Figure 1.
LPS induces increased expression of iNOS/COX-2/mPGES-1/PU.1 in BMDM.
Primary cultured mouse BMDM was treated with or without 1 µg/ml LPS for various time points from 2 to 24 hrs. A. The protein expression of targeted proteins in BMDM cell lysates was detected by Western blot assays. Representative blots and the densitometry of iNOS/COX-2/mPGES-1/PU.1 protein expression are showed from 5 independent experiments. Protein expression of COX-2 and iNOS was detected within 2 to 4 hrs post-LPS treatment; whereas increased protein expression of mPGES-1 and PU.1 was detected after 8 hrs of LPS treatment. In contrast, the protein expression of COX-1, mPGES-2, c-PGES or H-PGDS was not affected by LPS treatment. The protein loading in each experiment was normalized by β-actin. B–G. The LPS-stimulated (16 hrs) mRNA expression of COX-2 (B), iNOS (C), PU.1 (D), mPGES-1 (E), mPGES-2 (F), or c-PGES (G) in BMDM was confirmed by real-time quantitative RT-PCR. The mRNA of each gene was normalized to the β-actin mRNA expression level in the same sample. The results shown are mean of at least 4 independent experiments for each gene, * p<0.05 vs. control.
Figure 2.
Confocol fluorescent microscopy detects the expression and intracellular distribution of iNOS/COX-2/PU.1/mPGES-1 induced by LPS treatment.
Primary cultured mouse BMDM was treated with or without 1 µg/ml LPS for 16 hrs. The protein expression of iNOS (A), COX-2 (B), PU.1 (C), and mPGES-1 (D) was determined by immunostaining followed by confocol fluorescent microscopy. In each sample group, BMDM was stained with the green fluorescent-labeled antibody for the targeted protein and the blue fluorescent-labeled DAPI for nucleus; the overlay image of each targeted protein and nucleus are shown (magnification: ×200, scale bar: 10 µM). LPS stimulation significantly increased cytosolic expression of iNOS, COX-2, mPGES-1 and PU.1 in BMDM, suggesting newly synthesized protein expression in cytosol. PU.1 also showed strong nuclear staining (i.e., nuclear translocation) after LPS treatment; whereas both COX-2 and mPGES-1 showed similar enhanced perinuclear localization in LPS-treated groups. The results shown are representative images from 3 independent experiments.
Figure 3.
LPS stimulates PGE2 and PGD2 production in BMDM.
Equal amount of BMDM was treated with or without 1 µg/ml LPS for various time points from 2 to 24 hrs. The BMDM culture medium was collected and the concentrations of PGE2 and PGD2 in the sample media were quantified using LC-MS-MS. LPS induced both PGE2 and PGD2 production at a similar lower level in the first 8 hrs of treatment. In contrast, the production of PGE2 in BMDM continuously increased after LPS treatment for 12 hrs; whereas the production of PGD2 kept at a relatively stable level after 12 hrs of LPS treatment (n = 5).
Figure 4.
mPGES-1-selective inhibitor CAY10526 concentration-dependently attenuated LPS-induced PGE2 production in BMDM.
BMDM was pretreated with mPGES-1-selective inhibitor CAY10526 at 5, 10, or 20 µM for 0.5 hr prior to LPS (1 µg/ml) treatment for either 16 hrs or 7.5 hrs. CAY10526 concentration-dependently inhibited the LPS-induced PGE2 production at 16 hrs (A) and 7.5 hrs (F) as measured by LC-MS-MS assay. Results are the mean of at least 3 independent experiments.. Western blot assays showed CAY10526 concentration-dependently attenuated the LPS-induced mPGES-1 and iNOS protein expression at either 16 hrs (B) or 7.5 hrs (E) in BMDM, but had no inhibitory effect on the protein expression of mPGES-2, c-PGES or COX-2. Representative blots and the densitometry of iNOS/mPGES-1/COX-2 protein expression were showed from at least 3 independent experiments. C–D. Real time RT-PCR results showed the efforts of CAY10526 pretreatment (10 µM, 0.5 hr prior to LPS treatment) significantly attenuated the LPS-induced (16 hrs) iNOS mRNA expression in BMDM (D); whereas LPS-induced (16 hrs) mPGES-1 mRNA expression in BMDM was significantly increased after CAY10526 treatment (C), confirming that CAY10526 selectively inhibited the translation step of mPGES-1 mRNA into proteins. Results shown were mean from 4 independent experiments.
Figure 5.
Selective siRNA inhibition of mPGES-1, but not mPGES-2 or c-PGES, attenuated LPS-induced late-phase PGE2 production.
BMDM were transfected with siRNA's for mPGES-1, mPGES-2, c-PGES mRNA, or a control siRNA for 36 hrs, and were then treated with 1 µg/ml LPS for 16 hrs. A. The PGE2 level in culture medium was determined by LS-MC-MC. mPGES-1 siRNA significantly attenuated LPS-induced PGE2 production at 16 hrs in BMDM compared to that of BMDM transfected with the control siRNA. In contrast, the siRNA for either mPGES-2 or c-PGES did not affect the LPS-induced PGE2 production in BMDM compared to the control siRNA group. B. Western blot results showed that mPGES-1 siRNA not only selectively inhibit the protein expression of mPGES-1, but also that of iNOS. C. In contrast, transfection of BMDM with siRNA's for either mPGES-2 or c-PGES selectively attenuated the expression of its targeted protein expression accordingly, but had no inhibitory effect on LPS-induced expression of iNOS, COX-2, or mPGES-1. D. Real-time RT-PCR result confirmed that mPGES-1 siRNA significantly attenuated the LPS-induced mRNA expression of iNOS in BMDM at 16 hrs. E–G. Real-time RT-PCR results showed that siRNA's for mPGES-1 (E), mPGES-2 (F), or c-PGES (G) specifically inhibited the mRNA expression of its targeted PGES isoform compared to the control siRNA group, but did not affect the mRNA expression of the other two PGES isoforms in BMDM.
Figure 6.
Selective inhibition of PU.1 didn't affect LPS-induced mPGES-1 expression or late-phase PGE2 production in BMDM.
BMDM was transfected with either PU.1 siRNA or a control siRNA for 36 hrs, and were then treated with or without 1 µg/ml LPS for 16 hrs. The concentration of PGE2 and PGD2 in culture medium was then determined by LS-MC-MC. The protein and mRNA expression in BMDM was determined by Western blot assay or real-time RT-PCR, respectively. A. PU.1 siRNA significantly prevented LPS-induced protein expression of PU.1 in BMDM, but had no inhibitory effect on the protein expression of iNOS, mPGES-1, mPGES-2, or c-PGES (representative blots and the densitometry of iNOS/mPGES-1/PU.1 protein expression were showed from 3 independent experiments). B. Real-time RT-PCR result confirmed that PU.1 siRNA significantly attenuated the PU.1 mRNA expression with or without LPS (16 hrs) treatment in BMDM. C. LPS-induced (16 hrs) PGE2 and PGD2 production in BMDM was not affected by PU.1 siRNA (n = 3).
Figure 7.
Selective inhibition of COX-2 abolished LPS-induced PGs production, but didn't affect LPS-induced mPGES-1 expression.
BMDM were pretreated with COX-2-selective inhibitor NS-398 (20 µM, 0.5 hr) prior to the treatment of 1 µg/ml LPS for 16 hrs, the PGE2 and PGD2 levels in culture medium were measured by LS-MC-MC. A. NS-398 completely prevented LPS-induced both PGE2 and PGD2 production (n = 3). B. NS-398 also partially inhibited LPS-induced protein expression of COX-2 and iNOS, but had no inhibitory effect on the expression of COX-1/PU.1/mPGES-1/mPGES-2/c-PGES by Western blot assay. The representative blots and the densitometry of iNOS/COX-2/mPGES-1/PU.1 protein expression were from 3 independent experiments.
Figure 8.
The enzyme activities of expressed COX-2 and mPGES-1 enzymes between early and late phase of LPS treatment were not different.
The induced COX-2 and mPGES-1 enzymes were immunoprecipitated (IP, 2 hrs at 4°C) separately from equal amount of BMDM lysates at either 8 or 16 hrs of LPS treatment under the same experimental condition. The IP COX-2 or mPGES-1 enzyme concentrations were determined, and equal amount of IP COX-2 or mPGES-1 enzymes from each time point was used to determine their enzyme activity of PGE2 production from their substrates (either arachidonic acid or PGH2) in vitro. No significant difference in either COX-2 or mPGES-1 enzyme activity of PGE2 production was detected between 8 and 16 hrs of LPS treatment (n = 3).