In vitro evidence that local and systemic skeletal effectors can regulate 3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory actions(s) of embryonic chick bone extract.

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.