Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Protein Arginine Methyltransferase 5 (PRMT5) Regulates Adipogenesis of 3T3L-1 Cells

단백질 아르기닌 메틸전이효소 5(PRMT5)에 의한 3T3L-1 세포의 지방세포 분화 조절

  • Jang, Min Jung (Department of Molecular Biology, Dankook University) ;
  • Yang, Ji Hye (Department of Molecular Biology, Dankook University) ;
  • Kim, Eun-Joo (Department of Molecular Biology, Dankook University)
  • 장민준 (단국대학교 분자생물학과) ;
  • 양지혜 (단국대학교 분자생물학과) ;
  • 김은주 (단국대학교 분자생물학과)
  • Received : 2018.02.21
  • Accepted : 2018.03.23
  • Published : 2018.07.30
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Abstract

Peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) is a key transcription factor that regulates adipogenesis, and epigenetic control of $PPAR{\gamma}$ is of great interest in obesity-inhibition research. Our previous study showed that CACUL1 (CDK2-associated cullin domain 1) acts as a corepressor that inhibits $PPAR{\gamma}$ transcriptional activity and adipocyte differentiation. Here, we investigated the roles of protein arginine methyltransferase 5 (PRMT5), a novel binding partner of CACUL1, in regulating $PPAR{\gamma}$. The interaction between PRMT5 and CACUL1 was shown by immunoprecipitation assay in vivo and GST pulldown assay in vitro. As shown by luciferase reporter assay, PRMT5 and CACUL1 cooperated to inhibit the transcriptional activity of $PPAR{\gamma}$. The suppressive role of PRMT5 in adipogenesis was examined by Oil Red O staining using 3T3-L1 cells, which stably overexpress or deplete PRMT5. Overexpression of PRMT5 suppresses $PPAR{\gamma}$-mediated adipogenesis, whereas PRMT5 knockdown increases lipid accumulation in 3T3-L1 cells. Consistently, PRMT5 attenuates the expression of Lpl and aP2, the target genes of $PPAR{\gamma}$, as demonstrated by RT-qPCR analysis. Overall, these results suggest that PRMT5 interacts with CACUL1 to impair the transcriptional activity of $PPAR{\gamma}$, leading to the inhibition of adipocyte differentiation. Therefore, the regulation of PRMT5 enzymatic activity may provide a clue to develop an anti-obesity drug.

$PPAR{\gamma}$는 지방세포의 분화를 조절하는 핵심적인 전사 인자로서 이를 조절하는 후성유전학적 조절 기전이 비만억제 연구에서 중요하게 주목 받고 있다. 선행연구에서 CACUL1이 $PPAR{\gamma}$의 전사 활성 및 지방세포의 분화를 억제하는 corepressor로서 작용함을 밝힌 바 있으며 본 연구에서는 CACUL1의 새로운 결합 단백질로 발굴된 protein arginine methyltransferase 5 (PRMT5)의 $PPAR{\gamma}$ 조절 기능을 분석하였다. PRMT5가 CACUL1과 결합함을 immunoprecipitation assay in vivo와 GST-pull down assay in vitro를 통하여 확인하였다. Luciferase reporter assay 결과로 두 단백질이 상호 협력하여 $PPAR{\gamma}$의 전사 활성을 억제함을 확인하였다. PRMT5가 안정적으로 과발현 또는 knockdown되는 3T3-L1 세포주를 제작하여 지방세포 분화에 미치는 영향을 분석한 결과, PRMT5가 3T3-L1세포의 지방세포 분화를 억제함을 증명하였다. 같은 맥락으로 PRMT5는 $PPAR{\gamma}$의 타겟 유전자인 Lpl과 aP2의 발현을 억제하는 것을 RT-qPCR로 확인하였다. 이상의 연구 결과로 PRMT5이 CACUL1과 결합하여 $PPAR{\gamma}$의 전사 활성을 방해, 나아가 지방세포의 분화를 억제하는 기존에 알려지지 않은 분자적 기전을 처음으로 밝혔다. 따라서, PRMT5 효소 활성의 조절은 비만 억제를 위한 약물 개발에 단서를 제공할 것이다.

Keywords

References

  1. Branscomebe, T. L., Frankel, A., Lee, J. H., Cook, J. R., Yang, Z., Pestka, S. and Clarke, S. 2001. PRMT5 (janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins. J. Biol. Chem. 276, 32971-32976. https://doi.org/10.1074/jbc.M105412200
  2. Chen, H. C. and Farese, R. V. 2005. Inhibition of triglyceride synthesis as a treatment strategy for obesity: lessons from DGAT1-deficient mice. Arterioscler. Thromb. Vasc. Biol. 25, 482-486. https://doi.org/10.1161/01.ATV.0000151874.81059.ad
  3. Cooke, D. and Bloom, S. 2006. The obesity pipeline: current strategies in the development of anti-obesity drugs. Nat. Rev. Drug Discov. 5, 919-1200. https://doi.org/10.1038/nrd2136
  4. Fabbrizio, E., El Messaoudi, S., Polanowska, J., Paul, C., Cook, J. R., Lee, J. H., Negre, V., Rousset, M., Pestka, S., Le Cam, A. and Sardet, C. 2002. Negative regulation of transcription by the type II arginine methyltransferase PRMT5. EMBO Rep. 3, 641-645. https://doi.org/10.1093/embo-reports/kvf136
  5. Goyal, A., Chavez, S. L. and Reijo Pera, R. A. 2013. Generation of human induced pluripotent stem cells using epigenetic regulators reveals a germ cell-like identity in partially reprogrammed colonies. PLoS One 8, e82838. https://doi.org/10.1371/journal.pone.0082838
  6. Gu, Z., Gao, S., Zhang, F., Wan,g Z., Ma, W. and Davis, R. E. 2012. Protein arginine methyltransferase 5 is essential for growth of lung cancer cells. Biochem. J. 446, 235-241. https://doi.org/10.1042/BJ20120768
  7. Jang, M. J., Park, U. H., Kim, J. W., Choi, H., Um, S. J. and Kim, E. J. 2017. CACUL1 reciprocally regulates SIRT1 and LSD1 to repress PPAR$\gamma$ and inhibit adipogenesis. Cell Death Dis. 8, 3201. https://doi.org/10.1038/s41419-017-0070-z
  8. Karkhanis, V., Hu, Y. J., Baiocchi, R. A., Imbalzano, A. N. and Sif, S. 2011. Versatility of PRMT5 induced methylation in growth control and development. Trends Biochem. Sci. 36, 633-641. https://doi.org/10.1016/j.tibs.2011.09.001
  9. Kopelman, P. G. 2000. Obesity as a medical problem. Nature 404, 635-643. https://doi.org/10.1038/35007508
  10. Liu, F., Cheng, G., Hamard, P. J., Greenblatt, S., Wang, L., Man, N., Perna, F., Xu, H., Tadi, M., Luciani, L. and Nimer, S. D. 2015. Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis. J. Clin. Invest. 125, 3532-3544. https://doi.org/10.1172/JCI81749
  11. Powers, M. A., Fay, M. M., Factor, R. E., Welm, A. L. and Ullman, K. S. 2011. Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 71, 5579-5587. https://doi.org/10.1158/0008-5472.CAN-11-0458
  12. Rosen, E. D. and MacDougald, O. A. 2006. Adipocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol. 7, 885-896. https://doi.org/10.1038/nrm2066
  13. Sugii, S. and Evans, R. M. 2011. Epigenetic codes of PPARgamma in metabolic disease. FEBS Lett. 585, 2121e2128. https://doi.org/10.1016/j.febslet.2011.05.007
  14. Tee, W. W., Pardo, M., Theunissen, T. W., Yu, L., Choudhary, J. S., Hajkova, P. and Surani, M. A. 2010. Prmt5 is essential for early mouse deelopment and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev. 24, 2772-2777. https://doi.org/10.1101/gad.606110
  15. Tontonoz, P., Hu, E. and Spiegelman, B. M. 1994. Stimulation of adipogenesis in fibroblasts by PPAR$\gamma$2, a lipid-activated transcription factor. Cell 79, 1147-1156. https://doi.org/10.1016/0092-8674(94)90006-X
  16. Tontonoz, P. and Spiegelman, B. M. 2008. Fat and beyond: the diverse biology of PPARgamma. Annu. Rev. Biochem. 77, 289-312. https://doi.org/10.1146/annurev.biochem.77.061307.091829
  17. Wei, T. Y., Juan, C. C., Hsia, J. Y., Su, L. J., Lee, Y. C., Chou, H. Y., Chen, J. M., Wu, Y. C., Chiu, S. C., Hsu, C. P., Liu, K. L. and Yu, C. T. 2012. PRMT5 is a potential oncoprotein that upregulates G1 cyclins/CDKs and the PI3K/AKT signaling cascade. Cancer Sci. 103, 1640-1650. https://doi.org/10.1111/j.1349-7006.2012.02367.x