EMD273316 & EMD95833, type 4 phosphodiesterase inhibitors, stimulate fibroblastic-colony formation by bone marrow cells via direct inhibition of PDE4 and the induction of endogenous prostaglandin synthesis

Abstract

Background: Type 4 phosphodiesterase (PDE4) inhibitors have been shown to stimulate bone formation in vivo and to stimulate osteoblastic differentiation in vitro. As one possible mechanism for the stimulation of bone formation is the recruitment of osteoprogenitor cells from the bone marrow, we have investigated the effect of the PDE4 inhibitors EMD273316, EMD95833, EMD249615 and EMD 219906 on fibroblastic colony formation by whole bone marrow cells and on the ability of these colonies to adopt an osteoblastic phenotype.

Results: All four agents stimulated colony formation in a concentration dependent manner, however, in the case of EMD273316 & EMD95833, the effect was evident at lower concentrations and the addition of prostaglandin E2 (PGE2) was not necessary for maximal stimulation. It was subsequently found that co-incubation with indomethacin reduced the stimulatory effects of EMD273316 & EMD95833 but had no effect on the actions of EMD249615 and EMD 219906 and that EMD273316 & EMD95833 stimulated the synthesis of endogenous PGE2 by whole bone marrow cells whereas EMD249615 and EMD 219906 had no significant effect.

Conclusions: These data suggest that EMD249615, EMD 219906, EMD273316 & EMD95833 can promote the recruitment of bone marrow osteoprogenitor cells leading to a stimulation of bone formation via their direct inhibitory effects on PDE4. The actions of EMD273316 & EMD95833 however, are augmented by their ability to stimulate endogenous prostanoids synthesis which acts synergistically with their direct effects on PDE4.

Figure 1

Synergistic interaction between (a). IBMX or (b). rolipram and PGE₂ on fibroblastic colony formation by whole bone marrow. Whole bone marrow was prepared and cultured in the CFU-f assay as described in the text in the presence of either (a). 10 μM IBMX or (b). 10 μM rolipram in combination with varying concentrations of PGE₂. The medium was changed for fresh, PDE inhibitor & PGE₂-free, medium after 5 d and thereafter twice weekly. The cultures were then stopped after 18 d, fixed with cold ethanol and stained for total colonies with methylene blue. They were then photographed and quantitated. Data presented are mean colony numbers per Petri dish ± S.D. * Denotes significant difference from control cultures, p < 0.05; # denotes significant difference from corresponding IBMX or rolipram-free cultures, p < 0.05.

Figure 2

Effect of PDE4 inhibitors on fibroblastic colony formation by whole bone marrow. Whole bone marrow was prepared and cultured in the CFU-f assay as described in the text and treated with varying concentrations of the PDE4 inhibitors EMD 273316, EMD95833, EMD249615 or EMD219906. Medium changes, fixation and staining were as in Fig. 1. 10^-7 M PGE₂ was used as a positive control in these experiments. Data presented are mean colony numbers per Petri dish ± S.D. * Denotes significant difference from control cultures, p < 0.05.

Figure 3

Effect of the PDE4 inhibitor EMD95833 on fibroblastic colony formation and osteoblastic differentiation. Whole bone marrow was prepared and cultured in the CFU-f assay as described in the text and treated with varying concentrations of EMD95833. In order to assess the effects of EMD95833 on osteoblastic differentiation, after fixation the cells were stained for alkaline phosphatase with naphtholphosphate/fast red, calcium deposition with alizarin red and total colonies with methylene blue. Data presented are mean colony numbers per Petri dish ± S.D. * Denotes significant difference from the appropriately stained control cultures, p < 0.05.

Figure 4

Interaction between PDE4 inhibitors and PGE₂. CFU-f cultures were carried out as described in Fig. 1 and the cells treated with 1 μM EMD273316, EMD95833, EMD249615 or EMD219906 in the presence or absence of 1 nM PGE₂. Data presented are mean colony numbers per Petri dish ± S.D. * Denotes significant difference from control cultures, p < 0.05; # denotes significant difference from corresponding PGE₂-free cultures, p < 0.05.

Figure 5

The role of endogenous prostanoid synthesis in PDE4 inhibitor-mediated stimulation of colony formation. To test the hypothesis that some of the PDE4 inhibitors were also acting via the stimulation of endogenous prostanoids synthesis, CFU-f cultures were challenged with either EMD95833, EMD273316, EMD219906 or EMD249615 (1 μM) in the presence or absence of 3 μM indomethacin (a.). Medium changes, fixation and staining were as in Fig. 1. In addition, immediately before the first medium change, 1 ml aliquots of medium from were removed and PGE₂ levels determined by ELISA (b.). Data presented are means ± S.D. * Denotes significant difference from control cultures, p < 0.05, # denotes significant difference from corresponding indomethacin-free cultures, p < 0.05.