications, 224, 431–437. https://doi.org/10.1006/bbrc.1996.1044.

    Article  CAS  PubMed  Google Scholar 

  18. Relic, B., Benoit, V., Franchimont, N., Kaiser, M. J., Hauzeur, J. P., Gillet, P., et al. (2006). Peroxisome proliferator-activated receptor-gamma1 is dephosphorylated and degraded during BAY 11-7085-induced synovial fibroblast apoptosis. Journal of Biological Chemistry, 281, 22597–225604. https://doi.org/10.1074/jbc.M512807200.

    Article  CAS  PubMed  Google Scholar 

  19. Guo, F., Ren, X., Don, Y., Hu, X., Xu, D., Zhou, H., et al. (2016). Constitutive expression of PPARγ inhibits proliferation and migration of gastric cancer cells and down-regulates Wnt/β-Catenin signaling pathway downstream target genes TERT and ENAH. Gene, 584, 31–37. https://doi.org/10.1016/j.gene.2016.03.003.

    Article  CAS  PubMed  Google Scholar 

  20. Jin, D., Sun, J., Huang, J., Yu, X., Yu, A., He, Y., et al. (2015). Peroxisome proliferator-activated receptor γ enhances adiponectin secretion via up-regulating DsbA-L expression. Molecular and Cellular Endocrinology, 411, 97–104. https://doi.org/10.1016/j.mce.2015.04.015.

    Article  CAS  PubMed  Google Scholar 

  21. Camp, H. S., Wise, S. C., Hong, Y. H., Frankowski, C. L., Shen, X., Vanbogelen, R., et al. (2000). Differential activation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosiglitazone. Diabetes, 49, 539–547. https://doi.org/10.2337/diabetes.49.4.539.

    Article  CAS  PubMed  Google Scholar 

  22. Ma, J., Zhang, T., Fang, N., Zou, Y., Gong, Q., Yu, L., et al. (2012). Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma (PPARγ). Journal of Pharmacy and Pharmacology, 64, 719–726. https://doi.org/10.1111/j.2042-7158.2012.01462.x.

    Article  CAS  PubMed  Google Scholar 

  23. Thomas, P., & Smart, T. G. (2005). HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51, 187–200. https://doi.org/10.1016/j.vascn.2004.08.014.

    Article  CAS  PubMed  Google Scholar 

  24. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402–408. https://doi.org/10.1006/meth.2001.1262.

    Article  CAS  PubMed  Google Scholar 

  25. Tachibana, K., Kobayashi, Y., Tanaka, T., Tagami, M., Sugiyama, A., Katayama, T., et al. (2005). Gene expression profiling of potential peroxisome proliferator-activated receptor (PPAR) target genes in human hepatoblastoma cell lines inducibly expressing different PPAR isoforms. Nuclear Receptors, 3, 3. https://doi.org/10.1186/1478-1336-3-3.

    Article  CAS  Google Scholar 

  26. Ju, Z., Su, M., Hong, J., Ullah, S., Kim, E. L., Zhao, C., et al. (2018). Design of PPAR-γ agonist based on algal metabolites and the endogenous ligand 15-deoxy-Δ12, 14-prostaglandin J2. European Journal of Medicinal Chemistry, 157, 1192–1201. https://doi.org/10.1016/j.ejmech.2018.08.090.

    Article  CAS  PubMed  Google Scholar 

  27. Rochel, N., Krucker, K., Coutos-Thévenot, L., Osz, J., Zhang, R., Guyon, E., et al. (2019). Recurrent activating mutations of PPARγ associated with luminal bladder tumors. Nature Communications, 10, 253–264. https://doi.org/10.1038/s41467-018-08157-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

SMM thanks CONACYT-MEXICO for the Grant [No. 307119] and also thank Elizeth Pioquinto-Avila for technical support.

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Authors and Affiliations

  1. Universidad Autonoma de Nuevo Leon, Genetic Engineering and Genomics Laboratory, School of Chemistry, San Nicolas de los Garza, Nuevo Leon, Mexico

    Sonia Montero-Molina, Eder Arredondo-Espinoza, Jorge Solís-Estrada & Isaías Balderas-Rentería

  2. Biological Models Unit, Biomedical Research Institute, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico

    Daniel Garzón-Cortés

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  1. Sonia Montero-Molina
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  2. Eder Arredondo-Espinoza
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Correspondence to Isaías Balderas-Rentería.

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Montero-Molina, S., Arredondo-Espinoza, E., Solís-Estrada, J. et al. Molecular Cloning and Transient Expression of Recombinant Human PPARγ in HEK293T Cells Under an Inducible Tet-on System. Mol Biotechnol 61, 427–431 (2019). https://doi.org/10.1007/s12033-019-00173-7

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  • DOI: https://doi.org/10.1007/s12033-019-00173-7

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