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. 1996 Sep 15;318(Pt 3):965–971. doi: 10.1042/bj3180965

A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells.

P H Scott 1, C M Belham 1, J al-Hafidh 1, E R Chilvers 1, A J Peacock 1, G W Gould 1, R Plevin 1
PMCID: PMC1217712  PMID: 8836145

Abstract

In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation.

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Selected References

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  1. Benjamin C. W., Jones D. A. Platelet-derived growth factor stimulates growth factor receptor binding protein-2 association with Shc in vascular smooth muscle cells. J Biol Chem. 1994 Dec 9;269(49):30911–30916. [PubMed] [Google Scholar]
  2. Brown E. J., Albers M. W., Shin T. B., Ichikawa K., Keith C. T., Lane W. S., Schreiber S. L. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994 Jun 30;369(6483):756–758. doi: 10.1038/369756a0. [DOI] [PubMed] [Google Scholar]
  3. Burgering B. M., Coffer P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995 Aug 17;376(6541):599–602. doi: 10.1038/376599a0. [DOI] [PubMed] [Google Scholar]
  4. Calvo V., Wood M., Gjertson C., Vik T., Bierer B. E. Activation of 70-kDa S6 kinase, induced by the cytokines interleukin-3 and erythropoietin and inhibited by rapamycin, is not an absolute requirement for cell proliferation. Eur J Immunol. 1994 Nov;24(11):2664–2671. doi: 10.1002/eji.1830241115. [DOI] [PubMed] [Google Scholar]
  5. Cheatham B., Vlahos C. J., Cheatham L., Wang L., Blenis J., Kahn C. R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol. 1994 Jul;14(7):4902–4911. doi: 10.1128/mcb.14.7.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen R. H., Sarnecki C., Blenis J. Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol. 1992 Mar;12(3):915–927. doi: 10.1128/mcb.12.3.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. doi: 10.1038/370071a0. [DOI] [PubMed] [Google Scholar]
  8. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  9. Edelmann H. M., Kühne C., Petritsch C., Ballou L. M. Cell cycle regulation of p70 S6 kinase and p42/p44 mitogen-activated protein kinases in Swiss mouse 3T3 fibroblasts. J Biol Chem. 1996 Jan 12;271(2):963–971. doi: 10.1074/jbc.271.2.963. [DOI] [PubMed] [Google Scholar]
  10. Han J. W., Pearson R. B., Dennis P. B., Thomas G. Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites. J Biol Chem. 1995 Sep 8;270(36):21396–21403. doi: 10.1074/jbc.270.36.21396. [DOI] [PubMed] [Google Scholar]
  11. Hawkins P. T., Jackson T. R., Stephens L. R. Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Nature. 1992 Jul 9;358(6382):157–159. doi: 10.1038/358157a0. [DOI] [PubMed] [Google Scholar]
  12. Homma Y., Sakamoto H., Tsunoda M., Aoki M., Takenawa T., Ooyama T. Evidence for involvement of phospholipase C-gamma 2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells. Biochem J. 1993 Mar 15;290(Pt 3):649–653. doi: 10.1042/bj2900649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kahan C., Seuwen K., Meloche S., Pouysségur J. Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. J Biol Chem. 1992 Jul 5;267(19):13369–13375. [PubMed] [Google Scholar]
  14. Knox A. J. Airway re-modelling in asthma: role of airway smooth muscle. Clin Sci (Lond) 1994 Jun;86(6):647–652. doi: 10.1042/cs0860647. [DOI] [PubMed] [Google Scholar]
  15. Kumagai N., Morii N., Fujisawa K., Nemoto Y., Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J Biol Chem. 1993 Nov 25;268(33):24535–24538. [PubMed] [Google Scholar]
  16. Lane H. A., Fernandez A., Lamb N. J., Thomas G. p70s6k function is essential for G1 progression. Nature. 1993 May 13;363(6425):170–172. doi: 10.1038/363170a0. [DOI] [PubMed] [Google Scholar]
  17. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  18. Ludwig S., Rapp U. R. Cascading towards vascular disorder gene therapy. Nat Med. 1995 Jun;1(6):513–515. doi: 10.1038/nm0695-513. [DOI] [PubMed] [Google Scholar]
  19. Malarkey K., Belham C. M., Paul A., Graham A., McLees A., Scott P. H., Plevin R. The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors. Biochem J. 1995 Jul 15;309(Pt 2):361–375. doi: 10.1042/bj3090361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Malarkey K., Chilvers E. R., Lawson M. F., Plevin R. Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells. Br J Pharmacol. 1995 Oct;116(4):2267–2273. doi: 10.1111/j.1476-5381.1995.tb15063.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McLees A., Graham A., Malarkey K., Gould G. W., Plevin R. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926. Biochem J. 1995 May 1;307(Pt 3):743–748. doi: 10.1042/bj3070743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Merrall N. W., Plevin R. J., Stokoe D., Cohen P., Nebreda A. R., Gould G. W. Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes. Biochem J. 1993 Oct 15;295(Pt 2):351–355. doi: 10.1042/bj2950351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ming X. F., Burgering B. M., Wennström S., Claesson-Welsh L., Heldin C. H., Bos J. L., Kozma S. C., Thomas G. Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature. 1994 Sep 29;371(6496):426–429. doi: 10.1038/371426a0. [DOI] [PubMed] [Google Scholar]
  24. Monfar M., Lemon K. P., Grammer T. C., Cheatham L., Chung J., Vlahos C. J., Blenis J. Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol Cell Biol. 1995 Jan;15(1):326–337. doi: 10.1128/mcb.15.1.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakanishi H., Brewer K. A., Exton J. H. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
  26. Okada T., Sakuma L., Fukui Y., Hazeki O., Ui M. Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J Biol Chem. 1994 Feb 4;269(5):3563–3567. [PubMed] [Google Scholar]
  27. Pagès G., Lenormand P., L'Allemain G., Chambard J. C., Meloche S., Pouysségur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8319–8323. doi: 10.1073/pnas.90.18.8319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  29. Reinhard C., Fernandez A., Lamb N. J., Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J. 1994 Apr 1;13(7):1557–1565. doi: 10.1002/j.1460-2075.1994.tb06418.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reinhard C., Thomas G., Kozma S. C. A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4052–4056. doi: 10.1073/pnas.89.9.4052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ruderman N. B., Kapeller R., White M. F., Cantley L. C. Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1411–1415. doi: 10.1073/pnas.87.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sabers C. J., Martin M. M., Brunn G. J., Williams J. M., Dumont F. J., Wiederrecht G., Abraham R. T. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem. 1995 Jan 13;270(2):815–822. doi: 10.1074/jbc.270.2.815. [DOI] [PubMed] [Google Scholar]
  33. Sale E. M., Atkinson P. G., Sale G. J. Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J. 1995 Feb 15;14(4):674–684. doi: 10.1002/j.1460-2075.1995.tb07046.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stoyanov B., Volinia S., Hanck T., Rubio I., Loubtchenkov M., Malek D., Stoyanova S., Vanhaesebroeck B., Dhand R., Nürnberg B. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 1995 Aug 4;269(5224):690–693. doi: 10.1126/science.7624799. [DOI] [PubMed] [Google Scholar]
  35. Susa M., Vulević D., Lane H. A., Thomas G. Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin. J Biol Chem. 1992 Apr 5;267(10):6905–6909. [PubMed] [Google Scholar]
  36. Thomas G. MAP kinase by any other name smells just as sweet. Cell. 1992 Jan 10;68(1):3–6. doi: 10.1016/0092-8674(92)90199-m. [DOI] [PubMed] [Google Scholar]
  37. Toker A., Meyer M., Reddy K. K., Falck J. R., Aneja R., Aneja S., Parra A., Burns D. J., Ballas L. M., Cantley L. C. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem. 1994 Dec 23;269(51):32358–32367. [PubMed] [Google Scholar]
  38. Wang J., Auger K. R., Jarvis L., Shi Y., Roberts T. M. Direct association of Grb2 with the p85 subunit of phosphatidylinositol 3-kinase. J Biol Chem. 1995 May 26;270(21):12774–12780. doi: 10.1074/jbc.270.21.12774. [DOI] [PubMed] [Google Scholar]
  39. Zhang J., Zhang J., Benovic J. L., Sugai M., Wetzker R., Gout I., Rittenhouse S. E. Sequestration of a G-protein beta gamma subunit or ADP-ribosylation of Rho can inhibit thrombin-induced activation of platelet phosphoinositide 3-kinases. J Biol Chem. 1995 Mar 24;270(12):6589–6594. doi: 10.1074/jbc.270.12.6589. [DOI] [PubMed] [Google Scholar]
  40. van Corven E. J., Hordijk P. L., Medema R. H., Bos J. L., Moolenaar W. H. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1257–1261. doi: 10.1073/pnas.90.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]

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