Expression Of Cystic Fibrosis Transmembrane Regulator Research Articles

Ivacaftor therapy post myocardial infarction augments systemic inflammation and evokes contrasting effects with respect to tissue inflammation in brain and lung

Acquired cystic fibrosis transmembrane regulator (CFTR) dysfunctions have been associated with several conditions, including myocardial infarction (MI). Here, CFTR is downregulated in brain, heart, and lung tissue and associates with inflammation and degenerative processes. Therapeutically increasing CFTR expression attenuates these effects. Whether potentiating CFTR function yields similar beneficial effects post-MI is unknown. The CFTR potentiator ivacaftor is currently in clinical trials for treatment of acquired CFTR dysfunction associated with chronic obstructive pulmonary disease and chronic bronchitis. Thus, we tested ivacaftor as therapeutic strategy for MI-associated target tissue inflammation that is characterized by CFTR alterations.MI was induced in male C57Bl/6 mice by ligation of the left anterior descending coronary artery. Mice were treated with ivacaftor starting ten weeks post-MI for two consecutive weeks.Systemic ivacaftor treatment ameliorates hippocampal neuron dendritic atrophy and spine loss and attenuates hippocampus-dependent memory deficits occurring post-MI. Similarly, ivacaftor therapy mitigates MI-associated neuroinflammation (i.e., reduces higher proportions of activated microglia). Systemically, ivacaftor leads to higher frequencies of circulating Ly6C+ and Ly6Chi cells compared to vehicle-treated MI mice. Likewise, an ivacaftor-mediated augmentation of MI-associated pro-inflammatory macrophage phenotype characterized by higher CD80-positivity is observed in the MI lung. In vitro, ivacaftor does not alter LPS-induced CD80 and tumor necrosis factor alpha mRNA increases in BV2 microglial cells, while augmenting mRNA levels of these markers in mouse macrophages and differentiated human THP-1-derived macrophages.Our results suggest that ivacaftor promotes contrasting effects depending on target tissue post-MI, which may be largely dependent on its effects on different myeloid cell types.

A Comprehensive and Updated Review of MicroRNA Pathogenesis and Gene Expression in Cystic Fibrosis

MicroRNAs (miRNAs) are short (~22 nucleotides) non-coding sequences that regulate gene expression. It has been recently identified that miR-145 regulates cystic fibrosis transmembrane regulator (CFTR) expression, and response to first-generation miRNAs is recognized as major biomedical research in CF. Abnormal miRNA activity causes numerous diseases, thus the critical role of miRNA is to focus on miRNA bindings to target proteins. miRNA classifications depend on their genomic location and gene structure, and they include intergenic and intronic regions. Intronic miRNA location is in the introns of annotated genes. Intergenic and intronic miRNA genes are controlled with their promoters. Some miRNAs negatively regulate the cystic fibrosis transmembrane conductance regulator (CFTR) gene expression (e.g., miR-101) in adult lung cells and have no effect on fetal epithelial cells. MiR-145, miR-223, and miR-494 directly regulate the CFTR gene expression. MiR-509-3p and miR-494 regulate CFTR more strongly than the other miRNAs. MiR-138 enhances CFTR channels on the cell surface of epithelial cells. Polymorphisms in the 3′-UTR region of CFTR decrease the expression of CFTR. Mutations in the CF (CFTR) gene, located on chromosome 7, diminish ion transport. The consequent loss of chloride and bicarbonate secretion and absorption lead to a multi-organ disease called CF marked by mucus inspissation, chronic inflammation, and tissue destruction. The most affected organs are the lung and pancreas, but the liver, bowel, sinuses, and vas deferens, among others, are prominently involved as well. The loss of CFTR also results in an altered miRNA profile that regulates the response to therapy and disease pathogenesis. This review summarizes miRNA alterations in the CF tissue, the role of miRNA in disease pathogenesis, and the therapeutic opportunity for oligotherapeutic manipulation to improve CF outcomes.

Restoring myocardial infarction-induced long-term memory impairment by targeting the cystic fibrosis transmembrane regulator.

Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial infarction (MI). MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using invitro and invivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention. Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) invitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression invitro rescues the LCM-mediated down-regulation of PSD-95. Invivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window. Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI. Knut and Alice Wallenberg Foundation [F 2015/2112]; Swedish Research Council [VR; 2017-01243]; the German Research Foundation [DFG; ME 4667/2-1]; Hjärnfonden [FO2021-0112]; The Crafoord Foundation; Åke Wibergs Stiftelse [M19-0380], NMMP 2021 [V2021-2102]; the Albert Påhlsson Research Foundation; STINT [MG19-8469], Lund University; Canadian Institutes of Health Research [PJT-153269] and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.

Cystic fibrosis transmembrane regulator correction attenuates heart failure-induced lung inflammation.

Heart failure (HF) affects 64 million people worldwide. Despite advancements in prevention and therapy, quality of life remains poor for many HF patients due to associated target organ damage. Pulmonary manifestations of HF are well-established. However, difficulties in the treatment of HF patients with chronic lung phenotypes remain as the underlying patho-mechanistic links are still incompletely understood. Here, we aim to investigate the cystic fibrosis transmembrane regulator (CFTR) involvement in lung inflammation during HF, a concept that may provide new mechanism-based therapies for HF patients with pulmonary complications. In a mouse model of HF, pharmacological CFTR corrector therapy (Lumacaftor (Lum)) was applied systemically or lung-specifically for 2 weeks, and the lungs were analyzed using histology, flow cytometry, western blotting, and qPCR. Experimental HF associated with an apparent lung phenotype characterized by vascular inflammation and remodeling, pronounced tissue inflammation as evidenced by infiltration of pro-inflammatory monocytes, and a reduction of pulmonary CFTR+ cells. Moreover, the elevation of a classically-activated phenotype of non-alveolar macrophages coincided with a cell-specific reduction of CFTR expression. Pharmacological correction of CFTR with Lum mitigated the HF-induced downregulation of pulmonary CFTR expression and increased the proportion of CFTR+ cells in the lung. Lum treatment diminished the HF-associated elevation of classically-activated non-alveolar macrophages, while promoting an alternatively-activated macrophage phenotype within the lungs. Collectively, our data suggest that downregulation of CFTR in the HF lung extends to non-alveolar macrophages with consequences for tissue inflammation and vascular structure. Pharmacological CFTR correction possesses the capacity to alleviate HF-associated lung inflammation.

Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure.

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P’s availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.

Pathophysiology of Hyperechogenic Bowel in Congenitally Human Cytomegalovirus Infected Fetuses.

Hyperechogenic bowel (HB) is a nonspecific ultrasound finding that can be associated with human cytomegalovirus (CMV) congenital infection. In this study, we investigated HB pathophysiology in CMV-infected fetuses. We examined small and large intestine as well as pancreas in 8 fetuses at 22 weeks of gestation with congenital CMV infection. Ultrasound findings showed 4 fetuses with HB and 4 without. As negative group, 4 fetuses without CMV infection and without HB were studied. Immunohistochemistry for CMV, lymphocytic infiltrate, B-cell leukemia/lymphoma-2 (bcl-2), CD-117, cystic fibrosis transmembrane regulator (CFTR) were performed. HB fetuses showed multiple and sequential CMV-positive ganglion cells of Auerbach’s myenteric plexus. In the ganglia, bcl-2 was weakly expressed representing a reduced neuronal functionality. CD-117 revealed a regular distribution of Cajal cells, the pacemakers of intestinal contractility. Pancreas showed normal CFTR staining, indicating a preserved exocrine secretion, thus unlikely a contributory factor in HB. In CMV-infected fetuses without HB, CMV-positive cells were scatteredly found in ganglion cells and bcl-2 was strongly expressed. Intestinal CD-117 and pancreatic CFTR expression were similar to fetuses with HB. In conclusion, fetal CMV infection of the bowel may lead to peristalsis impairment (paralytic ileus) due to intestinal plexus involvement, which at ultrasound appeared as HB.

Targeting cerebrovascular sphingosine‐1‐phosphate signaling to treat heart failure‐induced brain dysfunction

Chronic cardiovascular diseases increasingly emerge as risk factors for neurodegeneration. Heart failure for instance, induces a chronic reduction in cerebral perfusion. Such chronic brain perfusion deficits are frequently accompanied by cognitive decline, which not only seriously impacts the patients’ quality of life but also reduces treatment compliance. Statistically, cognitive impairment is 5 times more prevalent in heart failure patients relative to the age‐matched general population. We previously demonstrated that heart failure associates to augmented signaling of a potent bioactive phospholipid called sphingosine‐1‐phosphate (S1P). During heart failure, such enhanced S1P signaling roots from dysfunctional cystic fibrosis transmembrane regulator (CFTR) expression and activity. CFTR controls the bioavailability of cerebrovascular S1P and hence, its pro‐constrictive effects. In heart failure, reduced microvascular CFTR expression correlates with enhanced myogenic tone and reduced cerebral blood flow.In this study, we investigated the therapeutic potential of CFTR corrector compounds to normalize cerebrovascular S1P signaling and thereby, improve neurological function during experimental heart failure in mice.Systemic CFTR corrector treatment stabilized cerebrovascular CFTR expression, improved myogenic tone and rectified deficient cerebral perfusion in heart failure, without altering the systemic parameters of cardiac output or total peripheral resistance. This normalization associated to recovery of neuronal integrity (i.e., dendrite morphology and dendritic spine density) and cognitive function. The heart failure‐associated dendrite atrophy (i.e., a 22% reduction in dendrite length) was not altered in mice treated with CFTR corrector compound. Longitudinal pre‐treatment (i.e., at 10 weeks post‐infarction) and post‐treatment analyses of the same vehicle‐ and CFTR corrector‐treated HF mice confirmed that CFTR corrector treatment reversed the impaired rhino‐cortical‐dependent, short‐term retention of object familiarity caused by heart failure.Our results put forward a new mechanistic understanding of heart failure‐associated brain complications, and CFTR correctors as potential treatment option for heart failure‐associated cognitive decline.Support or Funding InformationThis work is supported by the Knut & Alice Wallenberg Foundation and the Swedish Research Council.

Cyclic compression increases F508 Del CFTR expression in ciliated human airway epithelium.

The mechanisms by which transepithelial pressure changes observed during exercise and airway clearance can benefit lung health are challenging to study. Here, we have studied 117 mature, fully ciliated airway epithelial cell filters grown at air-liquid interface grown from 10 cystic fibrosis (CF) and 19 control subjects. These were exposed to cyclic increases in apical air pressure of 15 cmH2O for varying times. We measured the effect on proteins relevant to lung health, with a focus on the CF transmembrane regulator (CFTR). Immunoflourescence and immunoblot data were concordant in demonstrating that air pressure increased F508Del CFTR expression and maturation. This effect was in part dependent on the presence of cilia, on Ca2+ influx, and on formation of nitrogen oxides. These data provide a mechanosensory mechanism by which changes in luminal air pressure, like those observed during exercise and airway clearance, can affect epithelial protein expression and benefit patients with diseases of the airways.

Testosterone enhances expression and functional activity of epithelial sodium channel (ENaC), cystic fibrosis transmembrane regulator (CFTR) and sodium hydrogen exchanger (NHE) in vas deferens of sex-steroid deficient male rats

Effects of testosterone on expression and functional activity of ENaC, CFTR and NHE in vas deferens were investigated. MethodsOrchidectomized, adult male rats were given 125 and 250 μg/kg/day testosterone subcutaneously, with or without flutamide and finasteride for seven consecutive days. At the end of the treatment, rats were anesthetized and vas deferens were perfused. Changes in vas deferens fluid secretion rate, pH, HCO3−, Cl− and Na+ concentrations were recorded in the presence of amiloride and Cftr inh-172. Rats were then sacrificed and vas deferens were harvested and subjected for molecular biological analysis. ResultsTestosterone treatment caused the fluid pH and HCO3− concentrations to decrease but secretion rate, Cl− and Na+ concentrations to increase, where upon amiloride administration, the pH and HCO3− concentration increased but Cl− and Na+ concentrations further increased. In testosterone-treated rats, administration of Cftr inh-172 caused all fluid parameters to decrease. In testosterone-treated rats co-administered with flutamide or finasteride, pH and HCO3− concentration increased but fluid secretion rate, Cl− and Na+ concentrations decreased and these parameters were not affected by amiloride or Cftr inh-172 administration. Under testosterone influence, CFTR and γ-ENaC were highly expressed at the apical membrane while NHE-1 and 4 were highly expressed at the basolateral membrane of vas deferens epithelium. Meanwhile, NHE-2 and 3 were highly expressed at the apical membrane. ConclusionsDifferential expression of ENaC, CFTR and NHE in vas deferens under testosterone influence indicated the important role of these transporters in creating optimal fluid microenvironment that is essential for preserving male fertility.

Abstract 4716: Stemness markers in colorectal cancer: Analysis in a racially-diverse population

Abstract The diagnosis of colorectal cancer (CRC) is based on tumor-node-metastasis staging, a weak staging system due to different genetic and epigenetic backgrounds. Biological markers improve early detection and guide clinicians in subsequent therapies. Stemness molecules have the potential to identify patients at high risk of developing aggressive cancers. Herein, Zinc finger E-box binding homeobox 1 (ZEB1), Trefoil factor 3 (TFF3), Hepatoma Up-Regulate Protein (HURP), Mucin 2 (MUC2), and Cystic fibrosis transmembrane regulator (CFTR) are assessed as potential prognostic biomarkers in CRC. There were a total of (N=56) cases included in this study which were assessed by a pathologist, out of them 32 were African Americans (AA) and 24 Caucasian Americans (CA). The median follow-up for 42 surviving patients were noted to be 4.5 years (range 2.5 - 7.5 years). A tissue tumor microarray (TMA) was created using tumor stage-matched CRC tissues from AA and CA. The TMA was stained with each biomarker using immunohistochemistry (IHC) and consisted of 132 cores including both controls and tumors. The individual staining score ranged from (0-3) for both area and intensity. The product of area and intensity was used as the final score which ranged from (0-9) with 0 defining no expression and 9 as high expression. We evaluated the association for expression of all the five biomarkers with disease-free survival (DFS) in AA and CA separately. Stratification of the cohort by race revealed that in CA, high ZEB1 expression (nuclear and cytoplasmic) was associated with poor DFS (p= 0.015 for cytoplasm, p= 0.116 for nucleus). ZEB1 expression was not found to be a significant predictor of DFS in AA who are usually known to have worse CRC outcomes. These findings suggest that expression of ZEB1 may represent a marker for CRC prognosis, particularly in CA. High nuclear TFF3 expression in AA was found to be marginally associated with poorer DFS (p=0.089). However, the cytoplasmic expression of TFF3 was not significantly associated with DFS. Both the cytoplasmic and nuclear expression of HURP, MUC2, and CFTR among the AA and CA did not significantly correlate with DFS. Overall, we have identified two stemness molecules, ZEB1 and TFF3, with potential as markers for aggressiveness in CRC. Our findings also provide evidence of a possible biological basis for ethnicity-related differences with regards to stemness and regulatory factors of CRC aggressiveness. ZEB1 can contribute to be a novel biomarker to predict the prognosis of CRC in CA, and TFF3 will be a prognostic marker for AA. Future goals include collecting additional samples and follow-up of the patients to accurately estimate the prognosis. In addition, we will expand the project to the cellular and molecular basis underlining our findings. Funding sources: PCRP W81XWH-14-1-0151, UMMC Medical Student Research Program, and UMMC Office of Research. Citation Format: Ingrid C. Espinoza, Amit Reddy, Xu Zhang, Eldrin Bhanat, Logan Fair, Joy King, Elizabeth Tarsi, Tara Craft, Jesus Monico, Charulochana Subramony, Xinchun Zhou, Roy Duhe, Christopher Lahr, Christian Gomez. Stemness markers in colorectal cancer: Analysis in a racially-diverse population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4716. doi:10.1158/1538-7445.AM2017-4716

An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

SUMMARYCystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients’ cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls – 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.

Isoflavone genistein inhibits estrogen-induced chloride and bicarbonate secretory mechanisms in the uterus in rats.

We hypothesized that genistein could affect the chloride (Cl- ) and bicarbonate (HCO3- ) secretory mechanisms in uterus. Ovariectomized female rats were given estradiol or estradiol plus progesterone with 25, 50, or 100mg/kg/day genistein. Following completion of the treatment, uterine fluid Cl- and HCO3- concentrations were determined by in vivo uterine perfusion. Uteri were subjected for molecular biological analysis (Western blot, qPCR, and immunohistochemistry) to detect levels of expression of Cystic Fibrosis transmembrane regulator (CFTR), Cl- /HCO3- exchanger (SLC26a6), Na+ /HCO3- cotransporter (SLC4a4), and estrogen receptor (ER)-α and β. Coadministration of genistein resulted in decrease in Cl- and HCO3- concentrations and expression of CFTR, SLC26a6, SLC4a4, and ER-α and ER-β in the uteri of estradiol-treated rats. In estradiol plus progesterone-treated rats, a significant increase in the above parameters were observed following high-dose genistein treatment except for the SLC24a4 level. In conclusion, genistein-induced changes in the uterus could affect the reproductive processes that might result in infertility.

CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

Testosterone regulates levels of cystic fibrosis transmembrane regulator, adenylate cyclase, and cAMP in the seminal vesicles of orchidectomized rats

Secretions of chloride (Cl−)- and bicarbonate (HCO3−)-rich fluid by the seminal vesicles could involve cystic fibrosis transmembrane regulator (CFTR), which activity can be stimulated by cAMP generated from the reaction involving adenylate cyclase (AC). In this study, we investigated levels of CFTR, AC, and cAMP in the seminal vesicles under testosterone influence. Orchidectomized adult male rats received 7-day treatment with 125 or 250 μg/kg/day of testosterone with or without flutamide or finasteride. At the end of the treatment, animals were sacrificed and seminal vesicles were harvested for analyses of CFTR and AC protein expression level by Western blotting. Distribution of CFTR and AC in seminal vesicles was observed by immunohistochemistry. Levels of cAMP and dihydrotestosterone in seminal vesicle homogenates were measured by ELISA. Cystic fibrosis transmembrane regulator, AC, and cAMP levels increased with increasing doses of testosterone (P < 0.05 compared to nontreated orchidectomized rats). Cystic fibrosis transmembrane regulator and AC were expressed at the apical membrane of the epithelium lining the seminal vesicle lumen with higher expression levels observed in testosterone-treated rats than in non-treated orchidectomized rats (P < 0.05). The inhibitory effects of flutamide or finasteride on these parameters were greater in 250 μg/kg/day testosterone-treated rats than their effects in 125 μg/kg/day testosterone-treated rats. Higher dihydrotestosterone levels were observed in seminal vesicle homogenates after treatment with 250 μg/kg/day than with 125 μg/kg/day of testosterone (P < 0.05). Increased levels of CFTR, AC, and cAMP in seminal vesicles might contribute toward an increase in Cl− and HCO3− concentrations in the seminal fluid as reported under testosterone influence.

Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs.

Lung disease is a major cause of death in the United States, with current therapeutic approaches serving only to manage symptoms. The most common chronic and life-threatening genetic disease of the lung is cystic fibrosis (CF) caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). We have generated induced pluripotent stem cells (iPSCs) from CF patients carrying a homozygous deletion of F508 in the CFTR gene, which results in defective processing of CFTR to the cell membrane. This mutation was precisely corrected using CRISPR to target corrective sequences to the endogenous CFTR genomic locus, in combination with a completely excisable selection system, which significantly improved the efficiency of this correction. The corrected iPSCs were subsequently differentiated to mature airway epithelial cells where recovery of normal CFTR expression and function was demonstrated. This isogenic iPSC-based model system for CF could be adapted for the development of new therapeutic approaches.

Possible Recovery of Manifestation of Prolactin Receptor and Some of Its Target Proteins in the Liver and Kidney Cells of Female Rats after Relief of Cholestasis Complicated and Not Complicated by Hyperprolactinemia.

Immunohistochemistry with semi-quantitative analysis of computer images showed that prolactin receptor and cystic fi brosis transmembrane regulator (CFTR) in cholangiocytes of female rats elevated in cholestasis quickly respond to its relief. The effect of hyperprolactinemia on the extent of return of these proteins to baseline was different. Decompression of the bile duct abolishes the negative effect of hyperprolactinemia on CFTR expression and its positive effect on mrp3 expression in the proximal renal tubules. In renal medulla, mrp2 expression decreased when cholestasis was induced against the background of hyperprolactinemia and increases after its removal. Prolactin receptors and CFTR in cholangiocytes are most susceptible to the decrease in bile duct pressure. Changes in the expression of the studied proteins after cholestasis relief are apparently associated with attenuated toxicity of the products removed by the kidneys, which abolishes the effects of prolactin.

Testosterone decreases fluid and chloride secretions in the uterus of adult female rats via down-regulating cystic fibrosis transmembrane regulator (CFTR) expression and functional activity

ObjectivesEstrogen is known to stimulate uterine fluid and Cl− secretion via CFTR. This study investigated testosterone effect on these changes in a rat model. MethodsOvariectomized adult female rats received estrogen for five days or estrogen for three days followed by two days peanut oil or testosterone either alone or in the presence of flutamide or finasteride. At the end of treatment, uteri were perfused with perfusate containing CFTRinh-172. The rate of fluid and Cl− secretion were determined. Dose-dependent effect of testosterone and effect of forskolin on fluid secretion rate were measured. Animals were sacrificed and uteri were removed for CFTR protein and mRNA expression analyses, histology and cAMP measurement. Morphology of uterus, levels of expression of CFTR protein and mRNA and distribution of CFTR protein were observed. ResultsEstrogen causes increase while testosterone causes decrease in uterine fluid and Cl− secretions. The effects of estrogen but not testosterone were antagonized by CFTRinh-172. Luminal fluid volume and apical expression of CFTR in the luminal epithelia were highest under estrogen and lowest under testosterone influences. Similar changes were observed in CFTR protein and mRNA expressions. Uterine cAMP level was highest under estrogen and lowest under testosterone influence. Forskolin increases fluid secretion rate in estrogen but not in testosterone-treated animals. Testosterone effects were dose-dependent and were antagonized by flutamide however, not finasteride. ConclusionsTestosterone inhibition of estrogen-induced uterine fluid and Cl− secretion occurs via inhibition of CFTR expression and functional activities. These changes could explain the adverse effects of testosterone on fertility.

Manifestation of cystic fibrosis transmembrane regulator (CFTR) in hepatic ductal structures and renal tubules of female rats with experimental cholestasis of pregnancy.

Studies by the immunohistochemical method with semiquantitative analysis of images showed that hyperprolactinemia stimulated CFTR protein manifestation in the bile ducts of female rats, which was clearly expressed in experimental cholestasis of pregnancy. The expression of CFTR in the renal tubules was reduced in hyperprolactinemia under conditions of normal liver function and in cholestasis of pregnancy. Significant positive correlations between CFTR, prolactin receptor, and multiple drug resistance protein 3 were detected in the bile ducts, but not in the renal tubules. Presumably, prolactin has a direct effect on CFTR expression in the bile ducts and indirect effect in the renal tubules. Changes in CFTR protein manifestation in the hepatic ductal structures and renal tubules in experimental pregnancy cholestasis could aggravate the disease.

Genistein-induced fluid accumulation in ovariectomised rats' uteri is associated with increased cystic fibrosis transmembrane regulator expression

OBJECTIVE:High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator.METHODS:Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry.RESULTS:The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780.CONCLUSION:Genistein-induced luminal fluid accumulation in ovariectomised rats' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after menopause.

S-nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface

S-nitrosothiols (SNOs) are endogenous signaling molecules with a broad spectrum of beneficial airway effects. SNOs are normally present in the airway, but levels tend to be low in cystic fibrosis (CF) patients. We and others have demonstrated that S-nitrosoglutathione (GSNO) increases the expression, maturation, and function of wild-type and mutant F508del cystic fibrosis transmembrane conductance regulator (CFTR) in human bronchial airway epithelial (HBAE) cells. We hypothesized that membrane permeable SNOs, such as S-nitrosoglutathione diethyl ester (GNODE) and S-nitroso-N-acetyl cysteine (SNOAC) may be more efficient in increasing the maturation of CFTR. HBAE cells expressing F508del CFTR were exposed to GNODE and SNOAC. The effects of these SNOs on the expression and maturation of F508del CFTR were determined by cell surface biotinylation and Western blot analysis. We also found for the first time that GNODE and SNOAC were effective at increasing CFTR maturation at the cell surface. Furthermore, we found that cells maintained at low temperature increased cell surface stability of F508del CFTR whereas the combination of low temperature and SNO treatment significantly extended the half-life of CFTR. Finally, we showed that SNO decreased the internalization rate of F508del CFTR in HBAE cells. We anticipate identifying the novel mechanisms, optimal SNOs, and lowest effective doses which could benefit cystic fibrosis patients.