Molecular Cloning and Expression of Forkhead Transcription Factor O1 Gene from Pig Sus scrofa

Pang, Weijun;Sun, Shiduo;Bai, Liang;Yang, Gongshe

  • Received : 2007.07.21
  • Accepted : 2007.12.13
  • Published : 2008.04.01


Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes preadipocyte differentiation of clonal cell lines from rodents. We isolated the full-length cDNA of porcine FoxO1 gene using RACE, confirmed by visual Northern blotting. The deduced amino acids indicated 94% and 90% identities with the corresponding human and mice aa. Analysis of the aa sequence, showed that it included a Forkhead domain (aa 167-247), a transmembrane structure domain (aa 90-113), a LXXLL motif (aa 469-473), and 51 Ser, 8 Thr, and 4 Tyr phosphorylation sites, indicating a potential important role for FoxO1 transcriptional activity in vivo. Using the IMpRH panel, we mapped FoxO1 gene to chromosome 11p13. Our data provide basic molecular information useful for the further investigation on the function of FoxO1 gene. Time-course analysis of FoxO1 expressions indicated that levels of mRNA and protein gradually increased from day 0 to 3, and it reached almost maximal level at day 3, then decreased from day 5 to 7 in porcine primary preadipocyte differentiation. After induction by IGF-1, GPDH activity and accumulation of lipid increased, however, expressions of FoxO1 mRNA and protein were inhibited in a dose dependent manner. These results suggest that FoxO1 takes part in porcine preadipocyte differentiation and expressions of FoxO1 were regulated by IGF-1.


Pig;Forkhead Transcription Factor O1;Gene Cloning;Expression;Preadipocyte;IGF-1


  1. Accili, D. and K. C. Arden. 2004. FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell. 117:421-426.
  2. Adomas, A., M. Eklund, M. Johansson and F. O. Asiegbu. 2006. Identification and analysis of differentially expressed cDNAs during nonself-competitive interaction between Phlebiopsis gigantea and Heterobasidion parviporum. FEMS. Microbiol. Ecol. 57:26-39.
  3. Altomonte, J., L. Cong, S. Harbaran, A. Richter, J. Xu, M. Meseck and H. H. Dong. 2004. Foxo1 mediates insulin action on apoCIII and triglyceride metabolism. J. Clin. Invest. 114:1493-1503.
  4. Anderson, M. J., C. S. Viars, S. Czekay, W. K. Cavenee and K. C. Arden. 1998. Cloning and characterization of three human forkhead genes that comp rise an FKHR-like gene subfamily. Genomics. 47:187-199.
  5. Birkenkamp, K. U. and P. J. Coffer. 2003. Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors. Biochem Soc Trans. 31: 292-297.
  6. Frosch, E. F., C. Schmid, J. Schwander and J. Zapf. 1985. Actions of insulin-like growth factors. Annu Rev. Physiol. 47:443-467.
  7. Furuyama, T., T. Nakazawa, I. Nakana and N. Mori. 2000. Identification of the differential distribution patterns of mRNAs and consensus binding sequences formouse DAF-16 homologues. Biochem J. 349:629-634.
  8. Gregoire, F. M., C. M. Smas and H. S. Sul. 1998. Understanding adipocyte differentiation. Physiol. Rev. 78:783-809.
  9. Hampsey, M. and D. Reinberg. 1999. RNA polymerase II as a control panel for multiple coactivator complexes. Curr. Opin. Genet. Dev. 9:132-139.
  10. Dijkers, P. F., K. U. Birkenkamp, E. W. Lam, N. S. Thomas, J. W. Lammers, L. Koenderman and P. J. Coffer. 2002. FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity. J. Cell. Biol. 156:531- 542.
  11. Franz, O., I. Bruchhaus and T. Roeder. 1999. Verification of differential gene transcription using virtual northern blotting. Nucleic Acids Res. 27:e3.
  12. Brunet, A., J. Park, H. Tran, L. S. Hu, B. A. Hemmings and M. E. Greenberg. 2001. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol. Cell. Biol. 21:952-965.
  13. Chen, H., M. Tini and R. M. Evans. 2001. HATs on and beyond chromatin. Curr. Opin. Cell Biol. 13:218-224.
  14. Coolican, S. A., D. S. Samuel, D. Z. Ewton, F. J. McWade and J. R. Florini. 1997. The mitogenic and myogenic actions of insulinlike growth factors utilize distinct signaling pathways. J. Biol. Chem. 272:6653-6662.
  15. Daitoku, H., K. Yamagata, H. Matsuzaki, M. Hatta and A. Fukamizu. 2003. Regulation of PGC-1 promoter activity by protein kinase B and the forkhead transcription factor FKHR. Diabetes 52:642-649.
  16. Nakae, J., Y. H. Cao, H. Daitoku, A. Fukamizu, W. Ogawa, Y. Yano and Y. Hayashi. 2006. The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity. J. Clin. Invest. 116:2473-2483.
  17. Nakae, J., T. Kitamura, D. L. Silver and D. Accili. 2001. The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J. Clin. Invest. 108:1359-1367
  18. Nakae, J., T. Kitamura, Y. Kitamura, W. H. Biggs, K. C. Arden and D. Accili. 2003. The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Dev. Cell. 4:119-129.
  19. McInerney, E. M., D. W. Rose, S. E. Flynn, S. Westin, T. M. Mullen, A. Krones, J. Inostroza, J. Torchia, R. T. Nolte, N. Assa-Munt, M. V. Milburn, C. K. Glass and M. G. Rosenfeld. 1998. Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes. Dev. 12: 3357-3368.
  20. Nakae, J., W. H. Biggs, T. Kitamura, W. K. Cavenee, C. V. Wright, K. C. Arden and D. Accili. 2002. Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1. Nat. Genet. 32:245-253.
  21. Kops, G. J., T. B. Dansen, P. E. Polderman, I. Saarloos, K. W. Wirtz, P. J. Coffer, T. T Huang, J. L. Bos, R. H. Medema and B. M. Burgering. 2002. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419:316- 321.
  22. Li, Y., R. H. Lu, G. F. Luo, W. J. Pang and G. S. Yang. 2006. Effects of different cryoprotectants on the viability and biological characteristics of porcine preadipocyte. Cryobiol. 53:240-247.
  23. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
  24. MacDougald, O. A. and M. D. Lane. 1995. Transcriptional regulation of gene expression during adipocyte differentiation. Annu. Rev. Biochem. 64:345-373.
  25. Heery, D. M., E. Kalkhoven, S. Hoare and M. G. Parker. 1997. A signature motif in transcriptional coactivators mediates binding to nuclear receptors. Nature 387:733-736.
  26. Jacquemin, V., G. S. Butler-Browne, D. Furling and V. Mouly. 2007. IL-13 mediates the recruitment of reserve cells for fusion during IGF-1-induced hypertrophy of human myotubes. J. Cell. Sci. 120:670-681.
  27. Kaestner, K. H., W. Knochel and D. E. Martinez. 2000. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev. 14:142-146.
  28. Peter, M. A., K. H. Winterthaler and M. Boni-Schnetzler. 1993. Regulation of insulin-like growth factor-I (IGF-I) and IGFbinding proteins by growth hormone in rate white adipose tissue. Endocrinol. 133:2624-2631.
  29. Rosen, E. D., C. J. Walkey, P. Puigserver and B. M. Spiegelman. 2000. Transcriptional regulation of adipogenesis. Genes. Dev. 14:1293-1307.
  30. Sara, V. R. and K. Hall. 1990. Insulin-like growth factors and their binding proteins. Physiological Review 70:591-614
  31. Stahl, M., P. F. Dijkers, G. J. Kops, S. M. Lens, P. J. Coffer, B. M. Burgering and R. H. Medema. 2002. The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2. J. Immunol. 168:5024-5031.
  32. Wabitsch, M., E. Heinze, K. M. Debatin and W. F. Blum. 2000. IGF-I and IGFBP-3 expression in cultured human preadipocytes and adipocytes. Hormone and Metabolic Research. 32:555-559.
  33. Wang, Y. F., Y. Li, B. Liu, M. Yu., B. Fan, M. J. Zhu, T. A. Xiong and K. Li. 2005. Partial molecular characterization, polymorphism and chromosomal localization of the porcine PSMD4 gene. J. Anim. Breed. Genet. 122:247-250.
  34. Wise, L. S. and H. Green. 1979. Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. J. Biol. Chem. 254:273-275.
  35. Wrede, C. E., L. M. Dickson, M. K. Lingohr, I. Briaud and C. J. Rhodes. 2002. Protein kinase B/Akt prevents fatty acidinduced apoptosis in pancreatic beta-cells (INS-1). J. Biol. Chem. 277:49676-49684.
  36. Yu, J., Q. Y. Zhou, M. J. Zhu, C. C. Li, B. Liu, B. Fan and S. H. Zhao. 2007. The porcine FoxO1, FoxO3a and FoxO4 genes: cloning, mapping, expression and association analysis with meat production traits. Asian-Aust. J. Anim. Sci. 20:627-632.
  37. Farmer, S. R. 2003. The forkhead transcription factor Foxo1: a possible link between obesity and insulin resistance. Mol. Cell. 11:6-8.