This study confirms and expands upon our previous observation that COX-2 produced PGs inhibit PTH-stimulated OB differentiation in BMSCs [26]. When COX-2 expression or PG production was absent, PTH markedly stimulated OB differentiation in BMSCs. The window for the stimulatory effect was the first week of culture, and this observation, in conjunction with similar effects of PTH on both OB and Selleck Kinase Inhibitor Library adipocyte differentiation, suggests that PTH was acting on OB precursors or MSCs, consistent with reported effects of PTH on OB precursors or MSCs in vivo [2] and [7]. Because PTH is stable in culture up to 72 h between medium changes [35],
our culture conditions provided continuous exposure of cells to PTH, which A-1210477 in most in vitro studies has resulted in inhibition of OB differentiation. Because intermittent PTH is anabolic in vivo but continuous PTH is catabolic, it is often assumed that PTH must be applied intermittently in vitro in order to be osteogenic. This assumption was strengthened by positive effects
on OB differentiation when cells had short, transient exposure to PTH [8], [10] and [45]. However, the brief duration of PTH exposure is usually accomplished by removing PTH-containing media and replacing with fresh media. Since this procedure also removes PGs that accumulate in the media, it is possible that the osteogenic effects in such experiments were PD184352 (CI-1040) really due to the removal of PGs that inhibited osteogenic effects of PTH. The inhibitory effects of PGs on OB formation did not occur in vehicle-treated BMSC cultures but only in PTH-treated BMSCs. In these cultures, OCLs were formed in response to PTH during the same “window” of time that PTH had its stimulatory effect. The inhibitory effects of PGs did not occur in POBs washed free of hematopoietic cells or in OPG-treated BMSCs. Co-cultures of POBs with BMMs or with CM from BMMs demonstrated that RANKL-treated BMMs were required to see the inhibitory effects of PGs.
The need for RANKL in order to see the inhibitory effects and the reversal by OPG suggest that the BMMs involved were committed to the OC lineage. Finally, using these same co-cultures, we showed that PGs acted on BMMs to cause them to produce a soluble factor or factors that then acted on OBs to suppress PTH-stimulated OB differentiation. We could find no precedent for a soluble factor produced in OC lineage cells in response to PGs that inhibited PTH-stimulated OB differentiation. A number of studies have shown that soluble factors produced by monocytes and non-resorbing OCs can regulate OB differentiation in a stimulatory, but not inhibitory, manner [46], [47], [48], [49], [50] and [51].