Disruption of Interleukin-1β Autocrine Signaling Rescues Complex I Activity and Improves ROS Levels in Immortalized Epithelial Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1B. We have previously shown that IL-1B, at low doses (~30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-KB signaling. However, at higher doses (>2.5 ng/ml, ~150 pM), IL-1B inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1B in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1B (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-KB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1B blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ~50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1B, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels.Fil: Clauzure, Mariangeles. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Valdivieso, Ángel Gabriel. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Massip Copiz, María Macarena. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Schulman, Gustavo. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Teiber, Maria Luz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Tomás A. Santa-Coloma. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels

The mitochondrial complex I activity is reduced in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function

Abstract: Cystic fibrosis (CF) is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I). Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA) of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines) compared with CFDE/ 6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR). Moreover, colon carcinoma T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh)-172 or GlyH101) or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells

The expression of the mitochondrial encoded gene ND4 is downregulated in cystic fibrosis

Abstract: Cystic fibrosis (CF) is a disease produced by mutations in the CFTR channel. We have previously reported that the CFTR chloride transport activity regulates the differential expression of several genes, including SRC. Here we report that MT-ND4, a mitochondrial gene encoding a subunit of the mitochondrial Complex I (mtCx-I), is also a CFTR-dependent gene. A reduced expression of MT-ND4 was observed in CFDE cells (derived from a CF patient) when compared to CFDE cells ectopically expressing wild type CFTR. The differential expression of MT-ND4 in CF was confirmed by PCR. In situ hybridizations of deparaffinized human lung tissue slices derived from wt-CFTR or CF patients also showed downregulation of ND4 in CF. In addition, glibenclamide or CFTR(inh)-172 (CFTR chloride transport inhibitors) reduced MT-ND4 expression in cells expressing wt CFTR. These results suggest that the CFTR chloride transport activity indirectly up-regulates MT-ND4 expression

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

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

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