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. 1996 Jul;16(7):3878–3883. doi: 10.1128/mcb.16.7.3878

C/EBPalpha regulation of the growth-arrest-associated gene gadd45.

C M Constance 1, J I Morgan 4th 1, R M Umek 1
PMCID: PMC231384  PMID: 8668205

Abstract

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is expressed in postmitotic, differentiated adipocytes and is required for adipose conversion of 3T3-L1 cells in culture. Temporal misexpression of C/EBPalpha in undifferentiated adipoblasts leads to mitotic growth arrest. We report here that growth arrest- and DNA damage-inducible gene 45 (gadd45) is preferentially expressed in differentiated 3T3-L1 adipocytes similar to phenotype-associated genes. Furthermore, C/EBPalpha transactivates a reporter plasmid containing 1.5 kb of the gadd45 promoter region. The proto-oncogene myc, which inhibits adipocyte differentiation, abrogates C/EBPalpha activation of gadd45. gadd45 is known to be a target of the tumor suppressor p53 in a G1 checkpoint activated by DNA damage. Immunoprecipitation of radiolabeled proteins with conformation-specific antibodies revealed that wild-type p53 is expressed throughout 3T3-L1 adipocyte development, including the postmitotic period characterized by the accumulation of gadd45 and C/EBPalpha. A stable 3T3-L1 subline was engineered to express a dominant negative p53, human p53(143ala). The p53(143ala) subline differentiated to adipocytes and showed appropriate developmental expression of gadd45. These findings suggest that postmitotic growth arrest is coupled to adipocyte differentiation via C/EBPalpha stimulation of growth arrest-associated and phenotype-associated genes.

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Selected References

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  1. Antoun G. R., Re G. G., Terry N. H., Zipf T. F. Molecular genetic evidence for a differentiation-proliferation coupling during DMSO-induced myeloid maturation of HL-60 cells: role of the transcription elongation block in the c-myc gene. Leuk Res. 1991;15(11):1029–1036. doi: 10.1016/0145-2126(91)90108-6. [DOI] [PubMed] [Google Scholar]
  2. Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
  3. Bernlohr D. A., Angus C. W., Lane M. D., Bolanowski M. A., Kelly T. J., Jr Expression of specific mRNAs during adipose differentiation: identification of an mRNA encoding a homologue of myelin P2 protein. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5468–5472. doi: 10.1073/pnas.81.17.5468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernlohr D. A., Bolanowski M. A., Kelly T. J., Jr, Lane M. D. Evidence for an increase in transcription of specific mRNAs during differentiation of 3T3-L1 preadipocytes. J Biol Chem. 1985 May 10;260(9):5563–5567. [PubMed] [Google Scholar]
  5. Birkenmeier E. H., Gwynn B., Howard S., Jerry J., Gordon J. I., Landschulz W. H., McKnight S. L. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev. 1989 Aug;3(8):1146–1156. doi: 10.1101/gad.3.8.1146. [DOI] [PubMed] [Google Scholar]
  6. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. doi: 10.1101/gad.5.9.1538. [DOI] [PubMed] [Google Scholar]
  7. Christy R. J., Yang V. W., Ntambi J. M., Geiman D. E., Landschulz W. H., Friedman A. D., Nakabeppu Y., Kelly T. J., Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Dev. 1989 Sep;3(9):1323–1335. doi: 10.1101/gad.3.9.1323. [DOI] [PubMed] [Google Scholar]
  8. Dang C. V., van Dam H., Buckmire M., Lee W. M. DNA-binding domain of human c-Myc produced in Escherichia coli. Mol Cell Biol. 1989 Jun;9(6):2477–2486. doi: 10.1128/mcb.9.6.2477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donehower L. A., Harvey M., Slagle B. L., McArthur M. J., Montgomery C. A., Jr, Butel J. S., Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature. 1992 Mar 19;356(6366):215–221. doi: 10.1038/356215a0. [DOI] [PubMed] [Google Scholar]
  10. Fornace A. J., Jr, Alamo I., Jr, Hollander M. C. DNA damage-inducible transcripts in mammalian cells. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8800–8804. doi: 10.1073/pnas.85.23.8800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fornace A. J., Jr, Nebert D. W., Hollander M. C., Luethy J. D., Papathanasiou M., Fargnoli J., Holbrook N. J. Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. Mol Cell Biol. 1989 Oct;9(10):4196–4203. doi: 10.1128/mcb.9.10.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Freytag S. O. Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1. Mol Cell Biol. 1988 Apr;8(4):1614–1624. doi: 10.1128/mcb.8.4.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Green H., Kehinde O. An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion. Cell. 1975 May;5(1):19–27. doi: 10.1016/0092-8674(75)90087-2. [DOI] [PubMed] [Google Scholar]
  15. Halevy O., Novitch B. G., Spicer D. B., Skapek S. X., Rhee J., Hannon G. J., Beach D., Lassar A. B. Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science. 1995 Feb 17;267(5200):1018–1021. doi: 10.1126/science.7863327. [DOI] [PubMed] [Google Scholar]
  16. Hermeking H., Wolf D. A., Kohlhuber F., Dickmanns A., Billaud M., Fanning E., Eick D. Role of c-myc in simian virus 40 large tumor antigen-induced DNA synthesis in quiescent 3T3-L1 mouse fibroblasts. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10412–10416. doi: 10.1073/pnas.91.22.10412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hinds P. W., Finlay C. A., Frey A. B., Levine A. J. Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol. 1987 Aug;7(8):2863–2869. doi: 10.1128/mcb.7.8.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaestner K. H., Ntambi J. M., Kelly T. J., Jr, Lane M. D. Differentiation-induced gene expression in 3T3-L1 preadipocytes. A second differentially expressed gene encoding stearoyl-CoA desaturase. J Biol Chem. 1989 Sep 5;264(25):14755–14761. [PubMed] [Google Scholar]
  19. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  20. Konigsberg I. R. Skeletal myoblasts in culture. Methods Enzymol. 1979;58:511–527. doi: 10.1016/s0076-6879(79)58166-x. [DOI] [PubMed] [Google Scholar]
  21. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491–7495. doi: 10.1073/pnas.89.16.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lassar A. B., Buskin J. N., Lockshon D., Davis R. L., Apone S., Hauschka S. D., Weintraub H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell. 1989 Sep 8;58(5):823–831. doi: 10.1016/0092-8674(89)90935-5. [DOI] [PubMed] [Google Scholar]
  23. Lin F. T., Lane M. D. Antisense CCAAT/enhancer-binding protein RNA suppresses coordinate gene expression and triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. Genes Dev. 1992 Apr;6(4):533–544. doi: 10.1101/gad.6.4.533. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Macleod K. F., Sherry N., Hannon G., Beach D., Tokino T., Kinzler K., Vogelstein B., Jacks T. p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev. 1995 Apr 15;9(8):935–944. doi: 10.1101/gad.9.8.935. [DOI] [PubMed] [Google Scholar]
  26. Nathans D., Lau L. F., Christy B., Hartzell S., Nakabeppu Y., Ryder K. Genomic response to growth factors. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):893–900. doi: 10.1101/sqb.1988.053.01.102. [DOI] [PubMed] [Google Scholar]
  27. Rolland V., Le Liepvre X., Houbiguian M. L., Lavau M., Dugail I. C/EBP alpha expression in adipose tissue of genetically obese Zucker rats. Biochem Biophys Res Commun. 1995 Feb 15;207(2):761–767. doi: 10.1006/bbrc.1995.1252. [DOI] [PubMed] [Google Scholar]
  28. Samuelsson L., Strömberg K., Vikman K., Bjursell G., Enerbäck S. The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development. EMBO J. 1991 Dec;10(12):3787–3793. doi: 10.1002/j.1460-2075.1991.tb04948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scott R. E., Florine D. L., Wille J. J., Jr, Yun K. Coupling of growth arrest and differentiation at a distinct state in the G1 phase of the cell cycle: GD. Proc Natl Acad Sci U S A. 1982 Feb;79(3):845–849. doi: 10.1073/pnas.79.3.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shugart E. C., Levenson A. S., Constance C. M., Umek R. M. Differential expression of gas and gadd genes at distinct growth arrest points during adipocyte development. Cell Growth Differ. 1995 Dec;6(12):1541–1547. [PubMed] [Google Scholar]
  31. Spiegelman B. M., Frank M., Green H. Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development. J Biol Chem. 1983 Aug 25;258(16):10083–10089. [PubMed] [Google Scholar]
  32. Stein G. H., Dulić V. Origins of G1 arrest in senescent human fibroblasts. Bioessays. 1995 Jun;17(6):537–543. doi: 10.1002/bies.950170610. [DOI] [PubMed] [Google Scholar]
  33. Student A. K., Hsu R. Y., Lane M. D. Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. J Biol Chem. 1980 May 25;255(10):4745–4750. [PubMed] [Google Scholar]
  34. 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]
  35. Vasseur-Cognet M., Lane M. D. Trans-acting factors involved in adipogenic differentiation. Curr Opin Genet Dev. 1993 Apr;3(2):238–245. doi: 10.1016/0959-437x(93)90029-o. [DOI] [PubMed] [Google Scholar]
  36. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. doi: 10.1038/366701a0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Zhan Q., Carrier F., Fornace A. J., Jr Induction of cellular p53 activity by DNA-damaging agents and growth arrest. Mol Cell Biol. 1993 Jul;13(7):4242–4250. doi: 10.1128/mcb.13.7.4242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]

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