Skip to main content
SpringerLink
Log in

5-HT Induction of c-fos gene expression requires reactive oxygen species and rac1 and ras GTPases

  • Original Article
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

Serotonin (5-HT) stimulates superoxide release, phosphorylation, of p42/p44 mitogen-activated protein kinase (MAPK), and DNA synthesis in bovine pulmonary artery smooth muscle cells. Both p42/p44 MAPK and reactive oxygen species (ROS) generation are required for 5-HT-induced growth in SMC. Agents that block the production of ROS, or ROS scavengers, block MAPK activation by 5-HT. However, specific signal transduction by 5-HT leading to proteins that control entrance into the cell cycle are not well defined in smooth muscle cells. Here, we show by Western blot that 5-HT upregulates c-Fos, an immediate early gene product known to regulate the entrance of quiescent cells into the cell cycle. Northern blots showed that c-fos mRNA is induced by 5-HT in 30 min. This induction is blocked by PD98059, indicating that activation of MAPK is required. 5-HT-induced expression of a 350 bp c-fos promoter in a luciferase reporter is blocked by PD98059 and diphenyliodonium (DPI). The GTPases Rac1 and Ras have been implicated in growth factor-induced generation of ROS. Overexpression of either dominant negative (DN) Rac1 or DN Ras inhibited 5-HT-mediated c-fos promoter activation. 5-HT also induced expression from a truncated c-fos promoter containing an isolated serum response element. This activation was blocked by DPI and PD98059. Overexpression of activated Ras and Rac1 were additive for activation of the serum response element promoter. Regulation of cyclin D1, a protein shown to be regulated by c-fos and required for entry into the cell cycle, is upregulated by 5-HT and is blocked by DPI and PD98059. Nuclear factor-κB, which can also regulate cyclin D1, was not activated. We conclude that 5-HT stimulates c-fos and cyclin D1 expression through a ROS-dependent mechanism that requires Ras, Rac1, and MAPK.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Fanburg, B. L. and Lee, S. L. (1997) A new role for an old molecule: serotonin as a mitogen. Am. J. Physiol. 272, L795-L806.

    PubMed  CAS  Google Scholar 

  2. Wieissman, J. T., Ma, J. N., Essex, A., Gao, Y., and Burnstein, E. S. (2004) G.-protein-coupled receptor-mediated activation of rap GTPases: characterization of a novel Galphai regulated pathway. Oncogene 23, 241–249.

    Article  CAS  Google Scholar 

  3. Banasr M., Hery, M., Printemp, R., and Daszuta, A. (2004) Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone. Neuropsychopharmacology., 29, 450–460.

    Article  PubMed  CAS  Google Scholar 

  4. Abdouh, M., Albert, P. R., Drobetsky, E., Filep, J. G., and Kouassi, E. (2004) 5-HT1A-mediated promotion of mitogen-activated T and B cell survival and proliferation is associated with increased translocation of NF-kappaB to the nucleus. Brain Behav. Immunol. 18, 24–34.

    Article  CAS  Google Scholar 

  5. Nebigil, C. G., Jaffre, F., Messaddeq, N., Hickel, P., Monassier, L., Launay, J. M., and Maroteaux, L. (2003) Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy. Circulation 107, 3223–3229.

    Article  PubMed  CAS  Google Scholar 

  6. Lee, S. L., Wang, W. W., Lanzillo, J. J., and Fanburg, B. L. (1994) Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture. Am. J. Physiol. 266, L46-L52.

    PubMed  CAS  Google Scholar 

  7. Lee, S. L., Wang, W. W., Lanzillo, J. J., and Fanburg, B. L. (1994) Regulation of serotonin-induced DNA synthesis of bovine pulmonary artery smooth muscle cells. Am. J. Physiol. 266, L53-L60.

    PubMed  CAS  Google Scholar 

  8. Eddahibi, S., Hanoun, N., Lanfumey, L., Lesch, K. P., Raffestin, B., Hamon, M., and Adnot, S. (2000) Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J. Clin. Invest. 105, 1555–1562.

    Article  PubMed  CAS  Google Scholar 

  9. Eddahibi, S., Humbert, M., Fadel, E., Raffestin, B., Darmon, M., Capron, F., Simonneau, G., Dartevelle, P., Hamon, M., and Adnot, S. (2001) Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J. Clin. Invest. 108, 1141–1150.

    Article  PubMed  CAS  Google Scholar 

  10. Launay, J. M., Herve, P., Peoc'h, K., Tournois, C., Callebert, J., Nebigil, C. G., Etienne, N., Drouet, L., Humbert, M., Simonneau, G., and Maroteaux, L. (2002) Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension., Nat. Med., 8, 1129–1135.

    Article  PubMed  CAS  Google Scholar 

  11. Watanabe, T., Pakala, R., Katagiri, T., and Benedict, C. R. (2001) Serotonin potentiates angiotensin II-induced vascular smooth muscle cell proliferation. Atherosclerosis 159, 269–279.

    Article  PubMed  CAS  Google Scholar 

  12. Lee, S. L., Wang, W. W., and Fanburg, B. L. (1998) Superoxide as an intermediate signal for serotonin-induced mitogenesis. Free Radic. Biol. Med. 24, 855–858.

    Article  PubMed  Google Scholar 

  13. Lee, S. L., Wang, W. W., Finlay, G. A., and Fanburg, B. L. (1999) Serotonin stimulates mitogen-activated protein kinase activity through the formation of superoxide anion. Am. J. Physiol. 277, L282-L291.

    PubMed  CAS  Google Scholar 

  14. Lee, S. L., Simon, A. R., Wang., W. W., and Fanburg, B. L. (2001) H(2)O(2) signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L646-L652.

    PubMed  CAS  Google Scholar 

  15. Piechaczyk, M., and Blanchard, J. M. (1994) c-Fos proto-oncogene regulation and function., Crit. Rev. Oncol. Hematol. 17, 93–131.

    Article  PubMed  CAS  Google Scholar 

  16. Angel, P. and Kann, M. (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta 1072, 129–157.

    PubMed  CAS  Google Scholar 

  17. Wang, Y. Y., Wu, S. X., Liu, X. Y., Wang, W. W., and Li, Y. Q. (2003) Effects of c-fos antisense oligodeoxynucleotide on 5-HT-induced upregulation of preprodynorphin, preproenkephalin, and glutamic acid decarboxylase mRNA expression in cultured rat spinal dorsal horn neurons. Biochem. Biophys. Res. Commun. 309, 631–636.

    Article  PubMed  CAS  Google Scholar 

  18. Doi-Saika, M., Tokunaga, A., and Senba, E. (1997) Intradermal 5-HT induces Fos expression in rat dorsal horn neurons not via 5-HT3 but via 5-HT2A receptors. Neurosci. Res. 29, 143–149.

    Article  PubMed  CAS  Google Scholar 

  19. Corson, M., Alexander, R., and Berk, B., (1992) 5-HT2 receptor mRNA is over-expressed in cultured rat aortic smooth muscle cells relative to normal aorta. Am. J. Physiol. 262, C309-C315.

    PubMed  CAS  Google Scholar 

  20. Moskowitz, M. (1993) Neurogenic inflammation in the pathophysiology and treatment of migraine. Neurology 43, S16-S20.

    PubMed  CAS  Google Scholar 

  21. Sharma, S., Del Rizzo, D., Zahradka, P., Bhangu, S., Werner, J., Kumamoto H., Takeda, N., and Dhalla, N. (2001) Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: implications for long-term graft patency. Ann. Thorac. Surg. 71, 1856–1864.

    Article  PubMed  CAS  Google Scholar 

  22. Nebigil, C. G., Launay, J. M., Hickel, P., Tournois, C., and Maroteaux, L. (2000) 5-hydroxytryptamine 2B receptor regulates cell-cycle progression: cross-talk with tyrosine kinase pathways. Proc. Natl. Acad. Sci. U. S. A. 97, 2591–2596.

    Article  PubMed  CAS  Google Scholar 

  23. Jacks, T., and Weinberg, R. A. (1998) The expanding role of cell cycle regulators. Science 280, 1035–1036.

    Article  PubMed  CAS  Google Scholar 

  24. Han, E., Ng, S., Arber, N., Begemann, M., and Weinstein, I. (1999) Roles of cyclin D1 and related genes in growth inhibition, senescence and apoptosis. Apoptosis 4, 213–219.

    Article  PubMed  CAS  Google Scholar 

  25. Cuevas, P., Diaz-Gonzalez, D., and Dujovny, M. (2003) Antiproliferative action of neomycin is associated with inhibition of cyclin D1 activation in glioma cells. Neurol. Res. 25, 691–693.

    Article  PubMed  CAS  Google Scholar 

  26. Takahashi, K., Ohmichi, M., Yoshida, M., Hisamoto, K., Mabuchi, S., Arimoto-Ishida, E., Mori, A., Tsutumi, S., Tasaka, K., Murata, Y., and Kurachi, H. (2003) Both estrogen and raloxifene cause G1 arrest of vascular smooth muscle cells. J. Endocrinol. 178, 319–329.

    Article  PubMed  CAS  Google Scholar 

  27. Page, K., Li, J., Hodge, J. A., Liu, P. T., Vanden Hoek, T. L., Becker, L. B., Pestell, R. G., Rosner, M. R., and Hershhenson, M. B. (1999) Characterization of a Rac1 signaling pathway to cyclin D1 in airway smooth muscle cells. J. Biol. Chem. 274, 22065–22071.

    Article  PubMed  CAS  Google Scholar 

  28. Page, K., Li, J., and Hershenson, M. B. (1999) Platelet-derived growth factor stimulation of mitogen-activated protein kinases and cyclin D1 promoter activity in cultured airway smoothmuscle cells. Role of Ras. Am. J. Respir. Cell. Mol. Biol. 20, 1294–1302.

    PubMed  CAS  Google Scholar 

  29. Albanese, C., D'Amico, M., Reutens, A. T., Fu, M., Watanabe, G., Lee, R. J., Kitsis, R. N., Henglein, B., Avantaggiati, M., Somasundaram, K., Thimmapaya, B., and Pestell, R. G. (1999) Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis. J. Biol. Chem. 274, 34186–34195.

    Article  PubMed  CAS  Google Scholar 

  30. Albanese, C., Johnson, J., Watanabe, G., Eklund, N., Vu, D., Arnold, A., and Pestell, R. G. (1995) Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J. Biol. Chem. 270, 23489–23597.

    Article  Google Scholar 

  31. Marshall, M. S. (1995) Ras target proteins in eukaryotic cells. FASEB J. 9, 1311–1318.

    PubMed  CAS  Google Scholar 

  32. Thannickal, V. J., Day, R. M., Klinz, S. G., Bastien, M. C., Larios, J. M., and Fanburg, B. L. (2000) Ras-dependent and-independent regulation of reactive oxygen species by mitogenic growth factors and TGF-betal. FASEB J. 14, 1741–1748.

    Article  PubMed  CAS  Google Scholar 

  33. Suzukawa, K., Miura, K., Mitsushita, J., Resau, J., Hirose, K., Crystal, R., and Kamata, T. (2000) Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species. J. Biol. Chem. 275, 13175–13178.

    Article  PubMed  CAS  Google Scholar 

  34. Joneson, T. and Bar-Sagi, D. (1998) A Rac1 effector site controlling mitogenesis through superoxide production. J. Biol. Chem. 273, 17991–17994.

    Article  PubMed  CAS  Google Scholar 

  35. Ross, R. (1971) The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J. Cell. Biol. 50, 172–186.

    Article  PubMed  CAS  Google Scholar 

  36. Kim, D. W., Cheriyah, B., Roy, A. L., and Cochran, B. H. (1998) TFII-I enhances activation of the c-fos promoter through interactions with upstream elements. Mol. Cell. Biol. 18, 3310–3320.

    PubMed  CAS  Google Scholar 

  37. Harvat, B. L. and Wharton, W. (1995) Serum response element and flanking sequences mediate the synergistic transcriptional activation of c-fos by 12-O-tetradecanoylphorbol-13-acetate and cholera toxin in AKR-2B cells. Cell Growth Differ. 6, 955–964.

    PubMed  CAS  Google Scholar 

  38. Subramaniam, M., Schmidt, L. J., Crutchfield, E. D., and Getz, M. J. (1989) Negative regulation of serum-responsive enhancer elements. Nature 340, 64–66.

    Article  PubMed  CAS  Google Scholar 

  39. Kim, D. W., and Cochran, B. H. (2000) Extracellular signal-regulated kinase binds to TFII-I and regulates its activation of the c-fos promoter. Mol. Biol. Cell 20, 1140–1148.

    Article  CAS  Google Scholar 

  40. Goi, T., Rusanescu, G., Urano, T., and Feig, L. (1999) Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth. Mol. Biol. Cell 19, 1731–1741.

    CAS  Google Scholar 

  41. Simon, A. R., Takahashi, S., Severgnini, M., Cochran, B. H., and Fanburg, B. L. (2002) The role of the Jak-STAT pathway in PDGR-stimulated proliferation of human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 282, L1296-L1304.

    PubMed  CAS  Google Scholar 

  42. Kim, J. H., Cho, Y. S., Kim, B. C., Kim, Y. S., and Lee, G. S. (1997) Role of Rho GTPase in the endothelin-1-induced nuclear signaling. Biochem. Biophys. Res. Commun. 232, 223–226.

    Article  PubMed  CAS  Google Scholar 

  43. Hipskind, R. A., Rao, V. N., Mueller, C. G., Reddy, E. S., and Nordheim, A. (1991) Etsrelated protein Elk-1 is homologous to the c-fos regulatory factor p62TCF. Nature 354, 531–534.

    Article  PubMed  CAS  Google Scholar 

  44. Wang, C., Fu, M. D'Amico, M., Albanese, C., Zhou, J. N., Brownlee, M., Lisanti, M. P., Chatterjee, V. K., Lazar, M. A., and Pestell, R. G., (2001) Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1. Mol. Biol. Cell 21, 3057–3070.

    Article  CAS  Google Scholar 

  45. Suzuki, E., Nagata, D., Yoshizumi., M., Kakoki, M., Goto, A., Omata, M., and Hirata, Y. (2000) Reentry into the cell cycle of contact-inhibited vascular endothelial cells by a phosphatase inhibitor. Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J. Biol. Chem. 275, 3637–3644.

    Article  PubMed  CAS  Google Scholar 

  46. Balmanno, K. and Cook, S. J. (1999) Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip 1 and a subset of AP-1 proteins in CCL39 cells. Oncogene 18, 3085–3097.

    Article  PubMed  CAS  Google Scholar 

  47. Fleischmann, A., Hafezi, F., Elliott, C., Reme, C., Ruther, U., and Wagner, E. (2000) Fra-1 replaces c-Fos-dependent functions in mice. Genes Dev. 14, 2695–2700.

    Article  PubMed  CAS  Google Scholar 

  48. Phuchareon, J., and Tokuhisa, T. (1995) Deregulated c-Fos/AP-1 modulates expression of the cyclin and the cdk gene in splenic B cells stimulated with lipopolysaccharide. Cancer Lett. 92, 203–208.

    Article  PubMed  CAS  Google Scholar 

  49. Brown, J. R., Nigh, E., Lee, R. J., Ye, H., Thompson, M. A., Saudou, F., Pestell, R. G., and Greenberg, M. E. (1998) Fos family members induce cell cycle entry by activating cyclin D1. Mol. Biol. Cell 18, 5609–5619.

    CAS  Google Scholar 

  50. Matsushime, H., Roussel, M., Ashumun, R., and Sherr, C. (2001) Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell 65, 701–713.

    Article  Google Scholar 

  51. Balasenthil, S., Sahin., A. A., Barnes, C. J., Wang, R. A., Pestell, R. G., Vadlamudi, R. K., and Kumar, R. (2004) p21-activated kinase-1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells. J. Biol. Chem. 279, 1422–1428.

    Article  PubMed  CAS  Google Scholar 

  52. Albrecht, H., Tschopp, J., and Jongeneel, C. V. (1994) Bcl-2 protects from oxidative damage and apoptotic cell death without interfering with activation of NF-kappa B by TNF., FEBS Lett. 351, 45–48.

    Article  PubMed  CAS  Google Scholar 

  53. Sulciner, D. J., Irani, K., Yu, Z. X., Ferrans, V. J., Goldschmidt-Clermont, P., and Finkel, T. (1996) Rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation. Mol. Cell. Biol. 16, 7115–7121.

    PubMed  CAS  Google Scholar 

  54. Sundaresan, M., Yu, Z. X., Ferrans, V. J., Sulciner, D. J., Gutkind, J. S., Irani, K., Goldschmidt-Clermont, P. J., and Finkel, T. (1996) Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem. J. 318, 379–382.

    PubMed  CAS  Google Scholar 

  55. Thannickal, V. J., Hassoun, P. M., White, A. C., and Fanburg, B. L. (1993) Enhanced rate of H2O2 release from bovine pulmonary artery endothelial cells induced by TGF-beta 1. Am. J. Physiol. 265, L622-L626.

    PubMed  CAS  Google Scholar 

  56. Hill, C. S., and Treisman, R. (1995) Differential activation of c-fos promoter elements by serum, lysophosphatidic acid, G proteins and polypeptide growth factors. EMBO J. 14, 5037–5047.

    PubMed  CAS  Google Scholar 

  57. Yoo, M. H., Woo, C. H., You, H. J., Cho, S. H., Kim, B. C., Choi, J. E., Chun, J. S., Jhun, B. H., Kim, T. S., and Kim, J. H. (2001) Role of the cytosolic phospholipase A2-linked cascade in signaling by an oncogenic, constitutively active Ha-Ras isoform. J. Biol. Chem. 276, 24645–24653.

    Article  PubMed  CAS  Google Scholar 

  58. Fromm, C., Coso, O. A., Montaner, S., Xu, N., and Gutkind, J. S. (1997) The small GTP-binding protein Rho links G protein-coupled receptors and Galpha12 to the serum response element and to cellular transformation. Proc. Natl. Acad. Sci. U. S. A. 94, 10098–10103.

    Article  PubMed  CAS  Google Scholar 

  59. Launay, J. M., Birraux, G., Bondoux, D., Callebert, J., Choi, D. S., Loric, S., and Maroteaux, L. (1996) Ras involvement in signal transduction by the serotonin 5-HT2B receptor. J. Biol. Chem. 271, 3141–3147.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Tufts-New England Medical Center, Tupper Research Institute, Pulmonary, Critical Care and Sleep Division, 750 Washington Street, NEMC #257, 02111, Boston, MA

    Amy R. Simon, Mariano Severgnini, Liliana Rozo, Bathsheeba Andrahbi & Barry L. Fanburg

  2. Department of Physiology, Tufts University School of Medicine, 136 Harrison Ave., 02111, Boston, MA

    Amy R. Simon, Mariano Severgnini, Satoe Takahashi, Liliana Rozo & Brent H. Cochran

  3. Department of Pharmacology, Uniformed Service University of the Health Sciences, 4301 Jones Bridge Rd., 20814, Bethesda, MD

    Abena Agyeman & Regina M. Day

Authors
  1. Amy R. Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mariano Severgnini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Satoe Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liliana Rozo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bathsheeba Andrahbi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Abena Agyeman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Brent H. Cochran
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Regina M. Day
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Barry L. Fanburg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Amy R. Simon.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Simon, A.R., Severgnini, M., Takahashi, S. et al. 5-HT Induction of c-fos gene expression requires reactive oxygen species and rac1 and ras GTPases. Cell Biochem Biophys 42, 263–276 (2005). https://doi.org/10.1385/CBB:42:3:263

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/CBB:42:3:263

Index Entries

Search

Navigation