Pertussis toxin (PT) inhibits glycochenodeoxycholic acid (GCDCA)-induced caspase-3 activity and nuclear fragmentation.

<p>(<b>a</b>) Primary rat hepatocytes were treated for 4 hours with 50 μmol/L of GCDCA, 200 nmol/L of PT or a combination of both. PT was added 30 min prior to the addition of GCDCA. * P<0.05 for GCDCA + PT vs. GCDCA alone. (<b>b</b>) Hepatocytes were treated with different concentrations of PT 30 min prior to the addition of 50 μmol/L of GCDCA for 4 hours. (<b>c</b>) PT (200 nmol/L) significantly inhibits GCDCA-induced caspase-3 activation. P<0.05 for GCDCA (50, 100, 200 μM) + PT vs GCDCA (50, 100, 200 μM) alone. (<b>d</b>) Acridine orange staining. Treatment with 50 μmol/L of GCDCA induces nuclear condensation and fragmentation (white arrows) which is blocked with 200 nmol/L PT at 15 hours after the addition of GCDCA. (<b>e</b>) Sytox green staining. PT does not induce necrosis in hepatocytes after 15 hours. Hepatocytes treated with 5 mmol/L H₂ O₂ were used as positive control.</p

Pertussis toxin (PT) does not inhibit apoptosis in HepG2-rNtcp cells and rat H-4-II-E cells.

<p>(<b>a</b>) Caspase-3 activity in HepG2-rNtcp cells treated with 200 μmol/L of GCDCA in the presence and absence of 200 nmol/L PT. (<b>b</b>) HepG2-rNtcp cells were treated for 16 hours with 20 ng/ml of TNFα in the presence of 200 ng/ml of ActD. 200 nmol/L of PT was added 30 min prior to the addition of TNFα/ActD. (<b>c</b>) Caspase-3 activity in rat H-4-II-E cells treated with 50–500 μmol/L of GCDCA in the presence and absence of 200 nmol/L PT. (<b>d</b>) Rat H-4-II-E cells were treated for 16 hours with 20 ng/ml of TNFα in the presence of 200 ng/ml of ActD. 200 nmol/L of PT was added 30 min prior to the addition of TNFα/ActD.</p

The protective effect of pertussis toxin (PT) is independent of the activation of specific kinases.

<p>(<b>a</b>) Caspase-3 activity in rat hepatocytes treated with 50 μmol/L of GCDCA in the presence and absence of 200 nmol/L PT and with or without the inhibitors of ERK1/2- MAPK (10 μmol/L of U0126; U0), p38 MAPK (10 μmol/L of SB 203580; SB), PI3K (50 μmol/L of LY 294002; LY), PKC inhibitors (1 μmol/L of calphostin-C, 1 μmol/L of BSM-I).</p

The protective effect of pertussis toxin (PT) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis.

<p>(<b>a</b>) Hepatocytes were pre-incubated with 200 nmol/L of PT for 15 hours after which cells were washed and exposed to 50 μmol/L of GCDCA alone (+/− PT) or with simultaneous addition of PT (+/+ PT). * P<0.05 for GCDCA + PT (+/−) and GCDCA + PT (+/+) vs. GCDCA 50 μM. (<b>b</b>) hepatocytes were stimulated with 50 μmol/L GCDCA for 4 hours (GCDCA 50 µM). PT (200 nmol/L) was added 30 minutes prior to (−30 min), simultaneous with (0 min) or 30 minutes (+30 min), 1 hour (+1 hr), 2 hours (+2 hrs), 3 hours (+3 hrs) after the addition of GCDCA.* P<0.05 for −30 min of PT, 0 min of PT, +30 min of PT, +1 hr of PT vs GCDCA alone.</p

The −122/−69 region is required for optimal FXR-ligand-mediated induction of the <i>SHP</i> promoter in DLD-1, HEK293 and HepG2 cells.

<p>The colon carcinoma (DLD-1), human embryonic kidney (HEK293) and hepatoma (HepG2.rNtcp) cell lines were transfected with the indicated <i>hSHP</i> promoter constructs and expression plasmids for hFXR and hRXRα. Cells were treated with or without 100 µmol/L CDCA (A) or 1 µmol/L GW4064 (B). Luciferase activity was measured to determine <i>SHP</i> promoter activity. Data are presented as means of ± SD; n≥3. Promoter activity in CDCA/GW4064-treated condition is significantly different from the −278/+10 construct in DLD-1 (a), HEK293 (b) or HepG2.rNtcp (c) cells. <i>P</i>≤0.05 in a pairwise comparison by Mann-Whitney <i>U</i> test.</p

A FXR/RXRα/CDCA-responsive element is located in the −122/−69 region of the <i>SHP</i> promoter.

<p>A) shows an overview of the different constructs used to localize the FXR-responsive element in the −278/−69 region of the <i>SHP</i> promoter. Relevant binding sites for other NRs are included. (B, C) DLD-1 cells were transfected with the indicated <i>hSHP</i> promoter constructs and expression plasmids for hFXR and hRXRα. Cells were treated with or without 100 µmol/L CDCA (B) or 1 µmol/L GW4064 (C). Luciferase activity was measured to determine <i>SHP</i> promoter activity. Data are presented as means of ± SD; n≥3. Significant differences are indicated when compared to CDCA/GW4064-treated −569/+10 (a); CDCA/GW4064-treated −303/+10 (b); CDCA/GW4064-treated −278/+10(c). <i>P</i>≤0.05 in a pairwise comparison by Mann-Whitney <i>U</i> test.</p

FXR binds to the LRH-1 responsive element in the <i>hSHP</i> promoter.

<p>FXR was precipitated from nuclear extracts of hFXR-overexpressing DLD-1 cells using a DNA probe containing the SHP −122/−69 region (“wild type”(WT) or “mutated”(mut)), the IR-1 from the <i>hBSEP</i> promoter (positive control), the LacI binding site (negative control) or empty beads (EB, negative control). A) DNA probes of <i>SHP</i> −122/−69 and <i>BSEP</i>-IR-1 bind FXR. Competition experiments were performed with 3-fold excess <i>hBSEP</i>-IR-1 or LacI lacking a biotin label. B) the <i>SHP</i> −122/−69 region with a mutated LRH-1 site failed to precipitate or compete for FXR binding. Competition experiments were performed with 3-fold excess wild type <i>SHP</i> −122/−69, mutated <i>SHP</i> −122/−69 or LacI lacking a biotin label.</p

The IR-1 at −291/−279 is required for 9cRA-, but not for CDCA-mediated induction of the human <i>SHP</i> promoter.

<p>DLD-1 cells were transfected with hFXR and hRXRα expression plasmids and various <i>hSHP</i> promoter constructs as indicated. Cells were treated with or without 100 µmol/L CDCA and/or 1 µmol/L 9cRA. The synergistic effect of the FXR ligand (CDCA) and RXRα ligand (9cRA) on <i>SHP</i> promoter activity depends on the previously identified IR-1 located at −291/−279. Mutation or deletion of this IR-1 sequence did not abolish <i>SHP</i> promoter activation by FXR/CDCA. Luciferase activity was measured to determine the <i>SHP</i> promoter activity. Data are presented as means ± SD; n≥3. Vehicle-treated conditions are set to 1. p≤0.05 for *) in a pairwise comparison by Mann-Whitney U test.</p

The LRH-1 site is required for FXR-induced expression of <i>SHP</i>.

<p>(A) shows the location of IR-1-like half sites and an LRH-1 site in the −122/−69 region of the <i>hSHP</i> promoter. The latter was previously identified in the murine <i>Shp</i> promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088011#pone.0088011-Lee3" target="_blank">[35]</a> and conserved in rat and human (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088011#pone.0088011.s006" target="_blank">Figure S6</a></b>). All 4 IR-1-like sites and the LRH-1 site were mutated and analyzed for the effect on FXR/CDCA-mediated induction of the −278/+10 <i>hSHP</i> promoter (B) mutating one of the IR-1 half-sites did not or only partially reduce FXR/CDCA-dependent activation of the −122/−69 <i>hSHP</i> promoter fragment, whereas mutations in the LRH-1 site strongly reduced the response of the −122/−69 <i>hSHP</i> promoter fragment to FXR/CDCA-stimulation. *) significantly different from CDCA treated 278/+10 WT (C) in the context of the −569/−10 <i>hSHP</i> promoter fragment the LRH-1 site is the dominant FXR/CDCA response element (over the previously identified IR-1). DLD-1 cells were transfected with hFXR and hRXRα expression plasmids and various <i>hSHP</i> promoter constructs as indicated. Cells were treated with or without 100 µmol/L CDCA. Luciferase activity was measured to determine the <i>SHP</i> promoter activity. a) significantly different from CDCA treated 569/+10 WT. b) significantly different from CDCA treated −569/+10 IR-1 KO. Data presented as means ± SD; n≥3. <i>P</i>≤0.05 for *), a) and b) in a pairwise comparison by Mann-Whitney <i>U</i> test.</p

Correlations of fecal calprotectin levels (mg/kg) with serum levels of all detected molecules (pg/mL) in patients with Crohn’s disease.

<p>Correlations of fecal calprotectin levels (mg/kg) with serum levels of all detected molecules (pg/mL) in patients with Crohn’s disease.</p