Graphical summary for IL-1β effects on mCx-I activity and mitochondrial ROS levels in IB3-1 and Caco-2/pRS26.

<p>The figure illustrates the interactions among the different proteins, kinases or small molecules involved in this work. The interactions were drawn by using the software Pathway Studio (v 9, Elsevier). Arrows with the+symbol represent stimulations and those with the -| symbol represent inhibition. Green ellipses: small molecules; red sickle-vertex: kinases; purple rectangle: disease (CF); blue star-vertex: shRNA specific for CFTR. The results obtained with IL-1β blocking Ab or with the receptor inhibitor IL1RN suggest that an autocrine IL-1β signaling is responsible for the reduced mCx-I activity and the increased ROS levels seen in IB3-1 CF cells or in Caco-2/pRS26 cells. Inhibition of NF-κB or p38 MAPK also resulted in increased mCx-I activity and decreased ROS levels. The inhibition of MEK1/2 or JNKs (AP-1 pathway) had no effects. The mechanisms by which CFTR increases IL-1β and IL1-β, p38 MAPKs or NF-κB inhibit mCx-I and increase ROS levels remain to be determined (dotted lines).</p

Effect of IL-1β on mitochondrial Complex I activity (mCx-I).

<p>IB3-1 and S9 cells pre-incubated in serum free media for 24 h were incubated for additional 24 h (plus CFTR-stimulating cocktail) in the presence or absence of IL-1β (5 ng/ml) and the mCx-I activity was determined. A: mCx-I in-gel activity (IGA) of treated and untreated S9 and IB3-1 cells; mCx-III correspond to the WB for the UQCRC1 subunit of the mitochondrial complex III used as internal standard. B: Densitometric quantification and statistical analysis of the results shown in panel A (for n = 3); IGA of mCx-I was calculated as the ratio mCx-I IGA/mCx-III WB. C: Spectrophotometric measurements of mitochondrial NADH-cytochrome c reductase activity for the same experiments shown in panel A and B. Results were expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal S9 cells.</p

ROS levels in IB3-1 and S9 cells.

<p>S9 and IB3-1 cells were preincubated 24 h in serum free media. Then, the media was replaced (serum free, in the presence of a CFTR-stimulating cocktail) and the cells incubated another 24 h. The IB3-1 cells were treated in this second incubation as indicated. IL-1β: IL-1β (5 ng/ml); Ab-IL-1β: anti-IL-1β blocking antibody (30 ng/ml); Ab-Ctrl: anti-Histone H1 monoclonal antibody as negative control for antibody incubation (30 ng/ml); p38 Inh: p38 inhibitor SB203580 (5 µM); IKK Inh: IKK inhibitor III (2 µM); JNK Inh: JNK inhibitor SP600125 (5 µM); MEK1/2 Inh: MAPK/AP-1 pathway inhibitor U0126 (5 µM). A: MitoSOX fluorescence (5 µM for 10 min in Hank’s buffer). B: DCF fluorescence (10 µM DCFH-DA for 40 min in Hank’s buffer); Ab-Ctrl: anti-JNK2 monoclonal antibody as negative control. Results were expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data are expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared to IB3-1 cells.</p

P38/MAPK1 inhibition.

<p>After 24-1 cells were incubated for 24 h with increasing concentrations of the p38 MAPK inhibitor SB203580 (1, 5, 10 and 20 µM) and mCx-I activity was measured by using BN-PAGE and spectrophotometry. A: mCx-I in-gel activity (IGA) and mCx-III (WB). B: Densitometric quantification and statistical analysis of the results shown in panel A. IGA of mCx-I was calculated as the ratio mCx-I (IGA)/mCx-III (WB). C: Spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity in the same experiments of panel A, expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal IB3-1 cells.</p

Recovery of mCx-I activity by incubation with anti-IL-1β blocking antibody or with IL-1β receptor antagonist (IL1RN).

<p>S9 and IB3-1 cells preincubated for 24 h in serum free media were additionally incubated for 24 h (in the presence of a CFTR-stimulating cocktail) with anti-IL-1β blocking antibody or IL-1 receptor antagonist. The mCx-I activity was determined by using spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity. A: IB3-1 cells were treated with anti-IL-1β blocking antibody (30 ng/ml) or IL-1 receptor antagonist (10 ng/ml). The Figure shows the activity expressed as percentage (%), considering the average activity of S9 cells as 100%. Ab-Ctrl: anti-JNK2 monoclonal antibody as negative control (30 ng/ml). B: Spectrophotometric measurements of mitochondrial NADH-cytochrome c reductase activity of IB3-1 cells treated with increasing concentrations of anti-IL-1β blocking antibody (0, 1, 5, 15 and 30 ng/ml). C: Spectrophotometric measurements of mitochondrial NADH-cytochrome c reductase activity of IB3-1 cells treated with increasing concentrations of IL-1 receptor antagonist (0, 1, 5, 10 and 50 ng/ml). Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal IB3-1 cells.</p

JNK inhibition.

<p>IB3-1 cells preincubated for 24 h in serum free media were additionally incubated for 24 h with increasing concentrations of the JNK inhibitor SP600125 (1, 5, 10 and 20 µM) and the mCx-I activity was measured by using BN-PAGE and spectrophotometry. A: mCx-I in-gel activity (IGA) and mCx-III (WB). B: Densitometric quantification and statistical analysis of the results shown in panel A. IGA of mCx-I was calculated as the ratio mCx-I (IGA)/mCx-III (WB). C: Spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity in samples obtained from the same experiments of panel A, expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal IB3-1 cells.</p

P38 and NF-κB activation in S9 and IB3-1 cells.

<p>Cells were preincubated 24-1β (5ng/ml), anti-IL-1β blocking antibody (Ab-IL-1β) (30 ng/ml), p38 inhibitor SB203580 (p38 Inh) (5 µM), or IKK inhibitor III (IKK Inh) (2 µM), for another 24 h in serum-free media. A: Representative WB of phospho-p38 (p-p38) and total p38 of whole cellular lysates from S9 an IB3-1 cells. B: Densitometric quantification and statistical analysis of p38 phosphorylation status (calculated as the p-p38/p38 ratios for each experimental condition). C: Representative WB of NF-κB p65 and histone H1 from nuclear extracts of S9 and IB3-1 cells. D: Densitometric quantification and statistical analysis of the results shown in panel C (calculated as p65/histone ratio for each experimental condition). E: Representative WB of IκB-α and actin of whole cellular lysates from S9 and IB3-1 cells. F: Densitometric quantification and statistical analysis of the results shown in panel C (calculated as IκB-α/actin ratio for each experimental condition). The results were expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data are expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared to basal IB3-1 cells.</p

Il-1β secretion, Mitochondrial NADH cytochrome c reductase activity and ROS levels in stable CFTR knock down cells.

<p>Caco-2/pRSctrl cells (transfected with pRS control) and Caco-2/pRS26 cells (transfected with the shRNA pRS26) were preincubated 24 h in serum free media. A: Immune-dot blotting quantification of the IL-1β present in culture media. The results were expressed as pg/ml. B: Cells were treated for additional 24 h, as indicated. IL-1β: IL-1β (5 ng/ml); Ab-IL-1β: anti-IL-1β blocking antibody (30 ng/ml); Ab-Ctrl: anti-Histone H1 monoclonal antibody as negative control (30 ng/ml); p38 Inh: p38 inhibitor SB203580 (5 µM); IKK Inh: IKK inhibitor III (2 µM); JNK Inh: JNK inhibitor SP600125 (5 µM); MEK1/2 Inh: MAPK/AP-1 pathway inhibitor U0126 (5 µM). Spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity, expressed as percentage (%) relative to Caco-2/pRS26 control values. C: Mitochondrial ROS levels in the same experiments of panel B; the figure shows the MitoSOX fluorescence (5 µM for 10 min in Hank’s buffer). Results were expressed as percentage (%) relative to Caco-2/pRSctrl control values. D: Cellular ROS levels in the same experiments of panel B; the figure shows the DCF fluorescence (DCFH-DA 10 µM for 40 min in Hank’s buffer). Results were expressed as percentage (%) relative to Caco-2/pRSctrl control values. Measurements were performed in triplicate and data are expressed as mean ± SE of two independent experiments (n = 2). *indicates p<0.05 compared to Caco-2/pRS26 cells.</p

MEK1/2 inhibition.

<p>After 24-1 cells were incubated for 24 h with increasing concentrations of the MAPK/AP-1 pathway inhibitor U0126 (1, 5, 10 and 20 µM) and the mCx-I activity was measured by using BN-PAGE and spectrophotometry. A: mCx-I in-gel activity (IGA) and mCx-III Western blotting (WB). B: Densitometric quantification and statistical analysis of the results shown in panel A, calculated from three independent experiments (n = 3). IGA of mCx-I was calculated as the ratio mCx-I (IGA)/mCx-III (WB). C: Spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity in the same experiments of panel A, expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal IB3-1 cells.</p

Mitochondrial complex I in-gel activity (IGA) measured in cells expressing wt-CFTR.

<p>A: IGA of mitochondrial extracts from T84 cells after 24 h of treatment with 100 µM glibenclamide or 5 µM CFTR(inh)-172. B: Densitometric quantification of the results shown in panel A, expressed as % ratio of (mCx-I activity)/(protein load). C: IGA of mitochondrial extracts from T84 cells after 48 h of treatment with 100 µM glibenclamide or 5 µM CFTR(inh)-172. D: Densitometric queantification of C. E: IGA of the mCx-I from Caco-2 cells after 48 h of treatment with 5 µM GlyH101 or 5 µM CFTR(inh)-172, and WB of the mCx-III subunit UQCRC1, as internal standard. F: Densitometric quantification of the results shown in E expressed as % ratio of (mCx-I IGA)/UQCRC1(a.u.). The activity of mCx-I in T84 and Caco-2 cells treated with the same amount of DMSO (0.1%) was considered as 100%. Measurements in T84 cells were performed in duplicate and data are expressed as mean ± SE of three independent experiments (n = 3). Caco-2 cells results were obtained in triplicate and expressed as mean ± SE of three independent experiments (n = 3). * indicates p<0.05, as compared with control cells (ANOVA and Turkey's test).</p