Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Mar;15(3):1737–1746. doi: 10.1128/mcb.15.3.1737

Transcription-independent turnover of I kappa B alpha during monocyte adherence: implications for a translational component regulating I kappa B alpha/MAD-3 mRNA levels.

A K Lofquist 1, K Mondal 1, J S Morris 1, J S Haskill 1
PMCID: PMC230398  PMID: 7532282

Abstract

We identified I kappa B alpha/MAD-3 as an immediate-early gene in human monocytes that is expressed in response to a variety of signals, including adhesion, lipopolysaccharide, and phorbol myristate acetate. Within 5 min of monocyte adhesion, the level of the I kappa B alpha protein is markedly diminished but is rapidly replaced in a cycloheximide-sensitive manner within 20 min. Accompanying the rapid turnover of the I kappa B alpha protein is simultaneous translocation of NF-kappa B-related transcription factors to nuclei of adhered monocytes. The demonstration that NF-kappa B can regulate I kappa B alpha/MAD-3 gene transcription in other cell types suggested that the rapid increase in steady-state I kappa B alpha/MAD-3 mRNA levels we observed within 30 min of monocyte adherence would result from NF-kappa B-dependent transcriptional stimulation of the I kappa B alpha/MAD-3 gene. Nuclear run-on analyses indicated that, instead, while several immediate-early cytokine genes, such as the interleukin 1 beta (IL-1 beta) gene, were transcriptionally activated during monocyte adhesion, the rate of I kappa B alpha/MAD-3 gene transcription remained constant. The adherence-dependent increase in I kappa B alpha/MAD-3 mRNA levels was also not a consequence of mRNA stabilization events. Interestingly, while increases in both IL-1 beta and I kappa B alpha/MAD-3 mRNA levels were detected in nuclei of adherent monocytes, cytoplasmic levels of IL-1 beta mRNA increased during adherence whereas those of I kappa B alpha/MAD-3 mRNA did not. Taken together, our data suggest that two interactive mechanisms regulate monocytic I kappa B alpha/MAD-3 mRNA levels. We propose that adherent monocytes regulate nuclear processing (or decay) of I kappa B alpha/MAD-3 mRNA, thereby increasing mRNA levels without stimulating I kappa B alpha/MAD-3 gene transcription. Moreover, since inhibition of protein synthesis leads to accumulation of I kappa B alpha/MAD-3 mRNA without stimulating I kappa B alpha/MAD-3 gene transcription, we suggest that low cytoplasmic levels of I kappa B alpha/MAD-3 mRNA are maintained by a translation-dependent degradation mechanism.

Full Text

The Full Text of this article is available as a PDF (530.1 KB).

Selected References

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

  1. Aharon T., Schneider R. J. Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism. Mol Cell Biol. 1993 Mar;13(3):1971–1980. doi: 10.1128/mcb.13.3.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albelda S. M., Buck C. A. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed] [Google Scholar]
  3. Arend W. P., Gordon D. F., Wood W. M., Janson R. W., Joslin F. G., Jameel S. IL-1 beta production in cultured human monocytes is regulated at multiple levels. J Immunol. 1989 Jul 1;143(1):118–126. [PubMed] [Google Scholar]
  4. Arend W. P., Joslin F. G., Thompson R. C., Hannum C. H. An IL-1 inhibitor from human monocytes. Production and characterization of biologic properties. J Immunol. 1989 Sep 15;143(6):1851–1858. [PubMed] [Google Scholar]
  5. Baumann H., Gauldie J. The acute phase response. Immunol Today. 1994 Feb;15(2):74–80. doi: 10.1016/0167-5699(94)90137-6. [DOI] [PubMed] [Google Scholar]
  6. Beekhuizen H., van Furth R. Monocyte adherence to human vascular endothelium. J Leukoc Biol. 1993 Oct;54(4):363–378. [PubMed] [Google Scholar]
  7. Beg A. A., Baldwin A. S., Jr The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. Genes Dev. 1993 Nov;7(11):2064–2070. doi: 10.1101/gad.7.11.2064. [DOI] [PubMed] [Google Scholar]
  8. Beg A. A., Finco T. S., Nantermet P. V., Baldwin A. S., Jr Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993 Jun;13(6):3301–3310. doi: 10.1128/mcb.13.6.3301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brown K., Park S., Kanno T., Franzoso G., Siebenlist U. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2532–2536. doi: 10.1073/pnas.90.6.2532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  11. Campanero M. R., Pulido R., Ursa M. A., Rodríguez-Moya M., de Landázuri M. O., Sánchez-Madrid F. An alternative leukocyte homotypic adhesion mechanism, LFA-1/ICAM-1-independent, triggered through the human VLA-4 integrin. J Cell Biol. 1990 Jun;110(6):2157–2165. doi: 10.1083/jcb.110.6.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chandler L. A., Ehretsmann C. P., Bourgeois S. A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event. Mol Cell Biol. 1994 May;14(5):3085–3093. doi: 10.1128/mcb.14.5.3085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chiao P. J., Miyamoto S., Verma I. M. Autoregulation of I kappa B alpha activity. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):28–32. doi: 10.1073/pnas.91.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  15. Clark B. D., Collins K. L., Gandy M. S., Webb A. C., Auron P. E. Genomic sequence for human prointerleukin 1 beta: possible evolution from a reverse transcribed prointerleukin 1 alpha gene. Nucleic Acids Res. 1986 Oct 24;14(20):7897–7914. doi: 10.1093/nar/14.20.7897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cordle S. R., Donald R., Read M. A., Hawiger J. Lipopolysaccharide induces phosphorylation of MAD3 and activation of c-Rel and related NF-kappa B proteins in human monocytic THP-1 cells. J Biol Chem. 1993 Jun 5;268(16):11803–11810. [PubMed] [Google Scholar]
  17. Decker C. J., Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci. 1994 Aug;19(8):336–340. doi: 10.1016/0968-0004(94)90073-6. [DOI] [PubMed] [Google Scholar]
  18. Dhawan J., Lichtler A. C., Rowe D. W., Farmer S. R. Cell adhesion regulates pro-alpha 1(I) collagen mRNA stability and transcription in mouse fibroblasts. J Biol Chem. 1991 May 5;266(13):8470–8475. [PubMed] [Google Scholar]
  19. Eierman D. F., Johnson C. E., Haskill J. S. Human monocyte inflammatory mediator gene expression is selectively regulated by adherence substrates. J Immunol. 1989 Mar 15;142(6):1970–1976. [PubMed] [Google Scholar]
  20. Frantz B., Nordby E. C., Bren G., Steffan N., Paya C. V., Kincaid R. L., Tocci M. J., O'Keefe S. J., O'Neill E. A. Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. EMBO J. 1994 Feb 15;13(4):861–870. doi: 10.1002/j.1460-2075.1994.tb06329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fraser I., Hughes D., Gordon S. Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature. 1993 Jul 22;364(6435):343–346. doi: 10.1038/364343a0. [DOI] [PubMed] [Google Scholar]
  22. Gerritsen M. E., Bloor C. M. Endothelial cell gene expression in response to injury. FASEB J. 1993 Apr 1;7(6):523–532. doi: 10.1096/fasebj.7.6.8472891. [DOI] [PubMed] [Google Scholar]
  23. Gillis P., Malter J. S. The adenosine-uridine binding factor recognizes the AU-rich elements of cytokine, lymphokine, and oncogene mRNAs. J Biol Chem. 1991 Feb 15;266(5):3172–3177. [PubMed] [Google Scholar]
  24. Gilmore T. D., Morin P. J. The I kappa B proteins: members of a multifunctional family. Trends Genet. 1993 Dec;9(12):427–433. doi: 10.1016/0168-9525(93)90106-r. [DOI] [PubMed] [Google Scholar]
  25. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  26. Haskill S., Beg A. A., Tompkins S. M., Morris J. S., Yurochko A. D., Sampson-Johannes A., Mondal K., Ralph P., Baldwin A. S., Jr Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. Cell. 1991 Jun 28;65(7):1281–1289. doi: 10.1016/0092-8674(91)90022-q. [DOI] [PubMed] [Google Scholar]
  27. Haskill S., Johnson C., Eierman D., Becker S., Warren K. Adherence induces selective mRNA expression of monocyte mediators and proto-oncogenes. J Immunol. 1988 Mar 1;140(5):1690–1694. [PubMed] [Google Scholar]
  28. Haskill S., Peace A., Morris J., Sporn S. A., Anisowicz A., Lee S. W., Smith T., Martin G., Ralph P., Sager R. Identification of three related human GRO genes encoding cytokine functions. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7732–7736. doi: 10.1073/pnas.87.19.7732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hemler M. E., Sanchez-Madrid F., Flotte T. J., Krensky A. M., Burakoff S. J., Bhan A. K., Springer T. A., Strominger J. L. Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines. J Immunol. 1984 Jun;132(6):3011–3018. [PubMed] [Google Scholar]
  30. Henkel T., Machleidt T., Alkalay I., Krönke M., Ben-Neriah Y., Baeuerle P. A. Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature. 1993 Sep 9;365(6442):182–185. doi: 10.1038/365182a0. [DOI] [PubMed] [Google Scholar]
  31. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  32. Isaacs K. L., Sartor R. B., Haskill S. Cytokine messenger RNA profiles in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification. Gastroenterology. 1992 Nov;103(5):1587–1595. doi: 10.1016/0016-5085(92)91182-4. [DOI] [PubMed] [Google Scholar]
  33. Juliano R. L., Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993 Feb;120(3):577–585. doi: 10.1083/jcb.120.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kieran M., Blank V., Logeat F., Vandekerckhove J., Lottspeich F., Le Bail O., Urban M. B., Kourilsky P., Baeuerle P. A., Israël A. The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product. Cell. 1990 Sep 7;62(5):1007–1018. doi: 10.1016/0092-8674(90)90275-j. [DOI] [PubMed] [Google Scholar]
  35. Kiledjian M., Kadesch T. Post-transcriptional regulation of the human liver/bone/kidney alkaline phosphatase gene. J Biol Chem. 1991 Mar 5;266(7):4207–4213. [PubMed] [Google Scholar]
  36. Kishimoto T., Taga T., Akira S. Cytokine signal transduction. Cell. 1994 Jan 28;76(2):253–262. doi: 10.1016/0092-8674(94)90333-6. [DOI] [PubMed] [Google Scholar]
  37. Le Bail O., Schmidt-Ullrich R., Israël A. Promoter analysis of the gene encoding the I kappa B-alpha/MAD3 inhibitor of NF-kappa B: positive regulation by members of the rel/NF-kappa B family. EMBO J. 1993 Dec 15;12(13):5043–5049. doi: 10.1002/j.1460-2075.1993.tb06197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lenardo M. J., Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989 Jul 28;58(2):227–229. doi: 10.1016/0092-8674(89)90833-7. [DOI] [PubMed] [Google Scholar]
  39. Malim M. H., Cullen B. R. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol Cell Biol. 1993 Oct;13(10):6180–6189. doi: 10.1128/mcb.13.10.6180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mellits K. H., Hay R. T., Goodbourn S. Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B. Nucleic Acids Res. 1993 Nov 11;21(22):5059–5066. doi: 10.1093/nar/21.22.5059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Osipovich O. A., Fegeding K. V., Misuno N. I., Kolesnikova T. S., Savostin I. K., Sudarikov A. B., Voitenok N. N. Differential action of cycloheximide and activation stimuli on transcription of tumor necrosis factor-alpha, IL-1 beta, IL-8, and P53 genes in human monocytes. J Immunol. 1993 Jun 1;150(11):4958–4965. [PubMed] [Google Scholar]
  42. Read M. A., Whitley M. Z., Williams A. J., Collins T. NF-kappa B and I kappa B alpha: an inducible regulatory system in endothelial activation. J Exp Med. 1994 Feb 1;179(2):503–512. doi: 10.1084/jem.179.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rice N. R., Ernst M. K. In vivo control of NF-kappa B activation by I kappa B alpha. EMBO J. 1993 Dec;12(12):4685–4695. doi: 10.1002/j.1460-2075.1993.tb06157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rice N. R., MacKichan M. L., Israël A. The precursor of NF-kappa B p50 has I kappa B-like functions. Cell. 1992 Oct 16;71(2):243–253. doi: 10.1016/0092-8674(92)90353-e. [DOI] [PubMed] [Google Scholar]
  45. Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
  46. Savant-Bhonsale S., Cleveland D. W. Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex. Genes Dev. 1992 Oct;6(10):1927–1939. doi: 10.1101/gad.6.10.1927. [DOI] [PubMed] [Google Scholar]
  47. Schwartz M. A. Transmembrane signalling by integrins. Trends Cell Biol. 1992 Oct;2(10):304–308. doi: 10.1016/0962-8924(92)90120-c. [DOI] [PubMed] [Google Scholar]
  48. Schütze S., Potthoff K., Machleidt T., Berkovic D., Wiegmann K., Krönke M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell. 1992 Nov 27;71(5):765–776. doi: 10.1016/0092-8674(92)90553-o. [DOI] [PubMed] [Google Scholar]
  49. Scott M. L., Fujita T., Liou H. C., Nolan G. P., Baltimore D. The p65 subunit of NF-kappa B regulates I kappa B by two distinct mechanisms. Genes Dev. 1993 Jul;7(7A):1266–1276. doi: 10.1101/gad.7.7a.1266. [DOI] [PubMed] [Google Scholar]
  50. Shaw G., Kamen R. A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell. 1986 Aug 29;46(5):659–667. doi: 10.1016/0092-8674(86)90341-7. [DOI] [PubMed] [Google Scholar]
  51. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  52. Song M. K., Dozin B., Grieco D., Rall J. E., Nikodem V. M. Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone. J Biol Chem. 1988 Dec 5;263(34):17970–17974. [PubMed] [Google Scholar]
  53. Sporn S. A., Eierman D. F., Johnson C. E., Morris J., Martin G., Ladner M., Haskill S. Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J Immunol. 1990 Jun 1;144(11):4434–4441. [PubMed] [Google Scholar]
  54. Sun S. C., Ganchi P. A., Ballard D. W., Greene W. C. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science. 1993 Mar 26;259(5103):1912–1915. doi: 10.1126/science.8096091. [DOI] [PubMed] [Google Scholar]
  55. Sun S. C., Ganchi P. A., Béraud C., Ballard D. W., Greene W. C. Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1346–1350. doi: 10.1073/pnas.91.4.1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Ulmer A. J., Flad H. D. Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium. J Immunol Methods. 1979;30(1):1–10. doi: 10.1016/0022-1759(79)90268-0. [DOI] [PubMed] [Google Scholar]
  57. Wallis W. J., Beatty P. G., Ochs H. D., Harlan J. M. Human monocyte adherence to cultured vascular endothelium: monoclonal antibody-defined mechanisms. J Immunol. 1985 Oct;135(4):2323–2330. [PubMed] [Google Scholar]
  58. Wang A. M., Doyle M. V., Mark D. F. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9717–9721. doi: 10.1073/pnas.86.24.9717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wright S. Regulation of eukaryotic gene expression by transcriptional attenuation. Mol Biol Cell. 1993 Jul;4(7):661–668. doi: 10.1091/mbc.4.7.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yurochko A. D., Liu D. Y., Eierman D., Haskill S. Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9034–9038. doi: 10.1073/pnas.89.19.9034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. de Martin R., Vanhove B., Cheng Q., Hofer E., Csizmadia V., Winkler H., Bach F. H. Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B. EMBO J. 1993 Jul;12(7):2773–2779. doi: 10.1002/j.1460-2075.1993.tb05938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES