Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 May 15;324(Pt 1):237–242. doi: 10.1042/bj3240237

NF-kappaB-dependent expression of nitric oxide synthase is required for membrane fusion of chick embryonic myoblasts.

K H Lee 1, D G Kim 1, N Y Shin 1, W K Song 1, H Kwon 1, C H Chung 1, M S Kang 1
PMCID: PMC1218422  PMID: 9164862

Abstract

The activity of nitric oxide synthase (NOS) has recently been shown to increase transiently but dramatically in chick embryonic myoblasts that are competent for fusion and that NO acts as a messenger for the cell fusion. Here we show that the rise in NOS activity is tightly correlated with an increase in NOS protein level, and its synthesis is under transcriptional control. In addition, a partial cDNA sequence of NOS obtained by reverse transcription PCR on total RNA from chick myoblasts was found to be identical with that of the inducible type of NOS (iNOS) from chick macrophage. Thus chick myoblast NOS must belong to the family of iNOS. Consistently, pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor kappaB (NF-kappaB), prevented the expression of myoblast NOS. Furthermore the antioxidant also strongly inhibited cell fusion, and its inhibitory effect was reversed by treatment with sodium nitroprusside, an NO-generating agent. In addition, nuclear extracts obtained from myoblasts that were competent for fusion, but not those from proliferating cells or from fully differentiated myotubes, were capable of binding to the consensus NF-kappaB site in the promoter region of the gene encoding iNOS. These results suggest that NF-kappaB-dependent expression of NOS is an important step in membrane fusion of chick embryonic myoblasts.

Full Text

The Full Text of this article is available as a PDF (276.8 KB).

Selected References

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

  1. Ahn J. Y., Hong S. O., Kwak K. B., Kang S. S., Tanaka K., Ichihara A., Ha D. B., Chung C. H. Developmental regulation of proteolytic activities and subunit pattern of 20 S proteasome in chick embryonic muscle. J Biol Chem. 1991 Aug 25;266(24):15746–15749. [PubMed] [Google Scholar]
  2. Baek H. J., Jeon Y. J., Kim H. S., Kang M. S., Chung C. H., Ha D. B. Cyclic AMP negatively modulates both Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein and membrane fusion of chick embryonic myoblasts. Dev Biol. 1994 Sep;165(1):178–184. doi: 10.1006/dbio.1994.1244. [DOI] [PubMed] [Google Scholar]
  3. Bedoya F. J., Flodström M., Eizirik D. L. Pyrrolidine dithiocarbamate prevents IL-1-induced nitric oxide synthase mRNA, but not superoxide dismutase mRNA, in insulin producing cells. Biochem Biophys Res Commun. 1995 May 25;210(3):816–822. doi: 10.1006/bbrc.1995.1731. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burkly L., Hession C., Ogata L., Reilly C., Marconi L. A., Olson D., Tizard R., Cate R., Lo D. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature. 1995 Feb 9;373(6514):531–536. doi: 10.1038/373531a0. [DOI] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Choi S. W., Baek M. Y., Kang M. S. Involvement of cyclic GMP in the fusion of chick embryonic myoblasts in culture. Exp Cell Res. 1992 Mar;199(1):129–133. doi: 10.1016/0014-4827(92)90470-s. [DOI] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Eberhardt W., Kunz D., Pfeilschifter J. Pyrrolidine dithiocarbamate differentially affects interleukin 1 beta- and cAMP-induced nitric oxide synthase expression in rat renal mesangial cells. Biochem Biophys Res Commun. 1994 Apr 15;200(1):163–170. doi: 10.1006/bbrc.1994.1429. [DOI] [PubMed] [Google Scholar]
  10. Herrmann H., Heywood S. M., Marchok A. C. Reconstruction of muscle development as a sequence of macromolecular synthesis. Curr Top Dev Biol. 1970;5:181–234. [PubMed] [Google Scholar]
  11. Kamijo R., Harada H., Matsuyama T., Bosland M., Gerecitano J., Shapiro D., Le J., Koh S. I., Kimura T., Green S. J. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science. 1994 Mar 18;263(5153):1612–1615. doi: 10.1126/science.7510419. [DOI] [PubMed] [Google Scholar]
  12. Kang S. J., Shin K. S., Song W. K., Ha D. B., Chung C. H., Kang M. S. Involvement of transglutaminase in myofibril assembly of chick embryonic myoblasts in culture. J Cell Biol. 1995 Sep;130(5):1127–1136. doi: 10.1083/jcb.130.5.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Knowles R. G., Merrett M., Salter M., Moncada S. Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat. Biochem J. 1990 Sep 15;270(3):833–836. doi: 10.1042/bj2700833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Knowles R. G., Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994 Mar 1;298(Pt 2):249–258. doi: 10.1042/bj2980249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lee K. H., Baek M. Y., Moon K. Y., Song W. K., Chung C. H., Ha D. B., Kang M. S. Nitric oxide as a messenger molecule for myoblast fusion. J Biol Chem. 1994 May 20;269(20):14371–14374. [PubMed] [Google Scholar]
  17. Lin A. W., Chang C. C., McCormick C. C. Molecular cloning and expression of an avian macrophage nitric-oxide synthase cDNA and the analysis of the genomic 5'-flanking region. J Biol Chem. 1996 May 17;271(20):11911–11919. doi: 10.1074/jbc.271.20.11911. [DOI] [PubMed] [Google Scholar]
  18. Marsden P. A., Heng H. H., Scherer S. W., Stewart R. J., Hall A. V., Shi X. M., Tsui L. C., Schappert K. T. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem. 1993 Aug 15;268(23):17478–17488. [PubMed] [Google Scholar]
  19. Murre C., Voronova A., Baltimore D. B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits. Mol Cell Biol. 1991 Feb;11(2):1156–1160. doi: 10.1128/mcb.11.2.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nakane M., Schmidt H. H., Pollock J. S., Förstermann U., Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett. 1993 Jan 25;316(2):175–180. doi: 10.1016/0014-5793(93)81210-q. [DOI] [PubMed] [Google Scholar]
  21. Nathan C., Xie Q. W. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994 Sep 23;78(6):915–918. doi: 10.1016/0092-8674(94)90266-6. [DOI] [PubMed] [Google Scholar]
  22. Palombella V. J., Rando O. J., Goldberg A. L., Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell. 1994 Sep 9;78(5):773–785. doi: 10.1016/s0092-8674(94)90482-0. [DOI] [PubMed] [Google Scholar]
  23. Rosette C., Karin M. Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kappa B. J Cell Biol. 1995 Mar;128(6):1111–1119. doi: 10.1083/jcb.128.6.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmidt H. H., Walter U. NO at work. Cell. 1994 Sep 23;78(6):919–925. doi: 10.1016/0092-8674(94)90267-4. [DOI] [PubMed] [Google Scholar]
  25. Schreiber E., Matthias P., Müller M. M., Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res. 1989 Aug 11;17(15):6419–6419. doi: 10.1093/nar/17.15.6419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  27. Staudt L. M., Singh H., Sen R., Wirth T., Sharp P. A., Baltimore D. A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes. Nature. 1986 Oct 16;323(6089):640–643. doi: 10.1038/323640a0. [DOI] [PubMed] [Google Scholar]
  28. Thanos D., Maniatis T. NF-kappa B: a lesson in family values. Cell. 1995 Feb 24;80(4):529–532. doi: 10.1016/0092-8674(95)90506-5. [DOI] [PubMed] [Google Scholar]
  29. Williams G., Brown T., Becker L., Prager M., Giroir B. P. Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes. Am J Physiol. 1994 Oct;267(4 Pt 2):R1020–R1025. doi: 10.1152/ajpregu.1994.267.4.R1020. [DOI] [PubMed] [Google Scholar]
  30. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992 Apr 10;256(5054):225–228. doi: 10.1126/science.1373522. [DOI] [PubMed] [Google Scholar]
  31. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992 Apr 10;256(5054):225–228. doi: 10.1126/science.1373522. [DOI] [PubMed] [Google Scholar]
  32. Xie Q. W., Kashiwabara Y., Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994 Feb 18;269(7):4705–4708. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES