As expected, when KO POBs were co-cultured with KO BMMs, medium PGE2 was undetectable in vehicle or PTH-stimulated cultures [31] and [33]. WT BMMs (plated

at 10:1 ratio with POBs) made more PGE2 under basal conditions than WT POBs. The basal level of PGE2 production by POBs was likely due to the serum induction of COX-2 [34]. PTH stimulated PGE2 production 2- to 3-fold in co-cultures with WT POBs but had little effect in cultures with KO POBs, consistent with the expected absence of PTH receptors on BMMs. The small increase in PGE2 in the WT BMM, KO POB co-culture might be due to PTH-stimulated RANKL expression in the POBs, which subsequently Selleckchem Omipalisib induced COX-2 in BMMs [40]. In vehicle-treated cultures, the Osteocalcin levels decreased as PGE2 levels decreased ( Table 1). PTH-stimulated Osteocalcin mRNA expression was increased 20-fold relative to vehicle treatment in KO BMM-KO POB cultures, which had no detectable PGE2 production. In all other combinations, which contained WT POBs or WT BMMs and did produce measurable

PGE2, PTH-stimulated Osteocalcin expression was inhibited relative to the KO-KO combination. Hence, either POBs or BMMs expressing COX-2 were sufficient to prevent the PTH-stimulated www.selleckchem.com/products/pd-166866.html OB differentiation in this culture system. In many of our experiments in BMSC cultures (Fig. 1 and Fig. 3)

or in cultures with both POBs and BMMs (Table 1), but not in POBs cultured alone (Fig. 5), PTH given in the presence of COX-2 expression resulted in decreased Alp or Osteocalcin expression relative to vehicle-treated cultures. Since some of the OB differentiation in vehicle-treated cultures is explainable by the serum induction of COX-2 expression and endogenous PGE2 production ( Table 1) [34], this observation suggests that, in the presence of BMMs, the stimulatory effect of endogenous PGE2 on OB differentiation was suppressed in the presence of PTH. To look at 4��8C this possibility more directly, we treated BMSC cultures with PTH (10 nM), PGE2 (10 nM) and the combination (Fig. 6A). PGE2 stimulated Bone sialoprotein (Bsp) mRNA at 14 days in both WT and Cox-2 KO BMSCs. (The small but significant increase in the effects of PGE2 in KO cells has been seen before and may be due to down regulation of PGE2 receptors due to chronic exposure to endogenous PGE2 in WT cultures). Although both PTH and PGE2 individually stimulated Bsp mRNA expression in KO cultures, the combination of PTH and PGE2 had no stimulatory effect. To better understand the dose range over which these effects occurred, we treated Cox-2 KO BMSCs with PTH (10 nM) ± PGE2 (0.1 nM to 0.1 μM) for 14 days ( Fig. 6B).