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.

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 regulate 3[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 increase 3[H]-thymidine incorporation. In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 microM) and low-dose PTH (0.1 nM) stimulated 3[H]-thymidine incorporation, P less than .05 for each; isobutylmethylxanthine (IBMX--1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased 3[H]-thymidine incorporation; and PGE2 (1 microM) 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 on 3[H]-thymidine incorporation, being inhibitory after 4-8 hours of exposure and stimulatory after 20-24 hours (P less than .001 for each). The effects of calcitonin, fluoride, and low-dose PTH to increase 3[H]-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells. PTH inhibited 3[H]-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P less than .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 increase 3[H]-thymidine incorporation in calvarial cell cultures. We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase 3[H]-thymidine incorporation (P less than .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 on 3[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 decrease 3[H]-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase 3[H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)