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

Abstract

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.