NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl– concentration

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl–) accumulation. The anion Cl–, acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl– concentration, showing maximal expression and activity at 75 mM Cl–, in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl–. The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl–-stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl–, a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl– and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl– effects are mediated by SGK1, in which under Cl– modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling.Fil: Clauzure, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina. Universidad Nacional de La Pampa; ArgentinaFil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Dugour, Andrea Vanesa. Fundación Pablo Cassará; ArgentinaFil: Mori, Consuelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Massip Copiz, María Macarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Aguilar, María Á.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Sotomayor, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Asensio, Cristian Jorge Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Figueroa, Juan M.. Fundación Pablo Cassará; ArgentinaFil: Santa Coloma, Tomás Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; 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

Identification and characterization of human PEIG-1/GPRC5A as a 12-Otetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene

Abstract: Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca2+ chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation

CFTR chloride channel activity modulates the mitochondrial morphology in cultured epithelial cells

Abstract: The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl− ) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3− 1 CF cells, and S9 (IB3− 1 expressing wt-CFTR), and C38 (IB3− 1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3− 1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3− 1 CF cells treated with Mdivi-1, an inhibitor of mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF

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

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

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

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

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