mPGES-1-selective inhibitor CAY10526 concentration-dependently attenuated LPS-induced PGE₂ production in BMDM.

<p>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 (<b><i>A</i></b>) and 7.5 hrs (<i>F</i>) 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><i>B</i></b>) or 7.5 hrs (<i>E</i>) 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. <b><i>C–D.</i></b> 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 (<b><i>D</i></b>); whereas LPS-induced (16 hrs) mPGES-1 mRNA expression in BMDM was significantly increased after CAY10526 treatment (<b><i>C</i></b>), confirming that CAY10526 selectively inhibited the translation step of mPGES-1 mRNA into proteins. Results shown were mean from 4 independent experiments.</p

Selective inhibition of COX-2 abolished LPS-induced PGs production, but didn't affect LPS-induced mPGES-1 expression.

<p>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. <b><i>A</i></b>. NS-398 completely prevented LPS-induced both PGE₂ and PGD₂ production (n = 3). <b><i>B</i></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.</p

LPS induces increased expression of iNOS/COX-2/mPGES-1/PU.1 in BMDM.

<p>Primary cultured mouse BMDM was treated with or without 1 µg/ml LPS for various time points from 2 to 24 hrs. <b><i>A</i></b>. 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><i>B–G</i></b>. The LPS-stimulated (16 hrs) mRNA expression of COX-2 (<b><i>B</i></b>), iNOS (<b><i>C</i></b>), PU.1 (<b><i>D</i></b>), mPGES-1 (<b><i>E</i></b>), mPGES-2 (<b><i>F</i></b>), or c-PGES (<b><i>G</i></b>) 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, * <i>p</i><0.05 vs. control.</p

The enzyme activities of expressed COX-2 and mPGES-1 enzymes between early and late phase of LPS treatment were not different.

<p>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) <i>in vitro</i>. 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).</p

LPS stimulates PGE₂ and PGD₂ production in BMDM.

<p>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).</p

Confocol fluorescent microscopy detects the expression and intracellular distribution of iNOS/COX-2/PU.1/mPGES-1 induced by LPS treatment.

<p>Primary cultured mouse BMDM was treated with or without 1 µg/ml LPS for 16 hrs. The protein expression of iNOS (<b><i>A</i></b>), COX-2 (<b><i>B</i></b>), PU.1 (<b><i>C</i></b>), and mPGES-1 (<b><i>D</i></b>) 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.</p

Selective siRNA inhibition of mPGES-1, but not mPGES-2 or c-PGES, attenuated LPS-induced late-phase PGE₂ production.

<p>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. <b><i>A</i></b>. 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><i>B</i></b>. Western blot results showed that mPGES-1 siRNA not only selectively inhibit the protein expression of mPGES-1, but also that of iNOS. <b><i>C</i></b>. 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. <b><i>D</i></b>. Real-time RT-PCR result confirmed that mPGES-1 siRNA significantly attenuated the LPS-induced mRNA expression of iNOS in BMDM at 16 hrs. <b><i>E–G</i></b>. Real-time RT-PCR results showed that siRNA's for mPGES-1 (<b><i>E</i></b>), mPGES-2 (<b><i>F</i></b>), or c-PGES (<b><i>G</i></b>) 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.</p

Altered nicotine reward-associated behavior following α4 nAChR subunit deletion in ventral midbrain

<div><p>Nicotinic acetylcholine receptors containing α4 subunits (α4β2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 μg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.</p></div

Nicotine CPP is reduced in mice with vMB α4 deletion.

<p>a) CPP schematic. Prior to a 5-day, biased CPP procedure to establish nicotine CPP, mice were mildly food-restricted and handled. Drug-free pre-test and post-test days flanked 3 consecutive conditioning days that consisted of morning and afternoon saline (SAL) and nicotine (NIC) conditioning sessions. b) Nicotine CPP in C57Bl/6 WT mice. Groups of WT mice were conditioned with saline (n = 12), 0.25 mg/kg NIC (n = 12), or 0.5 mg/kg NIC (n = 12) to validate our CPP assay and identify a dose to be used subsequently in α4-flox mice. Mean (± SEM) place preference score is shown for the three drug doses. <i>p</i> values are for Dunnett’s multiple comparisons test. c) Nicotine CPP in α4-flox mice. AAV-GFP or AAV-Cre-GFP vectors were infused into vMB of α4-flox mice (GFP(+), n = 10; Cre(+), n = 10), and mice were subsequently conditioned with 0.25 mg/kg NIC. Mean (± SEM) place preference score is shown for both groups. <i>p</i> value is for unpaired t-test. d) Mean (± SEM) locomotor activity during the pre-test and post-test is shown for α4-flox mice conditioned with NIC. e) Mean (± SEM) locomotor activity during conditioning sessions 1, 2, and 3 is shown for α4-flox mice conditioned with NIC.</p

Functional deletion of α4 subunits in VTA.

<p>a) Recordings were made from α4-flox;Cre(-) and Cre(+) VTA DA neurons. ACh (1 mM) was applied by pressure ejection and inward currents were recorded. All recorded traces are shown in gray, and an averaged trace is shown in blue. Scale bar: 40 pA, 400 ms. b) Quantification of ACh-evoked currents in AAV-infected mice. Mean inward ACh-evoked currents from α4-flox;Cre(-) (n = 19 cells from n = 9 animals), α4-flox;Cre(+) (n = 14 cells from n = 5 animals), WT;Cre(-) (n = 7 cells from n = 2 animals), and WT;Cre(+) (n = 8 cells from n = 2 animals) cells ± SEM. ****<i>p</i><0.0001 (unpaired <i>t</i>-test, <i>t</i> = 8.788). c) Residual ACh-evoked currents in α4-flox;Cre(+) neurons are αCtxMII-sensitive. For Cre(+) responses in (B), αCtxMII (100 nM) was applied by superfusion and ACh was re-applied after 12–15 min. Before-after responses for n = 4 αCtxMII-treated neurons (n = 2 animals) are shown. **<i>p</i> = 0.0052 (paired <i>t</i>-test, <i>t</i> = 7.348).</p