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. 2010 Mar 22;285(21):15794–15804. doi: 10.1074/jbc.M109.074534

FIGURE 5.

FIGURE 5.

PGD2 and PGJ2 induce HA production in human orbital fibroblasts via DP1, but not DP2, activation. A, RT-PCR. Human orbital fibroblasts (OF1 and OF2) and T cells express DP1 and DP2 mRNA. Note the variability in the expression of DP2 between the two fibroblast strains. B, Western blot analysis revealed that orbital fibroblasts and T cells express both DP1 and DP2 protein. Membranes were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to ensure equal protein loading. C, pharmacological inhibition of DP1, but not DP2, blocks PGD2- and PGJ2-induced HA production. Orbital fibroblasts were left untreated (Vehicle) or were pretreated with 100 nm of the DP1 antagonist MK-0524 (MK) or DP2 antagonist ramatroban (RAM) for 1 h with or without PGD2 or PGJ2 for 18 h, and HA ELISA was performed. Both PGD2 and PGJ2 significantly increased HA production compared with untreated (p < 0.001 (###) and p < 0.01 (##), respectively). Pretreatment of orbital fibroblasts with MK significantly decreased the ability of PGD2 (**, p < 0.01) and PGJ2 (*, p < 0.05) to induce HA levels. RAM was not able to prevent PGD2- and PGJ2-increased HA levels (p < 0.05 (#) and p < 0.01 (##) compared with vehicle control, respectively). ns, not significant compared with untreated (vehicle). D, treatment with the DP1 agonist BW245C significantly increased the expression of HAS mRNA. Orbital fibroblasts were cultured in reduced serum for 3 days and exposed to BW245C (10 μm) for the indicated times. There was a significant increase in HAS1 (329 ± 128-fold increase; **, p < 0.01) and HAS2 (17 ± 0.14-fold increase; ***, p < 0.001) at 2 h compared with vehicle control. HAS3 mRNA increased by 6 h (4.8 ± 0.06; ***, p < 0.01). E, activation of DP1 by the selective agonist BW245C induces HA. There was a significant increase in HA when fibroblasts were treated with 5 and 10 μm BW245C (p < 0.05 (*) and p < 0.01 (**), respectively, compared with vehicle control. Samples were run in duplicate, utilizing three separate human orbital fibroblast strains (representative results are shown).