Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jan 1;108(1):159–167. doi: 10.1083/jcb.108.1.159

Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. I. A pertussis toxin- sensitive pathway is involved

PMCID: PMC2115354  PMID: 2536032

Abstract

Cells of the nonfusing muscle cell line BC3H1 stop proliferating and express a family of muscle-specific proteins when the FBS concentration is reduced from 20 to 0.5% (Munson, R., K.L. Caldwell, and L. Glaser. 1982. J. Cell Biol. 92:350-356). Several growth factors have been shown to block differentiation in this cell line. To begin to investigate the potential role of G proteins in signal transducing pathways from these receptors, we have examined the effects of cholera toxin (CT) and pertussis toxin (PT) on proliferation and differentiation in BC3H1 cells. PT specifically ADP ribosylates a protein with an apparent molecular mass of 40 kD in BC3H1 cell membranes, whereas CT specifically ADP ribosylates three proteins of 35-43 kD. When added to exponentially growing cells in 20% FBS, CT and PT inhibited [3H]thymidine incorporation by up to 75% in a dose-dependent fashion. We found the synthesis of creatine kinase (CK) and skeletal muscle myosin light chain was reversibly induced in cells in 20% FBS treated with PT, but no increased synthesis was seen in cells treated with CT or in control cells; Northern analysis indicated this induction was at the level of mRNA. In cells shifted to 0.5% FBS, CT inhibited the normally induced synthesis of CK whereas PT potentiated it by approximately 50%. Forskolin also inhibited growth in 20% FBS and differentiation in 0.5% FBS medium in a dose-dependent fashion. both forskolin and CT elevated cAMP levels compared with control or PT- treated cells, suggesting that CT is blocking proliferation and differentiation by elevating cAMP levels. These results establish that a PT-sensitive pathway is involved in regulating proliferation and differentiation in BC3H1 cells, and we postulate that PT functions by ADP ribosylating a G protein that transduces signals from growth factor receptors in these cells.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Anand-Srivastava M. B., Srivastava A. K., Cantin M. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. J Biol Chem. 1987 Apr 15;262(11):4931–4934. [PubMed] [Google Scholar]
  2. Aub D. L., Frey E. A., Sekura R. D., Cote T. E. Coupling of the thyrotropin-releasing hormone receptor to phospholipase C by a GTP-binding protein distinct from the inhibitory or stimulatory GTP-binding protein. J Biol Chem. 1986 Jul 15;261(20):9333–9340. [PubMed] [Google Scholar]
  3. Brass L. F., Laposata M., Banga H. S., Rittenhouse S. E. Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi. J Biol Chem. 1986 Dec 25;261(36):16838–16847. [PubMed] [Google Scholar]
  4. Chambard J. C., Paris S., L'Allemain G., Pouysségur J. Two growth factor signalling pathways in fibroblasts distinguished by pertussis toxin. Nature. 1987 Apr 23;326(6115):800–803. doi: 10.1038/326800a0. [DOI] [PubMed] [Google Scholar]
  5. Dexter T. M., Whetton A. D., Heyworth C. M. Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation. Blood. 1985 Jun;65(6):1544–1548. [PubMed] [Google Scholar]
  6. Entwistle A., Curtis D. H., Zalin R. J. Myoblast fusion is regulated by a prostanoid of the one series independently of a rise in cyclic AMP. J Cell Biol. 1986 Sep;103(3):857–866. doi: 10.1083/jcb.103.3.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Gill D. M. Mechanism of action of cholera toxin. Adv Cyclic Nucleotide Res. 1977;8:85–118. [PubMed] [Google Scholar]
  9. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  10. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  11. Gossett L. A., Zhang W., Olson E. N. Dexamethasone-dependent inhibition of differentiation of C2 myoblasts bearing steroid-inducible N-ras oncogenes. J Cell Biol. 1988 Jun;106(6):2127–2137. doi: 10.1083/jcb.106.6.2127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hildebrandt J. D., Hanoune J., Birnbaumer L. Guanine nucleotide inhibition of cyc- S49 mouse lymphoma cell membrane adenylyl cyclase. J Biol Chem. 1982 Dec 25;257(24):14723–14725. [PubMed] [Google Scholar]
  13. Hildebrandt J. D., Sekura R. D., Codina J., Iyengar R., Manclark C. R., Birnbaumer L. Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins. Nature. 1983 Apr 21;302(5910):706–709. doi: 10.1038/302706a0. [DOI] [PubMed] [Google Scholar]
  14. Hurley J. B., Simon M. I., Teplow D. B., Robishaw J. D., Gilman A. G. Homologies between signal transducing G proteins and ras gene products. Science. 1984 Nov 16;226(4676):860–862. doi: 10.1126/science.6436980. [DOI] [PubMed] [Google Scholar]
  15. Imboden J. B., Shoback D. M., Pattison G., Stobo J. D. Cholera toxin inhibits the T-cell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic free calcium. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5673–5677. doi: 10.1073/pnas.83.15.5673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jandreski M., Liew C. C. Characterization of neonatal cardiac myosin heavy chain mRNA and synthesis of complementary DNA. Can J Biochem Cell Biol. 1984 Apr;62(4):185–190. doi: 10.1139/o84-026. [DOI] [PubMed] [Google Scholar]
  17. Kassis S., Olasmaa M., Terenius L., Fishman P. H. Neuropeptide Y inhibits cardiac adenylate cyclase through a pertussis toxin-sensitive G protein. J Biol Chem. 1987 Mar 15;262(8):3429–3431. [PubMed] [Google Scholar]
  18. Kelvin D. J., Chance S., Shreeve M., Axelrad A. A., Connolly J. A., McLeod D. Interleukin 3 and cell cycle progression. J Cell Physiol. 1986 Jun;127(3):403–409. doi: 10.1002/jcp.1041270308. [DOI] [PubMed] [Google Scholar]
  19. Kelvin D. J., Simard G., Sue-A-Quan A., Connolly J. A. Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. II. Two signaling pathways distinguished by pertussis toxin and a potential role for the ras oncogene. J Cell Biol. 1989 Jan;108(1):169–176. doi: 10.1083/jcb.108.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kikuchi A., Kozawa O., Kaibuchi K., Katada T., Ui M., Takai Y. Direct evidence for involvement of a guanine nucleotide-binding protein in chemotactic peptide-stimulated formation of inositol bisphosphate and trisphosphate in differentiated human leukemic (HL-60) cells. Reconstitution with Gi or Go of the plasma membranes ADP-ribosylated by pertussis toxin. J Biol Chem. 1986 Sep 5;261(25):11558–11562. [PubMed] [Google Scholar]
  21. 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]
  22. Lathrop B., Olson E., Glaser L. Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line. J Cell Biol. 1985 May;100(5):1540–1547. doi: 10.1083/jcb.100.5.1540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lathrop B., Thomas K., Glaser L. Control of myogenic differentiation by fibroblast growth factor is mediated by position in the G1 phase of the cell cycle. J Cell Biol. 1985 Dec;101(6):2194–2198. doi: 10.1083/jcb.101.6.2194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lefkowitz R. J., Caron M. G. Adrenergic receptors. Models for the study of receptors coupled to guanine nucleotide regulatory proteins. J Biol Chem. 1988 Apr 15;263(11):4993–4996. [PubMed] [Google Scholar]
  25. Letterio J. J., Coughlin S. R., Williams L. T. Pertussis toxin-sensitive pathway in the stimulation of c-myc expression and DNA synthesis by bombesin. Science. 1986 Nov 28;234(4780):1117–1119. doi: 10.1126/science.3465038. [DOI] [PubMed] [Google Scholar]
  26. Lochrie M. A., Hurley J. B., Simon M. I. Sequence of the alpha subunit of photoreceptor G protein: homologies between transducin, ras, and elongation factors. Science. 1985 Apr 5;228(4695):96–99. doi: 10.1126/science.3856323. [DOI] [PubMed] [Google Scholar]
  27. Magnaldo I., L'Allemain G., Chambard J. C., Moenner M., Barritault D., Pouysségur J. The mitogenic signaling pathway of fibroblast growth factor is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblasts. J Biol Chem. 1986 Dec 25;261(36):16916–16922. [PubMed] [Google Scholar]
  28. Merrifield P. A., Payne M. R., Konigsberg I. R. Isoform specificity of monoclonal hybridoma antibodies to quail skeletal muscle myosin subunits. Biochem Biophys Res Commun. 1983 Jun 15;113(2):407–417. doi: 10.1016/0006-291x(83)91741-2. [DOI] [PubMed] [Google Scholar]
  29. Morris S. A., Bilezikian J. P. Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts. J Cell Physiol. 1986 Apr;127(1):28–38. doi: 10.1002/jcp.1041270105. [DOI] [PubMed] [Google Scholar]
  30. Moss J., Stanley S. J., Burns D. L., Hsia J. A., Yost D. A., Myers G. A., Hewlett E. L. Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein). J Biol Chem. 1983 Oct 10;258(19):11879–11882. [PubMed] [Google Scholar]
  31. Munson R., Jr, Caldwell K. L., Glaser L. Multiple controls for the synthesis of muscle-specific proteins in BC3H1 cells. J Cell Biol. 1982 Feb;92(2):350–356. doi: 10.1083/jcb.92.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oinuma M., Katada T., Ui M. A new GTP-binding protein in differentiated human leukemic (HL-60) cells serving as the specific substrate of islet-activating protein, pertussis toxin. J Biol Chem. 1987 Jun 15;262(17):8347–8353. [PubMed] [Google Scholar]
  33. Olson E. N., Caldwell K. L., Gordon J. I., Glaser L. Regulation of creatine phosphokinase expression during differentiation of BC3H1 cells. J Biol Chem. 1983 Feb 25;258(4):2644–2652. [PubMed] [Google Scholar]
  34. Olson E. N., Glaser L., Merlie J. P., Lindstrom J. Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line. J Biol Chem. 1984 Mar 10;259(5):3330–3336. [PubMed] [Google Scholar]
  35. Olson E. N., Glaser L., Merlie J. P., Sebanne R., Lindstrom J. Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line. J Biol Chem. 1983 Nov 25;258(22):13946–13953. [PubMed] [Google Scholar]
  36. Olson E. N., Spizz G., Tainsky M. A. The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation. Mol Cell Biol. 1987 Jun;7(6):2104–2111. doi: 10.1128/mcb.7.6.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Paris S., Chambard J. C., Pouysségur J. Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin. J Biol Chem. 1987 Feb 15;262(5):1977–1983. [PubMed] [Google Scholar]
  38. Paris S., Pouysségur J. Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts. EMBO J. 1986 Jan;5(1):55–60. doi: 10.1002/j.1460-2075.1986.tb04177.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Payne P. A., Olson E. N., Hsiau P., Roberts R., Perryman M. B., Schneider M. D. An activated c-Ha-ras allele blocks the induction of muscle-specific genes whose expression is contingent on mitogen withdrawal. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8956–8960. doi: 10.1073/pnas.84.24.8956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rasmussen C. D., Simmen R. C., MacDougall E. A., Means A. R. Methods for analyzing bovine papilloma virus-based calmodulin expression vectors. Methods Enzymol. 1987;139:642–654. doi: 10.1016/0076-6879(87)39117-7. [DOI] [PubMed] [Google Scholar]
  41. Ribeiro-Neto F. A., Mattera R., Hildebrandt J. D., Codina J., Field J. B., Birnbaumer L., Sekura R. D. ADP-ribosylation of membrane components by pertussis and cholera toxin. Methods Enzymol. 1985;109:566–572. doi: 10.1016/0076-6879(85)09115-7. [DOI] [PubMed] [Google Scholar]
  42. Roman D., Billadello J., Gordon J., Grace A., Sobel B., Strauss A. Complete nucleotide sequence of dog heart creatine kinase mRNA: conservation of amino acid sequence within and among species. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8394–8398. doi: 10.1073/pnas.82.24.8394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Scherer N. M., Toro M. J., Entman M. L., Birnbaumer L. G-protein distribution in canine cardiac sarcoplasmic reticulum and sarcolemma: comparison to rabbit skeletal muscle membranes and to brain and erythrocyte G-proteins. Arch Biochem Biophys. 1987 Dec;259(2):431–440. doi: 10.1016/0003-9861(87)90509-1. [DOI] [PubMed] [Google Scholar]
  44. Schubert D., Harris A. J., Devine C. E., Heinemann S. Characterization of a unique muscle cell line. J Cell Biol. 1974 May;61(2):398–413. doi: 10.1083/jcb.61.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schützle U. B., Wakelam M. J., Pette D. Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells. Biochim Biophys Acta. 1984 Oct 12;805(2):204–210. doi: 10.1016/0167-4889(84)90169-1. [DOI] [PubMed] [Google Scholar]
  46. Simard G., Connolly J. A. Membrane glycoproteins are involved in the differentiation of the BC3H1 muscle cell line. Exp Cell Res. 1987 Nov;173(1):144–155. doi: 10.1016/0014-4827(87)90340-5. [DOI] [PubMed] [Google Scholar]
  47. Spizz G., Roman D., Strauss A., Olson E. N. Serum and fibroblast growth factor inhibit myogenic differentiation through a mechanism dependent on protein synthesis and independent of cell proliferation. J Biol Chem. 1986 Jul 15;261(20):9483–9488. [PubMed] [Google Scholar]
  48. Strauch A. R., Offord J. D., Chalkley R., Rubenstein P. A. Characterization of actin mRNA levels during BC3H1 cell differentiation. J Biol Chem. 1986 Jan 15;261(2):849–855. [PubMed] [Google Scholar]
  49. Strauch A. R., Rubenstein P. A. Induction of vascular smooth muscle alpha-isoactin expression in BC3H1 cells. J Biol Chem. 1984 Mar 10;259(5):3152–3159. [PubMed] [Google Scholar]
  50. Stygall K., Mirsky R. The effect of cholera toxin on myogenesis in rat skeletal muscle cultures. Dev Biol. 1980 Jul;78(1):14–24. doi: 10.1016/0012-1606(80)90314-0. [DOI] [PubMed] [Google Scholar]
  51. Tanabe T., Nukada T., Nishikawa Y., Sugimoto K., Suzuki H., Takahashi H., Noda M., Haga T., Ichiyama A., Kangawa K. Primary structure of the alpha-subunit of transducin and its relationship to ras proteins. Nature. 1985 May 16;315(6016):242–245. doi: 10.1038/315242a0. [DOI] [PubMed] [Google Scholar]
  52. Teitelbaum I., Berl T. Effects of calcium on vasopressin-mediated cyclic adenosine monophosphate formation in cultured rat inner medullary collecting tubule cells. Evidence for the role of intracellular calcium. J Clin Invest. 1986 May;77(5):1574–1583. doi: 10.1172/JCI112473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wahrmann J. P., Winand R., Luzzati D. Effect of cyclic AMP on growth and morphological differentiation of an established myogenic cell line. Nat New Biol. 1973 Sep 26;245(143):112–113. doi: 10.1038/newbio245112a0. [DOI] [PubMed] [Google Scholar]
  55. Wang Y. C., Rubenstein P. A. Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells. J Cell Biol. 1988 Mar;106(3):797–803. doi: 10.1083/jcb.106.3.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Watkins D. C., Northup J. K., Malbon C. C. Regulation of G-proteins in differentiation. Altered ratio of alpha- to beta-subunits in 3T3-L1 cells. J Biol Chem. 1987 Aug 5;262(22):10651–10657. [PubMed] [Google Scholar]
  57. Wice B., Milbrandt J., Glaser L. Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate. J Biol Chem. 1987 Feb 5;262(4):1810–1817. [PubMed] [Google Scholar]
  58. Wojcikiewicz R. J., Kent P. A., Fain J. N. Evidence that thyrotropin-releasing hormone-induced increases in GTPase activity and phosphoinositide metabolism in GH3 cells are mediated by a guanine nucleotide-binding protein other than Gs or Gi. Biochem Biophys Res Commun. 1986 Aug 14;138(3):1383–1389. doi: 10.1016/s0006-291x(86)80436-3. [DOI] [PubMed] [Google Scholar]
  59. Zalin R. J., Montague W. Changes in adenylate cyclase, cyclic AMP, and protein kinase levels in chick myoblasts, and their relationship to differentiation. Cell. 1974 Jun;2(2):103–108. doi: 10.1016/0092-8674(74)90098-1. [DOI] [PubMed] [Google Scholar]
  60. Zalin R. J. The relationship of the level of cyclic amp to differentiation in primary cultures of chick muscle cells. Exp Cell Res. 1973 Mar 30;78(1):152–158. doi: 10.1016/0014-4827(73)90049-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES