In this investigation we have shown that a number of PDE4 inhibitors, including rolipram, IBMX, EMD219906, EMD249615, EMD273316 & EMD95833, stimulate colony formation by rat bone marrow cells in the CFU-f assay. In addition the proportion of colonies expressing alkaline phosphatase and depositing a calcified matrix, both markers associated with osteogenesis, increased proportionately. In the past we have suggested that positive activity in the CFU-f assay may have some value as a predictor of bone anabolic activity in vivo
. This is because a number of agents known to stimulate bone formation in vivo
such as PGE2
] or FGF-2 [13
] have also been found to be potent stimulators of colony formation in the CFU-f assay [15
]. These data therefore, suggest that the increased bone formation seen in animals treated with various PDE4 inhibitors [6
] may be mediated by the induction of recruitment and differentiation of bone marrow stromal cells.
These data are broadly in agreement with other reports on the effects of PDE4 inhibitors on osteoprogenitor cells. XT-44, Ro 20-1724 and denbufylline have all been shown to increase mineralised nodule formation in primary cultures of rat bone marrow cells [6
] and pentoxifylline and rolipram stimulated the expression of alkaline phosphatase in the bone marrow stromal cell line, ST2 [4
]. The experiments described in these investigations all used monolayer cultures of either cell lines or primary bone marrow cells. Although differentiation undoubtedly does take place, as shown by the results using cell lines, under these conditions it is not possible to say whether the increase in the numbers of mineralised nodules could be due to increases in the numbers of cells differentiating or due to the proliferation or recruitment of osteoprogenitor cells. In this study however, we have used CFU-f cultures which measure the numbers of proliferating stromal cells present in whole bone marrow. The increase in colony numbers seen after treatment with the PDE inhibitors, suggests that stromal cells, that would otherwise have remained quiescent, have been activated and induced to proliferate. As the proportion of colonies expressing alkaline phosphatase remained fairly constant throughout, it seems likely that the increase differentiation is due to an increase in the recruitment of precursor cells rather than a direct effect on differentiation.
The principle action of these drugs is to inhibit the enzyme Type 4 phosphodiesterase and thereby increase intracellular levels of cyclic AMP. It might therefore, be expected that they would only be active in the presence of agents such as PTH or PGE2, that act by stimulating the synthesis of cyclic AMP. Indeed a number of the agents tested, including IBMX, rolipram, EMD219906 and EMD249615 produced only modest effects when used in isolation. However, when used in combination with PGE2, synergistic effects were seen such that almost maximal stimulation of colony number was achieved at concentrations of PGE2 that do not normally have a significant effect in this system. The mechanism underlying the stimulation seen in response to these agents in the absence of exogenous prostanoids is open to speculation. It is possible that non-prostanoid agents present in the cultures are responsible for a basal level of cyclic AMP synthesis or alternatively, low levels of prostanoids, below the detection limit of the ELISA used, may be responsible.
In contrast EMD95833 and EMD273316 gave rise to significant increases in colony number in the absence of PGE2
and the addition of PGE2
had no further effect. This suggests that these agents stimulate the production of endogenous stimulators of cyclic AMP synthesis such as prostanoids. To support this hypothesis, inhibition of prostanoid synthesis by the addition of indomethacin to the cultures abrogated the stimulatory effects of the PDE inhibitors. Furthermore, direct measurement of PGE2
in the culture medium revealed that both EMD273316 and EMD95833 stimulated the production of PGE2
by whole bone marrow cells whereas EMD219906 and EMD249615 had no such effect. This is consistent with the effects of PDE4 inhibitors in other systems. PGE2
production has been shown to be stimulated by a number of specific PDE4 inhibitors in various cell types including bovine airway smooth muscle cells by Ro-20-1724 [19
], whole blood by RP73401 [20
] and human peripheral blood mononuclear cells by rolipram and CDP840 [21
]. It is known that prostaglandins are derived from arachidonic acid via the actions of two enzymes termed cyclooxygenase-1 (COX-1) and cylooxygenase-2 (COX-2). Broadly speaking COX-1 is thought to be present at relatively constant levels and is responsible for the housekeeping functions of prostanoids. In contrast, COX-2 expression is induced by a wide variety of cytokines, growth factors and other stimuli and is involved in the acute response to a number of pathological situations, in particular inflammation [22
]. The fact that PGE2
synthesis was increased after the addition of EMD273316 and EMD95833 would suggest the involvement of COX-2 however, this will need to be confirmed using specific inhibitors. Recently, Miyamoto et al [23
] demonstrated that the PDE4 inhibitor XT-611 also gave rise to bone-anabolic effects in an in vitro
bone marrow culture system. The system used was slightly different to that described above in that determined calcified nodule formation in confluent cultures of bone marrow cells. However, they did show that XT-611 stimulated PGE2
accumulation which potentiated the PDE4-related actions of XT-611. Furthermore they showed that this effect could be blocked by the addition of the COX-2 antagonists zaltoprofen and NS-398 [23
In summary, these data suggest that the bone anabolic effects of PDE4 inhibitors may be mediated by the recruitment and proliferation of quiescent bone marrow stromal cells followed by their osteoblastic differentiation. In the case of EMD219906 and EMD249615 this effect appears to be due to their direct effects on PDE4. EMD273316 and EMD95833 however, also stimulate the synthesis of endogenous PGE2 synthesis which then synergises with the PDE inhibitors enabling them to achieve maximal stimulation in the absence of exogenous stimulators of cyclic AMP synthesis.