Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvaria...
Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. This activity was not observed in the absence of calvarial osteoblasts, suggesting that calvarial osteoblasts are likely target cells of rolipram. Osteoclast formation by rolipram was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for the osteoclast differentiation factor, TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL). Northern blot analysis revealed the effect of rolipram to be associated with the increased expression of TRANCE mRNA in mouse calvarial osteoblasts. Collectively, these data indicate that PDE4 inhibitor up-regulates the TRANCE mRNA expression in osteoblasts, which in turn controls osteoclast formation.
Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. This activity was not observed in the absence of calvarial osteoblasts, suggesting that calvarial osteoblasts are likely target cells of rolipram. Osteoclast formation by rolipram was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for the osteoclast differentiation factor, TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL). Northern blot analysis revealed the effect of rolipram to be associated with the increased expression of TRANCE mRNA in mouse calvarial osteoblasts. Collectively, these data indicate that PDE4 inhibitor up-regulates the TRANCE mRNA expression in osteoblasts, which in turn controls osteoclast formation.
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,arial osteoblasts. Additional studies were performed to identify the mechanism by which rolipram induces osteoclastogenesis.
,arial cells was also examined. First, RT-PCR was performed to determine the mRNA expressions of the PDE4 subtypes in mouse cal.,arial cells, and compared these with the expression patterns in ST-2, a mouse bone-marrow stromal cell line able to differentiate into osteoblasts, and ATDC5, a mouse chondrogenic cell line. As shown in Fig 3A, cal.
The primers used for the mouse PDE4 subtypes, calcitonin receptor (CTR), cathepsin K and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) in this study are as blows: PDE4A, 5'-ggaactcacacacctgtcg-31 (forward), 5l-gttcttgtgctaagaggtcc-31 (reverse); PDE4B, 5l-tggaaatcctggctgccat-3 (forward), 5'-tccacagaagctgtgtgct-3' (reverse); PDE4C, 5-tggtatcagagtaggattcc-31 (forward), S'-ctctgtgtaaaccttggctg-S1 (reverse); PDE4D, 5'-cggaactcgctctgatgt-3* (forward), 5'-acagaggcgttgtgcttg-3* (reverse); CTR, 5-tttcaagaaccttagctgccagag-3' (forward), 5*-caaggcacggacaatgttgagaag-3' (reverse); cathepsin K, 5'-cttccaatacgtgcagcaga-3' (forward), 5Lacgcaccaatatcttgcacc-3' (reverse); and GAPDH, 5-gaaggtcggtgtgaacggatttggc-31 (forward), 5'-catgtaggccatgaggtccaccac-31 (reverse). The PCR program was as follows: 32(all mouse PDE4 subtypes, CTR and cathepsin K) or 28 (GAPDu) cycles, after an initial denaturation step at 94℃ for 3 minutes, then denaturation at 94℃ for 30 seconds, annealing at 58℃ for 45 seconds and extension at 72℃ for 60 seconds, with a final extension at 72℃ for 10 minutes.
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