IFN-γ and IL-10: seric and placental profile during pig gestation Seric and placental cytokines in pig gestation.

Concentration of interferon-gamma and interleukin-10 in maternal serum and in maternal and fetal porcine placental extracts from different gestation periods was determined. Crossbred pigs' placental samples of 17, 30, 60, 70, and 114 days gestation and non-pregnant uteri were used. Interferon-gamma concentration was increased at the placental interface at 17 days, in maternal and fetal placenta, and decreased significantly in the remaining gestation periods. Interferon-gamma showed a peak in serum at 60 days. Regarding interleukin-10, placental tissue concentrations were unaltered, there were no significant differences with non-gestating uteri samples. In serum interleukin-10 increased at 17, 60, and 114 days gestation. At 17 days there are uterus structural and molecular changes that allow the embryos implantation and placenta development. The presence of interferon-gamma found at this moment in the interface would favor that placental growth. Moreover, its significant increase in serum at 60 days, would generate a proinflammatory cytokine pattern that facility the placental remodeling characteristic of this moment of porcine gestation. On the other hand, a significant interleukin-10 increase in serum at 17, 60 and 114 days could indicate its immunoregulatory role at a systemic level during pig gestation.

Integrins and ligands, are correlated at pig placental interface during pregnancy?

In the present work, we emphasize the studies about integrins and their receptors in pig placental interface at different times of gestation. Uterine placental interface (n=24) of 17-, 30-, 60- and 70-days gestation (dg) and non-pregnant uterus (n=4) of crossbred sows were used. The presence of αvß3 and α5ß1 integrins, and their ligands fibronectin (FN), and osteopontin (OPN) were detected by immunohistochemistry, and the immunolabelled area percentage (IAP) and the optical density (OD) were determined. The integrins and its ligands analyzed have presented peaks of expression in early and mid-gestation, both in IAP and the OD area decreasing at 70 dg. These temporal changes showed us that the molecules studied in this work participate in embryo/feto-maternal attachment, variably. Besides, we found a significant correlation both in the intensity and in the extension of immunostaining for trophoblastic FN and endometrial αvß3, and trophoblastic OPN and endometrial α5ß1, throughout the entire pig pregnancy. At late gestation, take place a notable placental remodelation with subsequent removal or renewal of folds at the uterine-placental interface that results in the loss of focal adhesions. The decrease of the expression of some integrins and their ligands in late gestation, particularly at 70 dg, would demonstrate that there would be other adhesion molecules and other ligands that could be participating in the establishment of the maternal-fetal interface.

Histamine H4 Receptor Agonism Induces Antitumor Effects in Human T-Cell Lymphoma.

The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 µM) in combination with histamine (10 µM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses.

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

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.

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-1ß (IL-1ß), 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-1ß mRNA expression and partially the IL-1ß 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-1ß mRNA response to Cl- and the IL-1ß secretion, interrupting the autocrine IL-1ß loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1ß, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1ß itself, through autocrine signalling.

Epidermal growth factor receptor activity upregulates lactate dehydrogenase A expression, lactate dehydrogenase activity, and lactate secretion in cultured IB3-1 cystic fibrosis lung epithelial cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It has been postulated that reduced HCO3- transport through CFTR may lead to a decreased airway surface liquid pH. In contrast, others have reported no changes in the extracellular pH (pHe). We have recently reported that in carcinoma Caco-2/pRS26 cells (transfected with short hairpin RNA for CFTR) or CF lung epithelial IB3-1 cells, the mutation in CFTR decreased mitochondrial complex I activity and increased lactic acid production, owing to an autocrine IL-1ß loop. The secreted lactate accounted for the reduced pHe, because oxamate fully restored the pHe. These effects were attributed to the IL-1ß autocrine loop and the downstream signaling kinases c-Src and JNK. Here we show that the pHe of IB3-1 cells can be restored to normal values (∼7.4) by incubation with the epidermal growth factor receptor (EGFR, HER1, ErbB1) inhibitors AG1478 and PD168393. PD168393 fully restored the pHe values of IB3-1 cells, suggesting that the reduced pHe is mainly due to increased EGFR activity and lactate. Also, in IB3-1 cells, lactate dehydrogenase A mRNA, protein expression, and activity are downregulated when EGFR is inhibited. Thus, a constitutive EGFR activation seems to be responsible for the reduced pHe in IB3-1 cells.

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

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-1ß 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.

IL-1ß, IL-2 and IL-4 concentration during porcine gestation.

In pigs, given the type of epitheliochorial and non-invasive placenta, the trophoblast is in intimate contact with maternal tissues. The dialogue established between the conceptus and the endometrium involves, among others, the immune system, which minimizes the chances of rejection of the embryo and promotes the establishment of pregnancy. The aim of this work was to determine the concentration of IL-1ß, IL-2 and IL-4 in sera and in extracts of maternal and fetal placenta from sows of different gestational periods. Reproductive tracts from 23 crossbreed sows, between 30 and 114 days of gestation (dg), and from 8 non-pregnant sows were used. The concentration of the cytokines was determined by ELISA. IL-1ß, IL-2 and IL-4 demonstrated a similar pattern of concentration at the placental interface and serum; they were found elevated in tissues at 30 and 60-70 dg, and significantly decreased at term, period in which the cytokines were significantly increased in serum. These results show that IL-1ß, IL-2, and IL-4 are differentially modulated during pregnancy and at term, and suggest an important role of these cytokines in defining the proinflammatory stage of these periods.

Impairment of CFTR activity in cultured epithelial cells upregulates the expression and activity of LDH resulting in lactic acid hypersecretion.

Mutations in the gene encoding the CFTR chloride channel produce cystic fibrosis (CF). CF patients are more susceptible to bacterial infections in lungs. The most accepted hypothesis sustains that a reduction in the airway surface liquid (ASL) volume favor infections. Alternatively, it was postulated that a reduced HCO3- transport through CFTR leads to a decreased ASL pH, favoring bacterial colonization. The issue is controversial, since recent data from cultured primary cells and CF children showed normal pH values in the ASL. We have reported previously a decreased mitochondrial Complex I (mCx-I) activity in cultured cells with impaired CFTR activity. Thus, we hypothesized that the reduced mCx-I activity could lead to increased lactic acid production (Warburg-like effect) and reduced extracellular pH (pHe). In agreement with this idea, we report here that cells with impaired CFTR function (intestinal Caco-2/pRS26, transfected with an shRNA-CFTR, and lung IB3-1 CF cells) have a decreased pHe. These cells showed increased lactate dehydrogenase (LDH) activity, LDH-A expression, and lactate secretion. Similar effects were reproduced in control cells stimulated with recombinant IL-1ß. The c-Src and JNK inhibitors PP2 and SP600125 were able to increase the pHe, although the differences between control and CFTR-impaired cells were not fully compensated. Noteworthy, the LDH inhibitor oxamate completely restored the pHe of the intestinal Caco-2/pRS26 cells and have a significant effect in lung IB3-1 cells; therefore, an increased lactic acid secretion seems to be the key factor that determine a reduced pHe in these epithelial cells.

N-acetyl cysteine reverts the proinflammatory state induced by cigarette smoke extract in lung Calu-3 cells.

Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are lethal pulmonary diseases. Cigarette consumption is the main cause for development of COPD, while CF is produced by mutations in the CFTR gene. Although these diseases have a different etiology, both share a CFTR activity impairment and proinflammatory state even under sterile conditions. The aim of this work was to study the extent of the protective effect of the antioxidant N-acetylcysteine (NAC) over the proinflammatory state (IL-6 and IL-8), oxidative stress (reactive oxygen species, ROS), and CFTR levels, caused by Cigarette Smoke Extract (CSE) in Calu-3 airway epithelial cells. CSE treatment (100 µg/ml during 24 h) decreased CFTR mRNA expression and activity, and increased the release of IL-6 and IL-8. The effect on these cytokines was inhibited by N-acetyl cysteine (NAC, 5 mM) or the NF-kB inhibitor, IKK-2 (10 µM). CSE treatment also increased cellular and mitochondrial ROS levels. The cellular ROS levels were normalized to control values by NAC treatment, although significant effects on mitochondrial ROS levels were observed only at short times (5´) and effects on CFTR levels were not observed. In addition, CSE reduced the mitochondrial NADH-cytochrome c oxidoreductase (mCx I-III) activity, an effect that was not reverted by NAC. The reduced CFTR expression and the mitochondrial damage induced by CSE could not be normalized by NAC treatment, evidencing the need for a more specific reagent. In conclusion, CSE causes a sterile proinflammatory state and mitochondrial damage in Calu-3 cells that was partially recovered by NAC treatment.

Epiregulin (EREG) is upregulated through an IL-1ß autocrine loop in Caco-2 epithelial cells with reduced CFTR function.

CFTR is a cAMP-regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl- concentration, which in turn produces an increased interleukin-1ß (IL-1ß) secretion. The secreted IL-1ß then induces an autocrine positive feedback loop, further stimulating IL-1ß priming and secretion. Since IL-1ß can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco-2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco-2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR-impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL-1ß and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL-1ß loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco-2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco-2 CFTR-shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL-1ß autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl- , and IL-1ß.

CFTR modulates RPS27 gene expression using chloride anion as signaling effector.

In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Cl- concentrations ([Cl-]i), we observed several Cl--dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl- might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl-]i (using the Cl- fluorescent probe SPQ). The [Cl-]i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl- accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl- behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1ß receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1ß and JNK signaling downstream of Cl- in RPS27 modulation.

CFTR impairment upregulates c-Src activity through IL-1ß autocrine signaling.

Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to 'CFTR-corrected' S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1ß receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1ß→c-Src and IL-1ß→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1ß constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity.

Intracellular Chloride Concentration Changes Modulate IL-1ß Expression and Secretion in Human Bronchial Epithelial Cultured Cells.

Cystic fibrosis (CF) is caused by mutations in the CFTR gene, which encodes a cAMP-regulated chloride channel. Several cellular functions are altered in CF cells. However, it is not clear how the CFTR failure induces those alterations. We have found previously several genes differentially expressed in CF cells, including c-Src, MUC1, MTND4, and CISD1 (CFTR-dependent genes). Recently, we also reported the existence of several chloride-dependent genes, among them GLRX5 and RPS27. Here, varying the intracellular chloride concentration [Cl- ]i of IB3-1 CF bronchial epithelial cells, we show that IL-1ß mRNA expression and secretion are also under Cl- modulation. The response to Cl- is biphasic, with maximal effects at 75 mM Cl- . The regulation of the IL-1ß mRNA expression involves an IL-1ß autocrine effect, since in the presence of the IL-1ß receptor antagonist IL1RN or anti-IL-1ß blocking antibody, the mRNA response to Cl- disappeared. Similar effects were obtained with the JNK inhibitor SP600125, the c-Src inhibitor PP2 and the IKK inhibitor III (BMS-345541). On the other hand, the IL-1ß secretion is still modulated by Cl- in the presence of IL-1RN, IL-1ß blocking antibody, or cycloheximide, suggesting that Cl- is affecting the IL-1ß maturation/secretion, which in turn starts an autocrine positive feedback loop. In conclusion, the Cl- anion acts as a second messenger for CFTR, modulating the IL-1ß maturation/secretion. The results also imply that, depending on its intracellular concentration, Cl- could be a pro-inflammatory mediator. J. Cell. Biochem. 118: 2131-2140, 2017. © 2016 Wiley Periodicals, Inc.

The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes. METHODS: Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile. RESULTS: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM). CONCLUSION: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.

Disruption of interleukin-1ß 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-1ß. We have previously shown that IL-1ß, 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-1ß 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-1ß in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1ß (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-1ß 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-1ß 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-1ß, 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.

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.

Measurement of cystic fibrosis transmembrane conductance regulator activity using fluorescence spectrophotometry.

Cystic fibrosis (CF) is a frequent autosomal recessive disease caused by mutations that impair the CF transmembrane conductance regulator (CFTR) protein function. CFTR is a chloride channel activated by cyclic AMP (cAMP) via protein kinase A (PKA) and ATP hydrolysis. We describe here a method to measure CFTR activity in a monolayer of cultured cells using a fluorescence spectrophotometer and the chloride-sensitive probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Modifying a slice holder, the spectrophotometer quartz cuvette was converted in a perfusion chamber, allowing measurement of CFTR activity in real time, in a monolayer of T84 colon carcinoma cells. The SPQ Stern-Volmer constant (K(Cl(-))) for chloride in water solution was 115.0 ± 2.8M(-1), whereas the intracellular (K(Cl(-))) was 17.8 ± 0.8 M(-1), for T84 cells. A functional analysis was performed by measuring CFTR activity in T84 cells. The CFTR transport inhibitors CFTR(inh)-172 (5 µM) and glibenclamide (100 µM) showed a significant reduction (P<0.05) in CFTR activity. This simple method allows measuring CFTR activity in a very simple, reproducible, and sensitive way.