FIGURE 5.

mPGES-1 is essential for osteoclast formation and PGE₂ production induced by co-culture of B16 melanoma with mouse bone marrow cells and osteoblasts. Mouse bone marrow cells and osteoblasts from mPges1^−/− or wild-type mice were co-cultured with or without fixed B16 cells for 7 days. A, representative fields of the staining for tartrate-resistant acid phosphatase, a specific marker of osteoclasts. B, the number of tartrate-resistant acid phosphatase-positive osteoclasts, multinucleated cells containing three or more nuclei. Data are mean ± S.E. of four wells. *, p < 0.001. C, expression of RANKL mRNA determined by RT-PCR. Total RNA collected from the adherent cells on day 4 in the co-cultures was used for RT-PCR analysis. The graph shows the relative intensity of RANKL expression compared with the wild type. D, the concentration of PGE₂ in conditioned medium determined by enzyme immunoassay. Data are mean ± S.E. of four wells. *, p < 0.001. E, osteoblasts from mPges1^−/− or wild-type mice were added to a layer of fixed B16 cells or culture plates. The total RNA was extracted after 6 h and used to measure the expression of mPGES-1, mPGES-2, and cytosolic PGES by RT-PCR. The graphs show the relative intensity of each gene expression compared with the wild-type control.