Skip to main content
NCBI home page
Cell Regulation logoLink to Cell Regulation
. 1991 Nov;2(11):889–896. doi: 10.1091/mbc.2.11.889

Protein kinase A-dependent inhibition of alkaline phosphatase release by SaOS-2 human osteoblastic cells: studies in new mutant cell lines that express a cyclic AMP-resistant phenotype.

S Fukayama 1, A K Kearns 1, R M Skurat 1, A H Tashjian Jr 1, F R Bringhurst 1
PMCID: PMC361887  PMID: 1667091

Abstract

We have established mutant SaOS-2 cell lines that express a cyclic AMP (cAMP)-resistant phenotype to investigate the regulation and functional importance of orthophosphoric-monoester phosphohydrolase alkaline optimum (ALPase) in the action of parathyroid hormone (PTH). Cells were stably transfected with a plasmid that directs the synthesis of a mutant form of the type I regulatory subunit of protein kinase A (PKA) under the control of the metallothionein promotor. There was no significant difference between parental SaOS-2 cells and the mutant lines in the affinity or number of receptors for 125I-Nle8,18Tyr34bPTH1-34NH2, either in the absence or presence of Zn2+. When cAMP-dependent gene transcription was examined using transient transfection with a somatostatin promoter-chloramphenicol acetyl transferase (CAT) reporter plasmid, CAT activity stimulated by human PTH and dibutyryl cAMP (DBcAMP) was inhibited by greater than 90% in the presence of Zn2+ in the mutant cell lines. In contrast, activation by a phorbol ester of a pentameric collagenase promoter/CAT construct containing five tandem copies of the AP-1 response element (5x-TRE-CAT) was unaffected in Zn(2+)-treated mutant cells. The inhibitory actions of PTH and DBcAMP on ALPase release were blunted by up to 80-90% in the mutant cell lines in the presence of Zn2+; there were no significant differences in the magnitude of inhibitory effects between these agonists. We conclude that the inhibitory action of PTH on ALPase release in SaOS-2 cells is mediated via activation of PKA. These cAMP-resistant cell lines will be especially useful in elucidating signal transduction mechanism(s) for PTH in human osteoblastic cells.

Full text

PDF
889

Selected References

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

  1. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  2. Boland C. J., Fried R. M., Tashjian A. H., Jr Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones. Endocrinology. 1986 Mar;118(3):980–989. doi: 10.1210/endo-118-3-980. [DOI] [PubMed] [Google Scholar]
  3. Bringhurst F. R., Zajac J. D., Daggett A. S., Skurat R. N., Kronenberg H. M. Inhibition of parathyroid hormone responsiveness in clonal osteoblastic cells expressing a mutant form of 3',5'-cyclic adenosine monophosphate-dependent protein kinase. Mol Endocrinol. 1989 Jan;3(1):60–67. doi: 10.1210/mend-3-1-60. [DOI] [PubMed] [Google Scholar]
  4. Chase L. R., Aurbach G. D. The effect of parathyroid hormone on the concentration of adenosine 3',5'-monophosphate in skeletal tissue in vitro. J Biol Chem. 1970 Apr 10;245(7):1520–1526. [PubMed] [Google Scholar]
  5. Civitelli R., Hruska K. A., Shen V., Avioli L. V. Cyclic AMP-dependent and calcium-dependent signals in parathyroid hormone function. Exp Gerontol. 1990;25(3-4):223–231. doi: 10.1016/0531-5565(90)90056-8. [DOI] [PubMed] [Google Scholar]
  6. Civitelli R., Reid I. R., Westbrook S., Avioli L. V., Hruska K. A. PTH elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line. Am J Physiol. 1988 Nov;255(5 Pt 1):E660–E667. doi: 10.1152/ajpendo.1988.255.5.E660. [DOI] [PubMed] [Google Scholar]
  7. Dietrich J. W., Canalis E. M., Maina D. M., Raisz L. G. Hormonal control of bone collagen synthesis in vitro: effects of parathyroid hormone and calcitonin. Endocrinology. 1976 Apr;98(4):943–949. doi: 10.1210/endo-98-4-943. [DOI] [PubMed] [Google Scholar]
  8. Donahue H. J., Fryer M. J., Eriksen E. F., Heath H., 3rd Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells. J Biol Chem. 1988 Sep 25;263(27):13522–13527. [PubMed] [Google Scholar]
  9. Dunlay R., Hruska K. PTH receptor coupling to phospholipase C is an alternate pathway of signal transduction in bone and kidney. Am J Physiol. 1990 Feb;258(2 Pt 2):F223–F231. doi: 10.1152/ajprenal.1990.258.2.F223. [DOI] [PubMed] [Google Scholar]
  10. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  11. Fukayama S., Tashjian A. H., Jr Direct modulation by androgens of the response of human bone cells (SaOS-2) to human parathyroid hormone (PTH) and PTH-related protein. Endocrinology. 1989 Oct;125(4):1789–1794. doi: 10.1210/endo-125-4-1789. [DOI] [PubMed] [Google Scholar]
  12. Fukayama S., Tashjian A. H., Jr Direct modulation by estradiol of the response of human bone cells (SaOS-2) to human parathyroid hormone (PTH) and PTH-related protein. Endocrinology. 1989 Jan;124(1):397–401. doi: 10.1210/endo-124-1-397. [DOI] [PubMed] [Google Scholar]
  13. Fukayama S., Tashjian A. H., Jr Stimulation by parathyroid hormone of 45Ca2+ uptake in osteoblast-like cells: possible involvement of alkaline phosphatase. Endocrinology. 1990 Apr;126(4):1941–1949. doi: 10.1210/endo-126-4-1941. [DOI] [PubMed] [Google Scholar]
  14. Genge B. R., Sauer G. R., Wu L. N., McLean F. M., Wuthier R. E. Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization. J Biol Chem. 1988 Dec 5;263(34):18513–18519. [PubMed] [Google Scholar]
  15. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hohmann E. L., Tashjian A. H., Jr Functional receptors for vasoactive intestinal peptide on human osteosarcoma cells. Endocrinology. 1984 Apr;114(4):1321–1327. doi: 10.1210/endo-114-4-1321. [DOI] [PubMed] [Google Scholar]
  17. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  18. Light D. B., Schwiebert E. M., Karlson K. H., Stanton B. A. Atrial natriuretic peptide inhibits a cation channel in renal inner medullary collecting duct cells. Science. 1989 Jan 20;243(4889):383–385. doi: 10.1126/science.2463673. [DOI] [PubMed] [Google Scholar]
  19. Löwik C. W., van Leeuwen J. P., van der Meer J. M., van Zeeland J. K., Scheven B. A., Herrmann-Erlee M. P. A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP. Cell Calcium. 1985 Aug;6(4):311–326. doi: 10.1016/0143-4160(85)90002-8. [DOI] [PubMed] [Google Scholar]
  20. McLean F. M., Keller P. J., Genge B. R., Walters S. A., Wuthier R. E. Disposition of preformed mineral in matrix vesicles. Internal localization and association with alkaline phosphatase. J Biol Chem. 1987 Aug 5;262(22):10481–10488. [PubMed] [Google Scholar]
  21. Miksicek R., Heber A., Schmid W., Danesch U., Posseckert G., Beato M., Schütz G. Glucocorticoid responsiveness of the transcriptional enhancer of Moloney murine sarcoma virus. Cell. 1986 Jul 18;46(2):283–290. doi: 10.1016/0092-8674(86)90745-2. [DOI] [PubMed] [Google Scholar]
  22. Montminy M. R., Bilezikjian L. M. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature. 1987 Jul 9;328(6126):175–178. doi: 10.1038/328175a0. [DOI] [PubMed] [Google Scholar]
  23. Montminy M. R., Sevarino K. A., Wagner J. A., Mandel G., Goodman R. H. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6682–6686. doi: 10.1073/pnas.83.18.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murray E., Provvedini D., Curran D., Catherwood B., Sussman H., Manolagas S. Characterization of a human osteoblastic osteosarcoma cell line (SAOS-2) with high bone alkaline phosphatase activity. J Bone Miner Res. 1987 Jun;2(3):231–238. doi: 10.1002/jbmr.5650020310. [DOI] [PubMed] [Google Scholar]
  25. Nakamura T., Gold G. H. A cyclic nucleotide-gated conductance in olfactory receptor cilia. 1987 Jan 29-Feb 4Nature. 325(6103):442–444. doi: 10.1038/325442a0. [DOI] [PubMed] [Google Scholar]
  26. Nakamura T., Nakamura K., Stinson R. A. Release of alkaline phosphatase from human osteosarcoma cells by phosphatidylinositol phospholipase C: effect of tunicamycin. Arch Biochem Biophys. 1988 Aug 15;265(1):190–196. doi: 10.1016/0003-9861(88)90384-0. [DOI] [PubMed] [Google Scholar]
  27. Peck W. A. Cyclic AMP as a second messenger in the skeletal actions of parathyroid hormone: a decade-old hypothesis. Calcif Tissue Int. 1979 Nov;29(1):1–4. doi: 10.1007/BF02408047. [DOI] [PubMed] [Google Scholar]
  28. Pontén J., Saksela E. Two established in vitro cell lines from human mesenchymal tumours. Int J Cancer. 1967 Sep 15;2(5):434–447. doi: 10.1002/ijc.2910020505. [DOI] [PubMed] [Google Scholar]
  29. Register T. C., McLean F. M., Low M. G., Wuthier R. E. Roles of alkaline phosphatase and labile internal mineral in matrix vesicle-mediated calcification. Effect of selective release of membrane-bound alkaline phosphatase and treatment with isosmotic pH 6 buffer. J Biol Chem. 1986 Jul 15;261(20):9354–9360. [PubMed] [Google Scholar]
  30. Register T. C., Warner G. P., Wuthier R. E. Effect of L- and D-tetramisole on 32Pi and 45Ca uptake and mineralization by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. J Biol Chem. 1984 Jan 25;259(2):922–928. [PubMed] [Google Scholar]
  31. Register T. C., Wuthier R. E. Effect of vanadate, a potent alkaline phosphatase inhibitor, on 45Ca and 32Pi uptake by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. J Biol Chem. 1984 Mar 25;259(6):3511–3518. [PubMed] [Google Scholar]
  32. Reid I. R., Civitelli R., Halstead L. R., Avioli L. V., Hruska K. A. Parathyroid hormone acutely elevates intracellular calcium in osteoblastlike cells. Am J Physiol. 1987 Jul;253(1 Pt 1):E45–E51. doi: 10.1152/ajpendo.1987.253.1.E45. [DOI] [PubMed] [Google Scholar]
  33. Rodan S. B., Imai Y., Thiede M. A., Wesolowski G., Thompson D., Bar-Shavit Z., Shull S., Mann K., Rodan G. A. Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res. 1987 Sep 15;47(18):4961–4966. [PubMed] [Google Scholar]
  34. Seed B., Sheen J. Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
  35. Segal J. H., Pollock A. S. Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event. J Clin Invest. 1990 Nov;86(5):1442–1450. doi: 10.1172/JCI114860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shigeno C., Hiraki Y., Westerberg D. P., Potts J. T., Jr, Segre G. V. Photoaffinity labeling of parathyroid hormone receptors in clonal rat osteosarcoma cells. J Biol Chem. 1988 Mar 15;263(8):3864–3871. [PubMed] [Google Scholar]
  37. Wang H. Y., Ascoli M. Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase. Mol Endocrinol. 1990 Jan;4(1):80–90. doi: 10.1210/mend-4-1-80. [DOI] [PubMed] [Google Scholar]
  38. Warner G. P., Hubbard H. L., Lloyd G. C., Wuthier R. E. 32Pi- and 45Ca-metabolism by matrix vesicle-enriched microsomes prepared from chicken epiphyseal cartilage by isosmotic Percoll density-gradient fractionation. Calcif Tissue Int. 1983 May;35(3):327–338. doi: 10.1007/BF02405054. [DOI] [PubMed] [Google Scholar]
  39. Wong M., Schimmer B. P. Recovery of responsiveness to ACTH and cAMP in a protein kinase-defective adrenal cell mutant following transfection with a protein kinase gene. Endocr Res. 1989;15(1-2):49–65. doi: 10.1080/07435808909039088. [DOI] [PubMed] [Google Scholar]
  40. Woodford T. A., Correll L. A., McKnight G. S., Corbin J. D. Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation. J Biol Chem. 1989 Aug 5;264(22):13321–13328. [PubMed] [Google Scholar]
  41. Yamada H., Tsutsumi M., Fukase M., Fujimori A., Yamamoto Y., Miyauchi A., Fujii Y., Noda T., Fujii N., Fujita T. Effects of human PTH-related peptide and human PTH on cyclic AMP production and cytosolic free calcium in an osteoblastic cell clone. Bone Miner. 1989 Apr;6(1):45–54. doi: 10.1016/0169-6009(89)90022-6. [DOI] [PubMed] [Google Scholar]
  42. Yamaguchi D. T., Green J., Kleeman C. R., Muallem S. Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01. J Biol Chem. 1989 Mar 15;264(8):4383–4390. [PubMed] [Google Scholar]
  43. Yamaguchi D. T., Hahn T. J., Iida-Klein A., Kleeman C. R., Muallem S. Parathyroid hormone-activated calcium channels in an osteoblast-like clonal osteosarcoma cell line. cAMP-dependent and cAMP-independent calcium channels. J Biol Chem. 1987 Jun 5;262(16):7711–7718. [PubMed] [Google Scholar]
  44. Yamamoto I., Shigeno C., Potts J. T., Jr, Segre G. V. Characterization and agonist-induced down-regulation of parathyroid hormone receptors in clonal rat osteosarcoma cells. Endocrinology. 1988 Apr;122(4):1208–1217. doi: 10.1210/endo-122-4-1208. [DOI] [PubMed] [Google Scholar]

Articles from Cell Regulation are provided here courtesy of American Society for Cell Biology

RESOURCES