Summary
To further understand the mechanism of PTH effects on bone and bone cells, we have analyzed the effect of PTH on specific protein phosphorylation in cells isolated from neonatal mouse calvaria. Four populations of cells (I–IV), isolated by sequential digestion with chromatographically purified bacterial collagenase isozymes and neutral proteinase, were cultured overnight. Alkaline phosphatase activity was greater than acid phosphatase activity in all four populations. PTH stimulated cyclic AMP production in all four populations, although the effect was greatest in populations II and III. Cultured cells were treated with PTH for up to 15 minutes. Cytosolic and membrane fractions were obtained and assayed forin vitro protein phosphorylation. No hormonal effects were found in membrane fractions. In cytosol fractions, treatment of the population II cells for 10–15 minutes with 0.1 μM PTH decreased the subsequent protein phosphorylation of an 85,000 Mr protein. In contrast, PTH treatment increasedin vitro phosphorylation of both the 85,000 and 35,000 Mr proteins in population III cells. Phosphorylation of the 35,000 Mr protein was cyclic AMP-dependent. All of the phosphoproteins appeared to be phosphorylated solely on serine or threonine residues except the 85,000 Mr protein which may also contain significant amounts of phosphotyrosine. Therefore, some of the effects of PTH are cyclic AMP-mediated and other effects may be mediated through tyrosine phosphorylation. These data indicate that PTH has differential effects onin vitro protein phosphorylation in two separable populations of isolated neonatal mouse calvarial cells and support a hypothesis that multiple osteoblastlike cells existin vivo.
Similar content being viewed by others
References
Greengard P (1978) Phosphorylated proteins as physiological effectors. Science 199:146–152
Peck WA, Klahr S (1979) Cyclic nucleotides in bone and mineral metabolism. Adv Cyc Nucl Res 11:89–130
Hefley TJ (1987) Fast protein liquid chromatography of bacterial collagenase. Implications for the isolation of cells from bone. J Bone Min Res 2:505–515
Wong G, Cohn DV (1974) Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells. Nature 252:713–715
Wong GL, Cohn DV (1975) Target cells in bone for parathormone and calcitonin are different: enrichment of each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. Proc Nat Acad Sci 72:3167–3171
Hefley TJ, Stern PH, Brand JS (1983) Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum. Exp Cell Res 149:227–236
Layne E (1957) Spectrophotometric and turbidimetric methods for measuring proteins. Meth Enzymol 3:448–450
Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage T4. Nature 227:680–685
Martensen TH (1984) Chemical properties, isolation and analysis of O-phosphates in proteins. Meth Enzymol 107:3–23
Fleischmann RD, Pawelek JM (1985) Evidence that a 90-kDA phosphoprotein, an associated kinase, and a specific phosphatase are involved in the regulation of Cloudman melanoma cell proliferation by inslin. Proc Nat Acad Sci 82:1007–1011
Lowry OH (1957) Micromethods for assay of enzymes. Meth Enzymol 4:366–381
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent, J Biol Chem 193:265–275
Harper JF, Brooker CJ (1975) Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2′O acetylation by acetic anhydride in aqueous solution. J Cyc Nucl Res 12:207–218
Snedecor GW, Cochran WG (1980) Statistical methods. The Iowa State University Press, Ames, Iowa, USA
Rodan GA, Rodan SB (1984) Expression of the osteoblast phenotype. In: Peck WA (ed) Bone and mineral research, Annual 2. Elsevier, New York pp 244–285
Aparisi T, Stark A, Ericsson JL (1982) Human osteogenic sarcoma. Study of the ultrastructure, with specific notes on the localization of alkaline and acid phosphatase. Int Orthop 6:171–179
Cole AA, Walters LM (1987) Tartrate-resistant acid phosphatase in bone and cartilage following decalcification and cold-embedding in plastic. J Histochem Cytochem 35:203–206
Brown EM, Aurbach CD (1980) Role of cyclic nucleotides in secretory mechanisms and actions of parathyroid hormone and calcitonin. Vit Horm 38:205–250
Partridge NC, Kemp BE, Veroni MC, Martin TJ (1981) Activation of adenosine 3′,5′-monophosphate-dependent protein kinase in normal and malignant bone cells by parathyroid hormone, prostaglandin E2 and prostacyclin. Endocrinology 108:220–225
Habener JF, Rosenblatt M, Potts JT Jr (1984) Parathyroid hormone: biochemical aspects of biosynthesis, secretion, action and metabolism. Physiol Rev 64:985–1053
Wong GL (1986) Skeletal effects of parathyroid hormone. In: Peck WA (ed) Bone and mineral research, Annual 4. Elsevier, New York, pp 103–129
Lowik CWCM, van Leeuwen JPTH, van der Meer JK, Scheven BAA, Herrmann-Erlee MPM (1985) A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP. Cell Calcium 6:311–326
Rappaport MS, Stern PH (1986) Parathyroid hormone and calcitonin modify inositol phospholipid metabolism in fetal rat libm bones. J Bone Min Res 1:173–179
Nishizuka Y, Takai Y, Kishimoto A, Kikkawa U, Kaibuchi K (1984) Phospholipid turnover in hormone action. Rec Prog Horm Res 40:301–341
Hunter T, Cooper JA (1985) Protein-tyrosine kinases. Ann Rev Biochem 54:897–930
Tashjian AH Jr, Levine L (1978) Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria. Biochem Biophys Res Comm 85:966–975
Raisz LG, Simmons HA, Sandberg AL, Canalis E (1980) Direct stimulation of bone resorption by epidermal growth factor. Endocrinology 107:270–273
Hammerman MR, Hruska KA (1982) Cyclic AMP-dependent protein phosphorylation in canine renal brush border vesicles is associated with decreased phosphate transport. J Biol Chem 257:992–999
Hammerman MR, Hansen VA, Morrissey JJ (1983) Cyclic AMP-dependent protein phosphorylation and dephosphorylation alter phosphate transport in canine renal brush border vesicles. Biochim Biophys Acta 755:10–16
Takenawa T, Wada E, Tsumita T, Nasaki T, Filburn CR, Sacktor B (1984) Effect of parathyroid hormone, cyclic AMP and Ca++ on the phosphorylation of brush border membranes in rabbit kidney. Min Elect Metab 10:103–112
Noland TA Jr, Henry HL (1983) Protein phosphorylation in chick kidney—response to parathyroid hormone, cyclic AMP, calcium and phosphatidylserine. J Biol Chem 258:538–546
Ausiello DA, Rosenblatt M, Dayer JM (1980) Parathyroid hormone modulation of protein kinase in giant cell tumors of human bone. Am J Physiol 239:E144–149
Egan JJ, Rodan GA (1986) Parathyroid hormone reduces phosphorylation of osteoblast myosin light chain independent of calcium (abstract). J Bone Min Res 1:61
Stewart PJ, Stern PH (1987) Calcium/phosphatidylserine-stimulated protein phosphorylation in bone: Effect of parathyroid hormone. J Bone Min Res 2:281–287
Goldring SR, Mahaffey JE, Rosenblatt M, Dayer J-M, Potts JT Jr, Krane SM (1979) Parathyroid hormone inhibitors: comparisons of biological activity in bone- and skin-derived tissue. J Clin Endo Metab 48:655–659
Schmid C, Steiner T, Foresch ER (1983) Insulin-like growth factors stimulate synthesis of nucleic acids and glycogen in cultured calvaria cells. Calcif Tissue Int 35:578–585
Rodan GA, Martin TJ (1981) Role of osteoblasts in hormonal control of bone resorption—a hypothesis. Calcif Tissue Int 33:349–351
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Krieger, N.S., Hefley, T.J. Differential effects of parathyroid hormone on protein phosphorylation in two osteoblastlike cell populations isolated from neonatal mouse calvaria. Calcif Tissue Int 44, 192–199 (1989). https://doi.org/10.1007/BF02556564
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02556564