Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Dec;17(12):7353–7361. doi: 10.1128/mcb.17.12.7353

CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice.

N A Timchenko 1, T E Harris 1, M Wilde 1, T A Bilyeu 1, B L Burgess-Beusse 1, M J Finegold 1, G J Darlington 1
PMCID: PMC232591  PMID: 9372966

Abstract

CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Deng C., Zhang P., Harper J. W., Elledge S. J., Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell. 1995 Aug 25;82(4):675–684. doi: 10.1016/0092-8674(95)90039-x. [DOI] [PubMed] [Google Scholar]
  2. Descombes P., Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell. 1991 Nov 1;67(3):569–579. doi: 10.1016/0092-8674(91)90531-3. [DOI] [PubMed] [Google Scholar]
  3. Diehl A. M., Johns D. C., Yang S., Lin H., Yin M., Matelis L. A., Lawrence J. H. Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes. J Biol Chem. 1996 Mar 29;271(13):7343–7350. doi: 10.1074/jbc.271.13.7343. [DOI] [PubMed] [Google Scholar]
  4. Diehl A. M., Yang S. Q. Regenerative changes in C/EBP alpha and C/EBP beta expression modulate binding to the C/EBP site in the c-fos promoter. Hepatology. 1994 Feb;19(2):447–456. [PubMed] [Google Scholar]
  5. Fero M. L., Rivkin M., Tasch M., Porter P., Carow C. E., Firpo E., Polyak K., Tsai L. H., Broudy V., Perlmutter R. M. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell. 1996 May 31;85(5):733–744. doi: 10.1016/s0092-8674(00)81239-8. [DOI] [PubMed] [Google Scholar]
  6. Flodby P., Antonson P., Barlow C., Blanck A., Porsch-Hällström I., Xanthopoulos K. G. Differential patterns of expression of three C/EBP isoforms, HNF-1, and HNF-4 after partial hepatectomy in rats. Exp Cell Res. 1993 Sep;208(1):248–256. doi: 10.1006/excr.1993.1244. [DOI] [PubMed] [Google Scholar]
  7. Flodby P., Barlow C., Kylefjord H., Ahrlund-Richter L., Xanthopoulos K. G. Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha. J Biol Chem. 1996 Oct 4;271(40):24753–24760. doi: 10.1074/jbc.271.40.24753. [DOI] [PubMed] [Google Scholar]
  8. Flodby P., Liao D. Z., Blanck A., Xanthopoulos K. G., Hällström I. P. Expression of the liver-enriched transcription factors C/EBP alpha, C/EBP beta, HNF-1, and HNF-4 in preneoplastic nodules and hepatocellular carcinoma in rat liver. Mol Carcinog. 1995 Feb;12(2):103–109. doi: 10.1002/mc.2940120207. [DOI] [PubMed] [Google Scholar]
  9. Freytag S. O., Geddes T. J. Reciprocal regulation of adipogenesis by Myc and C/EBP alpha. Science. 1992 Apr 17;256(5055):379–382. doi: 10.1126/science.256.5055.379. [DOI] [PubMed] [Google Scholar]
  10. Freytag S. O., Paielli D. L., Gilbert J. D. Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev. 1994 Jul 15;8(14):1654–1663. doi: 10.1101/gad.8.14.1654. [DOI] [PubMed] [Google Scholar]
  11. Friedman A. D., Landschulz W. H., McKnight S. L. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells. Genes Dev. 1989 Sep;3(9):1314–1322. doi: 10.1101/gad.3.9.1314. [DOI] [PubMed] [Google Scholar]
  12. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  13. Hendricks-Taylor L. R., Darlington G. J. Inhibition of cell proliferation by C/EBP alpha occurs in many cell types, does not require the presence of p53 or Rb, and is not affected by large T-antigen. Nucleic Acids Res. 1995 Nov 25;23(22):4726–4733. doi: 10.1093/nar/23.22.4726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Juan T. S., Wilson D. R., Wilde M. D., Darlington G. J. Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2584–2588. doi: 10.1073/pnas.90.7.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kiyokawa H., Kineman R. D., Manova-Todorova K. O., Soares V. C., Hoffman E. S., Ono M., Khanam D., Hayday A. C., Frohman L. A., Koff A. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell. 1996 May 31;85(5):721–732. doi: 10.1016/s0092-8674(00)81238-6. [DOI] [PubMed] [Google Scholar]
  16. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  17. Landschulz W. H., Johnson P. F., McKnight S. L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science. 1989 Mar 31;243(4899):1681–1688. doi: 10.1126/science.2494700. [DOI] [PubMed] [Google Scholar]
  18. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  19. Lin F. T., Lane M. D. CCAAT/enhancer binding protein alpha is sufficient to initiate the 3T3-L1 adipocyte differentiation program. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8757–8761. doi: 10.1073/pnas.91.19.8757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin F. T., MacDougald O. A., Diehl A. M., Lane M. D. A 30-kDa alternative translation product of the CCAAT/enhancer binding protein alpha message: transcriptional activator lacking antimitotic activity. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9606–9610. doi: 10.1073/pnas.90.20.9606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maki C. G., Howley P. M. Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation. Mol Cell Biol. 1997 Jan;17(1):355–363. doi: 10.1128/mcb.17.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mischoulon D., Rana B., Bucher N. L., Farmer S. R. Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture. Mol Cell Biol. 1992 Jun;12(6):2553–2560. doi: 10.1128/mcb.12.6.2553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nakayama K., Ishida N., Shirane M., Inomata A., Inoue T., Shishido N., Horii I., Loh D. Y., Nakayama K. Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell. 1996 May 31;85(5):707–720. doi: 10.1016/s0092-8674(00)81237-4. [DOI] [PubMed] [Google Scholar]
  24. Noda A., Ning Y., Venable S. F., Pereira-Smith O. M., Smith J. R. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res. 1994 Mar;211(1):90–98. doi: 10.1006/excr.1994.1063. [DOI] [PubMed] [Google Scholar]
  25. Ossipow V., Descombes P., Schibler U. CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8219–8223. doi: 10.1073/pnas.90.17.8219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ramos R. A., Nishio Y., Maiyar A. C., Simon K. E., Ridder C. C., Ge Y., Firestone G. L. Glucocorticoid-stimulated CCAAT/enhancer-binding protein alpha expression is required for steroid-induced G1 cell cycle arrest of minimal-deviation rat hepatoma cells. Mol Cell Biol. 1996 Oct;16(10):5288–5301. doi: 10.1128/mcb.16.10.5288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995 May 15;9(10):1149–1163. doi: 10.1101/gad.9.10.1149. [DOI] [PubMed] [Google Scholar]
  28. Timchenko N. A., Wilde M., Nakanishi M., Smith J. R., Darlington G. J. CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes Dev. 1996 Apr 1;10(7):804–815. doi: 10.1101/gad.10.7.804. [DOI] [PubMed] [Google Scholar]
  29. Timchenko N., Wilson D. R., Taylor L. R., Abdelsayed S., Wilde M., Sawadogo M., Darlington G. J. Autoregulation of the human C/EBP alpha gene by stimulation of upstream stimulatory factor binding. Mol Cell Biol. 1995 Mar;15(3):1192–1202. doi: 10.1128/mcb.15.3.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Umek R. M., Friedman A. D., McKnight S. L. CCAAT-enhancer binding protein: a component of a differentiation switch. Science. 1991 Jan 18;251(4991):288–292. doi: 10.1126/science.1987644. [DOI] [PubMed] [Google Scholar]
  31. Wang N. D., Finegold M. J., Bradley A., Ou C. N., Abdelsayed S. V., Wilde M. D., Taylor L. R., Wilson D. R., Darlington G. J. Impaired energy homeostasis in C/EBP alpha knockout mice. Science. 1995 Aug 25;269(5227):1108–1112. doi: 10.1126/science.7652557. [DOI] [PubMed] [Google Scholar]
  32. Wu H., Wade M., Krall L., Grisham J., Xiong Y., Van Dyke T. Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev. 1996 Feb 1;10(3):245–260. doi: 10.1101/gad.10.3.245. [DOI] [PubMed] [Google Scholar]
  33. Yeh W. C., Cao Z., Classon M., McKnight S. L. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev. 1995 Jan 15;9(2):168–181. doi: 10.1101/gad.9.2.168. [DOI] [PubMed] [Google Scholar]
  34. el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES