ATP and Purinergic Receptor Agonists Stimulate the Mitogen-Activated Protein Kinase Pathway and DNA Synthesis in Mouse Mammary Epithelial Cells

  • Yuh In-Sub (College of Animal Resources Sciences, Kangwon National University)
  • 발행 : 2004.12.01

초록

The effects of adenosine 5'-triphosphate (ATP) and ATP analogs, P/sub 2y/ purinoceptor agonists, on growth of normal mouse mammary epithelial cells (NMuMG) were examined. Cells were plated onto 24 well plates in DMEM supplemented with 10 % fetal calf serum. After serum starvation for 24 hours, ATP, P/sub 2y/ purinoceptor agonists (AdoPP[NH]P, ATP-α-S, ATP-γ-S, β, γ-me-ATP and 2me-S-ATP), P/sub 2u/ purinoceptor agonist (UTP) and P/sub 2y/ purinoceptor antagonists (Reactive Blue 2, more selective to P/sub 2y/ receptor than PPADS; PPADS) were added. DNA synthesis was estimated as incorporation of 3H-thymidine into DNA (1 hour pulse with 1 μ Ci/ml, 18~19 hours after treatment). ATP, Adopp[NH]P, ATP-α-S or ATP-γ-S, significantly increased DNA synthesis at 1, 10 and 100 μM concentrations with dose-dependency (P<0.05), and the maximum responses of ATP and ATP analogs were shown at 100 μM concentration (P<0.05). The potency order of DNA synthesis was ATP≥ATP- γ -S>Adopp [NH]P>ATP-α-S. β, γ -me-ATP, 2me-S-ATP and UTP did not increase DNA synthesis. In autoradiographic analysis of percentage of S-phase cells, similar results were observed to those of DNA synthesis. Addition of 1, 10 or 100 μM Reactive Blue 2 or PPADS significantly decreased ATP (100 μM)-induced DNA synthesis, however, PPADS was less effective than Reactive Blue 2. In Elvax 40P implant experiment, ATP directly stimulated mammary endbud growth in situ suggesting the physiological regulator of ATP in mammary growth. ATP 100 μM rapidly increased MAPK activity, reaching a maximum at 5 min and then gradually decreasing to the base level in 30 min. ATP analogs, Adopp[NH]P and ATP-γ-S also increased MAPK activity, however, β, γ-me-ATP and 2me-S-ATP did not. The inhibitor of the upstream MAPK kinase (MEK), PD 98059 (25 μM), effectively reduced ATP (100 μM) or EGF(10 ng/ml, as positive control)-induced MAPK activity and DNA synthesis (P<0.05). These results indicate that ATP-induced DNA synthesis was prevented from the direct inhibition of MAPK kinase pathway. Overall results support the hypothesis that the stimulatory effects of normal mouse mammary epithelial growth by addition of ATP or ATP analogs are mediated through mammary tissue specific P/sub 2y/ purinoceptor subtype, and MAPK activation is necessary for the ATP-induced cell growth.

참고문헌

  1. Burnstock G, Kennedy C (1985): Is there a basis for distinguishing two types of $P_{2}$-purinoceptor ? Gen Pharmac 16:433-440
  2. Bumstock G, Warland JJ (1987): $P_{2}$-purinoceptors of two subtypes in the rabbit mesenteric artery: reactive blue 2 selectively inhibits responses mediated via the $P_{2y}$-but not the $P_{2x}$-purinoceptor. Br J Pharmacol 90: 383-391 https://doi.org/10.1111/j.1476-5381.1987.tb08968.x
  3. Harden TK, Boyer J, Nicholas RA (1995): $P_2$-purinoceptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35:541-579 https://doi.org/10.1146/annurev.pa.35.040195.002545
  4. Huwiler A, Pfeilschifter J (1994): Stimulation by extracellular ATP and UTP of the mitogen-activated protein kinase cascade and proliferation of rat renal mesangial cells. Br J Pharmacol 113:1455-1463 https://doi.org/10.1111/j.1476-5381.1994.tb17160.x
  5. Kunapuli SP, Daniel JL (1998): $P_2$ receptor subtypes in the cardiovascular system. Biochem J 336:513-523 https://doi.org/10.1042/bj3360513
  6. Sapag-Hagar M, Greenbaum AL (1973): Changes of the activities of adenylate cyclase and cAMP-phosphodiesterase and of the level of 3',5' cyclic adenosine monophosphate in rat mammary gland during pregnancy and lactation. Biochem Biophys Res Com 53:982-987 https://doi.org/10.1016/0006-291X(73)90188-5
  7. Wan, Y, Huang XY (1998): Ananlysis of the Gs/mitogen-activated protein kinase pathway in mutant S49 cells. J Biol Chem 273:14533-14537 https://doi.org/10.1074/jbc.273.23.14533
  8. Yu SM, Chen SF, Lau YT, Yang CM, Chen JC (1996): Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells. Molecular Pharmacol 50:1000-1009
  9. Ziganshin AU, Hoyle CHV, Xuenong B, Lambrecht G, Mutschler E, Baumert HG, Burnstock G (1993): PPADS selectively antagonizes $P_{2x}$-purinoreceptor-mediated responses in the rabbit urinary bladder. Br J Pharmacol 110:1491-1495 https://doi.org/10.1111/j.1476-5381.1993.tb13990.x
  10. Enomoto T, Tanuma S, Yamada M (1981): ATP requirement for the process of DNA replication in isolated HeLa nuclei. J Med Chem 20:907-911 https://doi.org/10.1021/jm00217a008
  11. Hoyle CH, Knight CE, Bumstock G (1990): Suramin antagonizes responses to $P_2$-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. Br J Pharmacol 99: 617-621 https://doi.org/10.1111/j.1476-5381.1990.tb12979.x
  12. Knowles MR, Clarke LL, Boucher RC (1991): Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl J Med 325:533-538 https://doi.org/10.1056/NEJM199108223250802
  13. Communin D, Govaerts C, Parmentier M, Boeynaems JM (1997): Cloning of a human purinergic $P_{2y}$ receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272:31969-31973 https://doi.org/10.1074/jbc.272.51.31969
  14. Huang NN, Wang DJ, Gonzalez FA, Heppel LA (1991): Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation. J Cell Physiol 146:483-494
  15. Huang NN, Wang DJ, Heppel LA (1989): Extra-cellular ATP is a mitogen for 3T3, 3T6 and A431 cells and acts synergistically with other growth factors. Proc Natl Acad Sci USA 86:7904-7908 https://doi.org/10.1073/pnas.86.20.7904
  16. Inoue K, Nakazawa K, Ohara-Imaizumi M, Obama T, Fujimori K, Takanaka A (1991): Antagonism by reactive blue 2 but not by brilliant blue G of extracellular ATP-evoked responses in PC12 phaeochromocytoma cells. Br J Pharmacol 102:851-854 https://doi.org/10.1111/j.1476-5381.1991.tb12265.x
  17. Sharoni Y, Feldman B, Teuerstein L, Levy J (1984): Protein kinase activity in the rat mammary gland during pregnancy, lactation and weaning: A correlation with growth but not with progesterone receptor levels. Endocrinology 115:1918-1924 https://doi.org/10.1210/endo-115-5-1918
  18. Gutkind JS (1998): The pathways connecting G-protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 1839-1842
  19. Churchill PC, Ellis VR (1993): Pharmacological characterization of the renovascular $P_{2}$ purinergic receptors. The J of Pharmacol and Exper Thera 265: 334-338
  20. Faure M, Voyno-Yasenetskaya TA, Bourne HR (1994): cAMP and $\beta\;\gamma$ subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem 269:7851-7854
  21. Harper S, Webb TE, Charlton SJ, Ng LL, Boarder MR (1998): Evidence that $P_2Y_4$ nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. Br J Pharmacol 124:703-710 https://doi.org/10.1038/sj.bjp.0701895
  22. Owens RB, Smith HS, Hackett AJ (1974): Epithelial cell cultures from normal glandular tissue of mice. J Natl Cancer Instit 53:261-265 https://doi.org/10.1093/jnci/53.1.261
  23. Rillema JA (1976): Cyclic nucleotides, adenylate cyclase, and cyclic AMP phosphodiesterase in mammary glands from pregnant and lactating mice. Proc Soc Exp Biol Med 151:748-751 https://doi.org/10.3181/00379727-151-39299
  24. Shamay A, Pines M, Waksman M, Gertler A (1990): Proliferation of bovine undifferentiated mammary epithelial cells in vitro is modulated by G-proteins. Mol Cell Endocrinol 69:217-226 https://doi.org/10.1016/0303-7207(90)90015-Z
  25. Hershey HV (1977): Effect of ATP analogs on DNA synthesis in isolated nuclei. Biochem Biophys Acta 479:256-264
  26. Houston DA, Burnstock G, Vanhoutte PM (1987): Different $P_2$-purinergic receptor subtypes of endothelium and smooth muscle in canine blood vessels. J Pharmac Exp Ther 241:501-506
  27. Martinez-Lacaci I, De Santis M, Karman S, Bianco C, Kim N, Wallace-Jones B, Wechselberger C, Ebert AD, Salomon DS (2002): Regulation of heparin-binding EGF-like growth factor expression in Ha-ras transformed human mammary epithelial cells. J of Cell Physiol 186(2):233-242
  28. Ray FR, Huang W, Slater M, Barden JA (2002): Purinergic receptor distribution in endothelial cells in blood vessels: a basis for selection of coronary artery grafts. Atherosclerosis 162:55-61
  29. Burnstock G, Fischer B, Hoyle CHV., Maillard M, Ziganshin AU, Brizzolara AL, Von Isacovics A, Boyer JL, Kendall-Harden T, Jacobson KA (1994): Structure activity relationships for derivatives of adenosine- 5'-triphosphate as agonists at $P_{2}$ purinoceptors: heterogeneity within $P_{2x}$ and $P_{2y}$ subtypes. Drug Develop Res 31:206-219 https://doi.org/10.1002/ddr.430310308
  30. Mason SJ, Paradiso AM, Boucher RC (1991): Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br J Pharmacol 103:1649-1656 https://doi.org/10.1111/j.1476-5381.1991.tb09842.x
  31. Osipchuk Y, Cahalan M (1992): Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells. Nature Lond 359:241-244 https://doi.org/10.1038/359241a0
  32. Dubyak GR, El-Moatassium C (1993): Signal transduction via $P_{2}$-purinergic receptors for extracellular ATP and other nucleotides. American J of Physiol 265:c577-606
  33. Murgia M, Hanau S, Pizzo P, Rippa M, Virgilio FD (1993): Oxidized ATP: An irreversible inhibitor of the macrophage purinergic $P_{2z}$ receptor. The J of Biol Chem 268:8199-8203
  34. Lambrecht G, Friebe T, Grimm U, Windscheif EB, Hildebrandt C, Baumert HG, SpatzKumbel G, Mutschler E (1992): PPADS, a novel functionally selective antagonist of $P_2$ purinoreceptor-mediated responses. Eur J Pharmacol 217:217-219 https://doi.org/10.1016/0014-2999(92)90877-7
  35. Manzini S, Hoyle CHV, Bumstock G (1986): An electrophysiological analysis of the effect of relative blue 2, a putative $P_2$-puriniceptor antagonist, on inhibitory junction potentials of rat caecum. Eur J Pharmacol 127:197-204 https://doi.org/10.1016/0014-2999(86)90364-X
  36. Abbracchio MP, Burnstock G (1994): Purinoceptors: are there families of $P_{2x}$ and $P_{2y}$ purinoceptors ? Pharmacol Ther 64:445-475
  37. Gordon L (1986): Extracellular ATP: effects, sources and fate. Biochem J 233:309-319
  38. Johnson JL, Fenton S, Sheffield LG (1996): Prolactin inhibits epidermal growth factor-induced Ras-MAPK signaling in mammary epithelial cells. J Biol Chem 271(35):21574-21578 https://doi.org/10.1074/jbc.271.35.21574
  39. Silberstein GB, Daniel CW (1987): Investigation of mouse mammary ductal growth regulation using slow-release plastic implants. J Dairy Sci 70:1981-1990 https://doi.org/10.3168/jds.S0022-0302(87)80240-0
  40. Gao Z, Chen T, Weber MJ, Linden J (1999): $A_{2B}$ adenosine and $P_{2y2}$ receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. J Biol Chem 274:5972-5980 https://doi.org/10.1074/jbc.274.9.5972
  41. Snedecor GW, Cochran WG (1980): In Statistical Methods, 7th ed., The Iowa State University Press, Ames
  42. Davis RJ (1994): MAPKs: new JNK expands the group. Trends Biochem Sci 19:470-473
  43. Boeynaems JM, Pearson JD (1990): $P_{2}$-Purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms. Trends Pharmacol Sci 11:34-37 https://doi.org/10.1016/0165-6147(90)90039-B
  44. Rapaport E (1983): Treatment of human tumor cells with ADP or ATP yields arrest of growth in the S phase of the cell cycle. J Cell Physiol 114:279-283