Summary
These investigations were intended to determine whether local and systemic skeletal effectors—3′5′-cyclic adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D), calcitonin, and NaF—could regulate3[H]-thymidine incorporation (i.e., into DNA) in serum-free, monolayer cultures of embryonic chick calvarial cells, and/or modulate the activity of embryonic chick bone extracts to increase3[H]-thymidine incorporation. In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 μM) and low-dose PTH (0.1 nM) stimulated3[H]-thymidine incorporation,P<.05 for each; isobutylmethylxanthine (IBMX-1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased3[H]-thymidine incorporation; and PGE2 (1 μM) had no effect. The stimulatory actions of calcitonin, fluoride, and low-dose PTH were inductive, and the inhibitory actions of IBMX and 1,25(OH)2D were acute. PTH had complex time-dependent actions on3[H]-thymidine incorporation, being inhibitory after 4–8 hours of exposure and stimulatory after 20–24 hours (P<.001 for each). The effects of calcitonin, fluoride, and low-dose PTH to increase3[H]-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells. PTH inhibited3[H]-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P<.005). The inhibitory actions of IBMX and 1,25(OH)2D were independent of cell differentiation. Additional studies further revealed that these local and systemic skeletal effectors could also modulate the activity of embryonic chick bone extracts to increase3[H]-thymidine incorporation in calvarial cell cultures. We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase3[H]-thymidine incorporation (P<.001 for each). These activations were noncompetitive, indicating (1) mechanistic differences between the stimulatory actions of the effectors and the chick bone extract (i.e., different rate-limiting steps for the effects of each on3[H]-thymidine incorporation); and (2) that neither calcitonin, fluoride, nor 0.1 nM PTH altered the apparent affinity of the cells for stimulatory activity(s) in the extract. High-dose PTH was a noncompetitive inhibitor with respect to bone extract activity, indicating that the effect of 10 nM PTH to decrease3[H]-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase3[H]-thymidine incorporation. Both IBMX and PGE2 were competitive inhibitors of bone extract-stimulated3[H]-thymidine incorporation (P<.001 for each), implying that these effectors (IBMX, PGE2, and embryonic chick bone extract) shared a common (or coincidentally equal) rate-limiting step. The effects of 1,25(OH)2D on bone extract-stimulated3[H]-thymidine incorporation were different at high and low doses. At a low concentration (1 nM), 1,25(OH)2D enhanced the effect of bone extract to increase3[H]-thymidine incorporation, but higher concentrations (e.g., 100 nM) were inhibitory (P<.01 for each). Together, these data demonstrate that local and systemic skeletal effectors can have direct effects on embryonic chick calvarial cells,in vitro, to regulate the basal rate of3[H]-thymidine incorporation, and to modulate the stimulatory action of an embryonic chick bone extract.
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References
Treharne RW (1981) Review of Wolff's law and its proposed means of operation. Orthopaedic Reviews 10:35–47
Cowin SC, Lanyon LE, Rodan GA (eds) (1984) Functional adaptation in bone tissue. Calcif Tissue In (Suppl 1):36
Baylink DJ, Morey ER, Ivey JL, Stauffer ME (1980) Vitamin D and bone. In: Norman AW (ed) Vitamin D: molecular biology and clinical nutrition, pp 387–453
Baylink DJ (1985) Differential responses of alveolar bone osteoclasts residing at different sites. In: Proc American Institute of Oral Biology, 42nd ann, meeting. Palm Springs, CA, pp 39–48
Briancon D, Meunier P (1981) Treatment of osteoporosis with fluoride, calcium and vitamin D. In: Frost HM (ed) Orthopaedic clinics of North America, Vol. 12, WB Saunders, Philadelphia, pp 629–648
Reutter FW, Olah AJ (1978) Bone biopsy and clinical observations in long-term treatment of osteoporosis with NaF and vitamin D3. In Courvoisier B, Donath A, and Baud CA (eds) Fluoride and bone, Hans Huber, Bern, pp 249–255
Klein DC, Raisz LG (1970) Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology 86:1436–1440
Rodan GA, Rodan SB, Majeska RJ (1982) Expression of the osteoblastic phenotype in osteosarcoma cell lines in culture. In: Silbermann M, and Slavkin HC (eds) Current advances in skeletogenesis, Excerpta Medica, Princeton, pp 315–320
Canalis E, Peck WA, Raisz LG (1980) Stimulation of DNA and collagen synthesis by autologous growth factor in cultured fetal rat calvaria. Science 210:1021–1023
Howard GA, Bottemiller BL, Baylink DJ (1981) Evidence for the coupling of bone formation to bone resorption in vitro. Metab Bone Disease & Rel Res 2:131–135
Drivdahl RH, Howard GA, Baylink DJ (1982) Extracts of bone contain a potent regulator of bone formation. Biochem Biophys Acta 714:26–33
Farley JR, Baylink DJ (1982) Purification of a skeletal growth factor from human bone. Biochemistry 21:3502–3507
Jennings J, Mohan S, Baylink DJ (1987) Purification of bovine skeletal growth factor. In: Barnes C and Sirbasku A (eds) Peptide growth factors. Methods in Enzymology. Academic Press, Orlando 146:281–294
Mohan S, Jennings JC, Linkhart TA, Baylink DJ (1986) Isolation and purification of a low molecular weight skeletal growth factor from human bone. Biochem Biophys Acta 884:234–242
Sampath TK, De Simone DP, Reddi AH (1982) Extracellular bone matrix-derived growth factors. Exp Cell Res 142:460–464
Howard GA, Bottemiller BL, Turner RT, Rader JI, Baylink DJ (1981) Parathyroid hormone stimulates bone formation and resorption in organ cultures: evidence for a coupling mechanism. Proc Natl Acad Sci USA 78:3204–3208
Farley JR, Tarbaux N, Murphy LA, Masuda T, Baylink DJ (in press) In vitro evidence that bone formation may be coupled to resorption by release of mitogen(s) from resorbing bone. Metabolism
Pfeilschafter J, D'Souza S, Mundy GR (1986) Transforming growth factor-beta is released from resorbing bone and stimulates osteoblast activity, Abstract #294. J Bone Mineral Res (Suppl 1):1
Farley J, Wergedal J, Baylink D (1983) Fluoride directly stimulates proliferation and alkaline phosphatase activity of bone-forming cells. Science 223:330–332
Farley JR, Masuda T, Wergedal JE, Baylink DJ (1982) Human skeletal growth factor: characterization of the mitogenic effect on bone cells in vitro. Biochemistry 21:3508–3513
Puzas JE, Drivdahl RH, Howard GA, Baylink DJ (1981) Endogenous inhibitor of bone cell proliferation. Proc Soc Exp Biol Med 166:113–122
Gruber H, Ivey J, Thompson E, Chestnut C, Baylink D (in press) osteoblast and osteoclast cell number and cell activity in postmenopausal osteoporosis. Mineral and Electrolyte Metabolism
Baylink DJ, Liu C-C (1979) The regulation of endosteal bone volume. J Periodontol (Special Issue) 50:43–49
Gospodarowicz D, Bailecki H, Greenburg C (1978) Purification of the fibroblast growth factor activity from bovine serum. J Biol Chem 253:3736–3741
Drivdahl RH, Puzas JE, Howard GA, Baylink DJ (1981) Regulation of DNA synthesis in chick calvarial cells by factors from bone organ culture. Proc Soc Exp Biol Med 168:143–150
Farley JR, Tarbaux N, Baylink DJ (1986) Skeletal alkaline phosphatase activity as a bone formation index in vitro. Metabolism 35:563–571
Linkhart T, Jennings J, Mohan S, Wakely G, Baylink D (1986) Characterization of mitogenic activities extracted from bovine bone matrix. Bone 7:479–487
Centrella M, Canalis E (1985) Transforming and non-transforming growth factors are present in medium conditioned by fetal rat calvaria. Proc Natl Acad Sci USA 82:7335–7339
Hauschka PV, Maurakos AE, Iafrati MD, Doleman SE, Klagsbrun M (1986) Growth factors in bone matrix: isolation of multiple types by affinity chromatography on Heparin-sepharose. J Biol Chem 261:12665–12674
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram amounts of protein, utilizing the principle of protein-dye binding. Anal Biochem 72:248–255
Farley JR, Fitzsimmons R, Taylor AK, Jorch UM, Lau K-HW (1985) Direct effects of ethanol on bone resorption and formation in vitro. Arch Biochem Biophys 238:305–314
Segel IH (1975) Enzyme kinetics. Wiley-Interscience, New York
Burger H, Boonekamp PM, Nijweide PJ (1986) Osteoblast and osteoclast precursors in primary cultures of calvarial bone cells. Anatomical Record 214:32–40
MacDonald BR, Gallagher JA, Russell RGG (1986) Parathyroid hormone stimulates the proliferation of cells derived from human bone. Endocrinology 118:2445–2449
Burch WM, Lebovitz HE (1983) Parathyroid hormone stimulates growth of embryonic chick pelvic cartilage in vitro. Calcif Tissue Int 35:526–530
Van der Plas A, Feyen JHM, Nijweide PJ (1985) Direct effect of parathyroid hormone on the proliferation of osteoblast-like cells: a possible involvement of cyclic AMP. Biochem Biophys Res Commun 129:918–925
Silberman M, Mirsky N, Levitan S, Weisman Y (1983) The effect of 1,25-dihydroxyvitamin D3 on cartilage growth in neonatal mice. Metab Bone Dis & Rel Res 4:337–345
Burch WM (1984) Calcitonin stimulates growth and maturation of embryonic chick pelvic cartilage in vitro. Endocrinology 114:1196–1202
Burch WM, Lebovitz HE (1981) Adenosine 3′,5′-monophosphate: a modulator of embryonic chick cartilage growth. J Clin Invest 68:1496–1502
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Farley, J.R., Tarbaux, N.M., Vermeiden, J.P.M. et al. In vitro evidence that local and systemic skeletal effectors can regulate3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory actions(s) of embryonic chick bone extract. Calcif Tissue Int 42, 23–33 (1988). https://doi.org/10.1007/BF02555835
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DOI: https://doi.org/10.1007/BF02555835