Several

studies have proposed OC-produced factors that, unlike our findings, are not specific for PTH-treated cultures but can inhibit OB differentiation in general. These factors include cardiotropin-1 [55], semaphorin 4D [56], and sclerostin [47]. We have done several microarray studies on the BMMs under our culture conditions and did not find differential expression of any of these factors by COX-2 expression/activity or PGE2 addition (data not shown), but this does not rule out their regulation at the protein level. The inhibition of PTH-stimulated differentiation mediated by endogenous PGs could be generated by addition of PGE2, but not other agonists for other PG receptors, to cultures. Moreover, production of the inhibitory CM required expression on BMMs of EP4, one of two receptors for PGE2 that activates cAMP signaling. Hence, it seems likely that Z-VAD-FMK purchase the endogenous PG mediating this website the inhibitory action under our conditions is PGE2. PGE2 is expected to have its major actions via cAMP/PKA signaling pathways similar to those stimulated by PTH. Exogenous PGE2 concentrations as low as 0.1 nM were sufficient to inhibit osteogenic effects of PTH, and levels ≥ 4 nM

were seen in vehicle-treated co-cultures of POBs and BMMs as long as one cell type expressed COX-2. PGE2 itself stimulates OB differentiation in vitro, as shown in the current studies. For a number of agents, such as TGFβ, BMP2, strontium ranelate and fresh serum [14], [17], [18] and [19], the induction of COX-2 expression

and PGE2 production enhances their stimulation of OB differentiation in vitro. In contrast to PTH, these agents all have major actions via signaling pathways other than cAMP/PKA. Hence, other agonists that act via cAMP signaling O-methylated flavonoid pathways might also be inhibited by PGE2 in this culture model. CM from COX-2 expressing BMMs did not block the stimulatory effects of endogenous PGs or exogenous PGE2 unless the cultures were also treated with PTH. In the absence of BMMs, the combination of PTH with PGE2 had additive effects on OB differentiation, as expected of two osteogenic agents. In contrast, in the presence of the as yet unidentified factor or factors secreted by BMMs, the stimulatory effect of the combination of PTH and PGE2 was abrogated. Assuming that the stimulatory effects of PTH and PGE2 on OBs are mediated via stimulation of cAMP, it is possible that the CM contains a factor that acts via Gαi to inhibit production of PTH- and PGE2-stimulated cAMP. PGE2 in WT CM can act via EP3, which is coupled to Gαi. However, it is unclear why this effect would only occur in the presence of PTH. The factor that blocks PTH-stimulated differentiation produced by BMMs is unlikely to be PGE2 itself because the addition of PGE2 to PTH, in the absence of BMMs or WT CM, resulted in additive stimulatory effects.