Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Aug 1;373(Pt 3):815–824. doi: 10.1042/BJ20030208

Extracellular ATP stimulates the early growth response protein 1 (Egr-1) via a protein kinase C-dependent pathway in the human osteoblastic HOBIT cell line.

Alex Pines 1, Milena Romanello 1, Laura Cesaratto 1, Giuseppe Damante 1, Luigi Moro 1, Paola D'andrea 1, Gianluca Tell 1
PMCID: PMC1223538  PMID: 12729460

Abstract

Extracellular nucleotides exert an important role in controlling cell physiology by activating intracellular signalling cascades. Osteoblast HOBIT cells express P2Y(1) and P2Y(2) G-protein-coupled receptors, and respond to extracellular ATP by increasing cytosolic calcium concentrations. Early growth response protein 1 (Egr-1) is a C(2)H(2)-zinc-finger-containing transcriptional regulator responsible for the activation of several genes involved in the control of cell proliferation and apoptosis, and is thought to have a central role in osteoblast biology. We show that ATP treatment of HOBIT cells increases Egr-1 protein levels and binding activity via a mechanism involving a Ca(2+)-independent protein kinase C isoform. Moreover, hypotonic stress and increased medium turbulence, by inducing ATP release, result in a similar effect on Egr-1. Increased levels of Egr-1 protein expression and activity are achieved at very early times after stimulation (5 min), possibly accounting for a rapid way for changing the osteoblast gene-expression profile. A target gene for Egr-1 that is fundamental in osteoblast physiology, COL1A2, is up-regulated by ATP stimulation of HOBIT cells in a timescale that is compatible with that of Egr-1 activation.

Full Text

The Full Text of this article is available as a PDF (302.8 KB).

Selected References

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

  1. Alexander Dorothea, Judex Martin, Meyringer Rotraud, Weis-Klemm Michaela, Gay Steffen, Müller-Ladner Ulf, Aicher Wilhelm K. Transcription factor Egr-1 activates collagen expression in immortalized fibroblasts or fibrosarcoma cells. Biol Chem. 2002 Dec;383(12):1845–1853. doi: 10.1515/BC.2002.208. [DOI] [PubMed] [Google Scholar]
  2. Beigi R., Kobatake E., Aizawa M., Dubyak G. R. Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am J Physiol. 1999 Jan;276(1 Pt 1):C267–C278. doi: 10.1152/ajpcell.1999.276.1.C267. [DOI] [PubMed] [Google Scholar]
  3. Boarder M. R., Weisman G. A., Turner J. T., Wilkinson G. F. G protein-coupled P2 purinoceptors: from molecular biology to functional responses. Trends Pharmacol Sci. 1995 Apr;16(4):133–139. doi: 10.1016/s0165-6147(00)89001-x. [DOI] [PubMed] [Google Scholar]
  4. Bowler W. B., Buckley K. A., Gartland A., Hipskind R. A., Bilbe G., Gallagher J. A. Extracellular nucleotide signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling. Bone. 2001 May;28(5):507–512. doi: 10.1016/s8756-3282(01)00430-6. [DOI] [PubMed] [Google Scholar]
  5. Bowler W. B., Dixon C. J., Halleux C., Maier R., Bilbe G., Fraser W. D., Gallagher J. A., Hipskind R. A. Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase. J Biol Chem. 1999 May 14;274(20):14315–14324. doi: 10.1074/jbc.274.20.14315. [DOI] [PubMed] [Google Scholar]
  6. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Buckley K. A., Wagstaff S. C., McKay G., Gaw A., Hipskind R. A., Bilbe G., Gallagher J. A., Bowler W. B. Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone. J Biol Chem. 2000 Dec 21;276(12):9565–9571. doi: 10.1074/jbc.M005672200. [DOI] [PubMed] [Google Scholar]
  8. Dekker L. V., Palmer R. H., Parker P. J. The protein kinase C and protein kinase C related gene families. Curr Opin Struct Biol. 1995 Jun;5(3):396–402. doi: 10.1016/0959-440x(95)80103-0. [DOI] [PubMed] [Google Scholar]
  9. Fang M. A., Glackin C. A., Sadhu A., McDougall S. Transcriptional regulation of alpha 2(I) collagen gene expression by fibroblast growth factor-2 in MC3T3-E1 osteoblast-like cells. J Cell Biochem. 2001;80(4):550–559. [PubMed] [Google Scholar]
  10. Gashler A., Sukhatme V. P. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol. 1995;50:191–224. doi: 10.1016/s0079-6603(08)60815-6. [DOI] [PubMed] [Google Scholar]
  11. Gerasimovskaya Evgenia V., Ahmad Shama, White Carl W., Jones Peter L., Carpenter Todd C., Stenmark Kurt R. Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor. J Biol Chem. 2002 Sep 18;277(47):44638–44650. doi: 10.1074/jbc.M203012200. [DOI] [PubMed] [Google Scholar]
  12. Gordon J. L. Extracellular ATP: effects, sources and fate. Biochem J. 1986 Jan 15;233(2):309–319. doi: 10.1042/bj2330309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grygorczyk R., Hanrahan J. W. CFTR-independent ATP release from epithelial cells triggered by mechanical stimuli. Am J Physiol. 1997 Mar;272(3 Pt 1):C1058–C1066. doi: 10.1152/ajpcell.1997.272.3.C1058. [DOI] [PubMed] [Google Scholar]
  14. Gschwendt M., Müller H. J., Kielbassa K., Zang R., Kittstein W., Rincke G., Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun. 1994 Feb 28;199(1):93–98. doi: 10.1006/bbrc.1994.1199. [DOI] [PubMed] [Google Scholar]
  15. Guenette D. K., Ritzenthaler J. D., Foley J., Jackson J. D., Smith B. D. DNA methylation inhibits transcription of procollagen alpha 2(I) promoters. Biochem J. 1992 May 1;283(Pt 3):699–703. doi: 10.1042/bj2830699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harden T. K., Boyer J. L., Nicholas R. A. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol. 1995;35:541–579. doi: 10.1146/annurev.pa.35.040195.002545. [DOI] [PubMed] [Google Scholar]
  17. Huang N., Wang D. J., Heppel L. A. Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7904–7908. doi: 10.1073/pnas.86.20.7904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jimenez S. A., Gaidarova S., Saitta B., Sandorfi N., Herrich D. J., Rosenbloom J. C., Kucich U., Abrams W. R., Rosenbloom J. Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts. J Clin Invest. 2001 Nov;108(9):1395–1403. doi: 10.1172/JCI12347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones S. J., Gray C., Boyde A., Burnstock G. Purinergic transmitters inhibit bone formation by cultured osteoblasts. Bone. 1997 Nov;21(5):393–399. doi: 10.1016/s8756-3282(97)00174-9. [DOI] [PubMed] [Google Scholar]
  20. Jorgensen N. R., Geist S. T., Civitelli R., Steinberg T. H. ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells. J Cell Biol. 1997 Oct 20;139(2):497–506. doi: 10.1083/jcb.139.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Keeting P. E., Scott R. E., Colvard D. S., Anderson M. A., Oursler M. J., Spelsberg T. C., Riggs B. L. Development and characterization of a rapidly proliferating, well-differentiated cell line derived from normal adult human osteoblast-like cells transfected with SV40 large T antigen. J Bone Miner Res. 1992 Feb;7(2):127–136. doi: 10.1002/jbmr.5650070203. [DOI] [PubMed] [Google Scholar]
  22. Koyama T., Oike M., Ito Y. Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells. J Physiol. 2001 May 1;532(Pt 3):759–769. doi: 10.1111/j.1469-7793.2001.0759e.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lazarowski E. R., Homolya L., Boucher R. C., Harden T. K. Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. J Biol Chem. 1997 Sep 26;272(39):24348–24354. doi: 10.1074/jbc.272.39.24348. [DOI] [PubMed] [Google Scholar]
  24. Levin L. G., Rudd A., Bletsa A., Reisner H. Expression of IL-8 by cells of the odontoblast layer in vitro. Eur J Oral Sci. 1999 Apr;107(2):131–137. doi: 10.1046/j.0909-8836.1999.eos107209.x. [DOI] [PubMed] [Google Scholar]
  25. Li-Weber M., Laur O., Krammer P. H. Novel Egr/NF-AT composite sites mediate activation of the CD95 (APO-1/Fas) ligand promoter in response to T cell stimulation. Eur J Immunol. 1999 Sep;29(9):3017–3027. doi: 10.1002/(SICI)1521-4141(199909)29:09<3017::AID-IMMU3017>3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  26. Lu Z., Liu D., Hornia A., Devonish W., Pagano M., Foster D. A. Activation of protein kinase C triggers its ubiquitination and degradation. Mol Cell Biol. 1998 Feb;18(2):839–845. doi: 10.1128/mcb.18.2.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Majeska R. J., Rodan S. B., Rodan G. A. Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. Endocrinology. 1980 Nov;107(5):1494–1503. doi: 10.1210/endo-107-5-1494. [DOI] [PubMed] [Google Scholar]
  28. McMahon A. P., Champion J. E., McMahon J. A., Sukhatme V. P. Developmental expression of the putative transcription factor Egr-1 suggests that Egr-1 and c-fos are coregulated in some tissues. Development. 1990 Feb;108(2):281–287. doi: 10.1242/dev.108.2.281. [DOI] [PubMed] [Google Scholar]
  29. Molnar G., Crozat A., Pardee A. B. The immediate-early gene Egr-1 regulates the activity of the thymidine kinase promoter at the G0-to-G1 transition of the cell cycle. Mol Cell Biol. 1994 Aug;14(8):5242–5248. doi: 10.1128/mcb.14.8.5242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ogata T. Fluid flow induces enhancement of the Egr-1 mRNA level in osteoblast-like cells: involvement of tyrosine kinase and serum. J Cell Physiol. 1997 Jan;170(1):27–34. doi: 10.1002/(SICI)1097-4652(199701)170:1<27::AID-JCP4>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
  31. Ohba M., Shibanuma M., Kuroki T., Nose K. Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol. 1994 Aug;126(4):1079–1088. doi: 10.1083/jcb.126.4.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oike M., Kimura C., Koyama T., Yoshikawa M., Ito Y. Hypotonic stress-induced dual Ca(2+) responses in bovine aortic endothelial cells. Am J Physiol Heart Circ Physiol. 2000 Aug;279(2):H630–H638. doi: 10.1152/ajpheart.2000.279.2.H630. [DOI] [PubMed] [Google Scholar]
  33. Osipchuk Y., Cahalan M. Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells. Nature. 1992 Sep 17;359(6392):241–244. doi: 10.1038/359241a0. [DOI] [PubMed] [Google Scholar]
  34. Ostrom R. S., Gregorian C., Insel P. A. Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J Biol Chem. 2000 Apr 21;275(16):11735–11739. doi: 10.1074/jbc.275.16.11735. [DOI] [PubMed] [Google Scholar]
  35. Romanello M., D'Andrea P. Dual mechanism of intercellular communication in HOBIT osteoblastic cells: a role for gap-junctional hemichannels. J Bone Miner Res. 2001 Aug;16(8):1465–1476. doi: 10.1359/jbmr.2001.16.8.1465. [DOI] [PubMed] [Google Scholar]
  36. Romanello M., Pani B., Bicego M., D'Andrea P. Mechanically induced ATP release from human osteoblastic cells. Biochem Biophys Res Commun. 2001 Dec 21;289(5):1275–1281. doi: 10.1006/bbrc.2001.6124. [DOI] [PubMed] [Google Scholar]
  37. Román J., Colell A., Blasco C., Caballeria J., Parés A., Rodés J., Fernández-Checa J. C. Differential role of ethanol and acetaldehyde in the induction of oxidative stress in HEP G2 cells: effect on transcription factors AP-1 and NF-kappaB. Hepatology. 1999 Dec;30(6):1473–1480. doi: 10.1002/hep.510300623. [DOI] [PubMed] [Google Scholar]
  38. Sabirov R. Z., Dutta A. K., Okada Y. Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release. J Gen Physiol. 2001 Sep;118(3):251–266. doi: 10.1085/jgp.118.3.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shaulian E., Karin M. AP-1 in cell proliferation and survival. Oncogene. 2001 Apr 30;20(19):2390–2400. doi: 10.1038/sj.onc.1204383. [DOI] [PubMed] [Google Scholar]
  40. Shimegi S. ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1). Calcif Tissue Int. 1996 Feb;58(2):109–113. doi: 10.1007/BF02529732. [DOI] [PubMed] [Google Scholar]
  41. Stula M., Orzechowski H. D., Gschwend S., Vetter R., von Harsdorf R., Dietz R., Paul M. Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells. Mol Cell Biochem. 2000 Jul;210(1-2):101–108. doi: 10.1023/a:1007126218740. [DOI] [PubMed] [Google Scholar]
  42. Taylor A. L., Kudlow B. A., Marrs K. L., Gruenert D. C., Guggino W. B., Schwiebert E. M. Bioluminescence detection of ATP release mechanisms in epithelia. Am J Physiol. 1998 Nov;275(5 Pt 1):C1391–C1406. doi: 10.1152/ajpcell.1998.275.5.C1391. [DOI] [PubMed] [Google Scholar]
  43. Tell G., Scaloni A., Pellizzari L., Formisano S., Pucillo C., Damante G. Redox potential controls the structure and DNA binding activity of the paired domain. J Biol Chem. 1998 Sep 25;273(39):25062–25072. doi: 10.1074/jbc.273.39.25062. [DOI] [PubMed] [Google Scholar]
  44. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  45. Törnquist K., Ekokoski E., Dugué B. Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells. J Cell Physiol. 1996 Feb;166(2):241–248. doi: 10.1002/(SICI)1097-4652(199602)166:2<241::AID-JCP1>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  46. Vincenti M. P., Brinckerhoff C. E. Early response genes induced in chondrocytes stimulated with the inflammatory cytokine interleukin-1beta. Arthritis Res. 2001 Sep 18;3(6):381–388. doi: 10.1186/ar331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Webb B. L., Lindsay M. A., Seybold J., Brand N. J., Yacoub M. H., Haddad E. B., Barnes P. J., Adcock I. M., Giembycz M. A. Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level. Biochem Pharmacol. 1997 Jul 1;54(1):199–205. doi: 10.1016/s0006-2952(97)00165-2. [DOI] [PubMed] [Google Scholar]
  48. You Jun, Jacobs Christopher R., Steinberg Thomas H., Donahue Henry J. P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells. J Biol Chem. 2002 Oct 9;277(50):48724–48729. doi: 10.1074/jbc.M209245200. [DOI] [PubMed] [Google Scholar]

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

RESOURCES