Downregulation Of Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor

Introduction and AimsCFTR, a Cl‐ channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals.MethodsMonolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively.ResultsTreatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p < 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p < 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl‐ secretory responses to the Ca2+‐ and cAMP‐dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt‐like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF‐κB, or inhibit its translocation to the nucleus. ChIP‐qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p <0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p < 0.01) and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p < 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist‐induced Cl‐ secretory responses across T84 cells mounted in Ussing chambers.ConclusionFXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease.

Trichomonas vaginalis infection impairs anion secretion in vaginal epithelium

Trichomonas vaginalis is a common protozoan parasite, which causes trichomoniasis associated with severe adverse reproductive outcomes. However, the underlying pathogenesis has not been fully understood. As the first line of defense against invading pathogens, the vaginal epithelial cells are highly responsive to environmental stimuli and contribute to the formation of the optimal luminal fluid microenvironment. The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel widely distributed at the apical membrane of epithelial cells, plays a crucial role in mediating the secretion of Cl− and HCO3−. In this study, we investigated the effect of T. vaginalis on vaginal epithelial ion transport elicited by prostaglandin E2 (PGE2), a major prostaglandin in the semen. Luminal administration of PGE2 triggered a remarkable and sustained increase of short-circuit current (ISC) in rat vaginal epithelium, which was mainly due to Cl− and HCO3− secretion mediated by the cAMP-activated CFTR. However, T. vaginalis infection significantly abrogated the ISC response evoked by PGE2, indicating impaired transepithelial anion transport via CFTR. Using a primary cell culture system of rat vaginal epithelium and a human vaginal epithelial cell line, we demonstrated that the expression of CFTR was significantly down-regulated after T. vaginalis infection. In addition, defective Cl− transport function of CFTR was observed in T. vaginalis-infected cells by measuring intracellular Cl− signals. Conclusively, T. vaginalis restrained exogenous PGE2-induced anion secretion through down-regulation of CFTR in vaginal epithelium. These results provide novel insights into the intervention of reproductive complications associated with T. vaginalis infection such as infertility and disequilibrium in vaginal fluid microenvironment.

CFTR plays an important role in the regulation of vascular resistance and high-fructose/salt-diet induced hypertension in mice

BackgroundThe pathophysiological roles of cystic fibrosis transmembrane-conductance regulator (CFTR) Cl− channels in the regulation of blood pressure (BP) remain controversial. Here we studied the function of CFTR Cl− channels in regulation of BP and in the high-fructose-salt-diet (HFSD) induced hypertension in mice. MethodsThe systolic, diastolic and mean BP (SBP, DBP and MBP, respectively) were continuously monitored from unrestricted conscious wild-type (cftr+/+) FVB and CFTR-knockout (cftr−/−) mice (8-week old, male). HFSD (64.7% fructose, 2% NaCl water) or control normal starch diet (CNSD, 58.9% corn starch, 0 NaCl water) was given for 8 weeks and vascular Doppler were performed. Real-time PCR and Western blot were used to examine mRNA and protein expression, respectively. ResultsThe aortic stiffness, daytime and nighttime SBP, DBP, and MBP of the cftr−/− mice were significantly higher than those in the age- and gender-matched cftr+/+ mice, which is consistent with the findings of increased vascular resistance in cystic fibrosis patients. The aortic stiffness, daytime and nighttime SBP, DBP, and MBP of cftr+/+ mice fed with HFSD were all significantly higher than those fed with CNSD. Importantly, HFSD caused a significant decrease in mRNA and protein expression of WINK1, WINK4 and CFTR in aorta and mesenteric arteries, but not in the kidney, corroborating that HSFD-induced downregulation of WINKs and loss of CFTR function specifically in the arteries may mediate the increased BP. ConclusionsCFTR regulates peripheral arterial resistance and BP in vivo. HFSD-induced CFTR downregulation specifically in the arteries may be a novel mechanism for hypertension.

Cystic Fibrosis, CFTR, and Colorectal Cancer.

Cystic fibrosis (CF), caused by biallelic inactivating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, has recently been categorized as a familial colorectal cancer (CRC) syndrome. CF patients are highly susceptible to early, aggressive colorectal tumor development. Endoscopic screening studies have revealed that by the age of forty 50% of CF patients will develop adenomas, with 25% developing aggressive advanced adenomas, some of which will have already advanced to adenocarcinomas. This enhanced risk has led to new CF colorectal cancer screening recommendations, lowering the initiation of endoscopic screening to age forty in CF patients, and to age thirty in organ transplant recipients. The enhanced risk for CRC also extends to the millions of people (more than 10 million in the US) who are heterozygous carriers of CFTR gene mutations. Further, lowered expression of CFTR is reported in sporadic CRC, where downregulation of CFTR is associated with poor survival. Mechanisms underlying the actions of CFTR as a tumor suppressor are not clearly understood. Dysregulation of Wnt/β-catenin signaling and disruption of intestinal stem cell homeostasis and intestinal barrier integrity, as well as intestinal dysbiosis, immune cell infiltration, stress responses, and intestinal inflammation have all been reported in human CF patients and in animal models. Notably, the development of new drug modalities to treat non-gastrointestinal pathologies in CF patients, especially pulmonary disease, offers hope that these drugs could be repurposed for gastrointestinal cancers.

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor

Introduction and AimsThe intestinal epithelium functions to transport nutrients, fluid and electrolytes, while at the same time acting as a barrier to the entry of harmful substances. CFTR is a transmembrane Cl− channel important in regulating intestinal fluid transport and is implicated in the pathogenesis of a number of intestinal diseases. Bile acids, classically known for their roles in lipid digestion, are now also recognised as important enteric hormones that regulate many aspects of epithelial function. Indeed, it is thought that bile acid metabolism is a primary mechanism by which the microbiota communicates with its human host. Here, we set out to investigate the role of the nuclear bile acid receptor, farnesoid x receptor (FXR), in regulating epithelial CFTR expression.MethodsT84 human colonic epithelial cells were cultured as polarised monolayers on permeable supports and treated bilaterally with the FXR agonist, GW4064 (5 μM), over a range of times. Levels of FGF19, an index of FXR activation, CFTR, FXR, FOXA1, and miR‐494 were measured by qRT‐PCR, western blotting, or ELISA.ResultsTreatment of the cells with GW4064 significantly increased FGF19 mRNA expression by 1235 ± 237.3 fold (n = 8; p < 0.05) and protein by 719.6 ± 92.0 pg/mL (n = 5; p < 0.01) after 48 hrs. Moreover, GW4064 downregulated CFTR mRNA to 0.47 ± 0.1 fold after 12 hrs (n = 8; p < 0.01) and protein levels to 0.36 ± 0.1 fold after 48 hrs, compared to vehicle‐treated controls (n = 4; p < 0.05). Transcriptomic analysis confirmed FXR‐induced downregulation of CFTR in primary human colonic enteroids. Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl− secretory responses to the Ca2+‐dependent agonist carbachol (CCh; 100 μM) and the cAMP‐dependent agonist, forskolin (10 μM) by 79.9 ± 7.5 % (n=4; p <0.01) and 74.2 ± 8.9 % (n=4; p < 0.01), respectively. Expression of miR‐494, which is known to target CFTR, was not increased by GW4064 treatment in T84 cells. However, mRNA expression of FOXA1, a transcription factor that regulates CFTR, was inhibited by 33.2 ± 5.2% after 3 hrs (n = 4; p <0.05).ConclusionThe nuclear bile acid receptor, FXR, regulates colonic epithelial CFTR expression. Such actions are likely to be important in the setting of microbial regulation of host intestinal physiology in health and disease. As such, FXR represents an excellent target for development of new drugs to treat intestinal diseases associated with dysregulated CFTR function.Support or Funding InformationThis work was funded by a Science Foundation Ireland Principal Investigator Award to SJK

42 CFTR Mediates Enteric Bacterial Translocation Post Severe Burn via ERK and Inflammatory Signaling

Abstract Introduction This research was to investigate the role of cystic fibrosis transmembrane conductance regulator (CFTR) in mediating enteric bacterial translocation (EBT) post severe burn as well as the signaling pathways involved. Methods A hypoxia treatment model of Caco-2 in vitro and a 30% TBSA full-thickness dermal burn mice model in vivo were established to explore the effect of major burn injury on intestinal epithelial barrier and the expression of CFTR and tight junction proteins. In addition, the DF508 mice (mice with F508del CFTR gene mutation) were used to analyze bacterial counts in extraintestinal organs and the expression of ZO-1, OCLN and E-cadherin. And CFTR specific inhibitor or down-regulation of CFTR in Caco-2 cells were used to explore the effect of CFTR on tight junction of intestinal cells. Furthermore, in the models above, MAPK pathway and inflammatory factors were detected to explore the mechanism. Finally, Vitamin D3 was used to pretreat mice before severe burn or Caco-2 cells before hypoxia. Bacterial counts in extraintestinal organs and the expression of CFTR, ZO-1, OCLN and P-ERK as well as the concentrations of inflammatory factors were detected. Results Our data showed a decrease of CFTR, concomitant with activation of ERK signaling, elevation of inflammatory factors (TNF-α, IL-1β and IL-8) and declined expression of ZO-1, occludin and E-cadherin, and a cellular mislocation of ZO-1 and OCLN post hypoxia. Furthermore, either administration of CFTR specific inhibitor or down-regulation of CFTR caused similar alterations as hypoxia did. Ileum of DF508 -/- and +/- mice exhibited increased inflammation, declined ZO-1, occludin and E-cadherin and EBT compared to +/+ mice. Moreover, vitamin D3 could reverse the junctional damage induced by hypoxia, thus preventing occurrence of EBT, possibly via up-regulating CFTR. Conclusions CFTR/ERK/inflammatory factors signaling was critical in modulating intestinal epithelial junction, accordingly regulating EBT. Vitamin D3 can protect intestinal epithelial barrier after severe burn, which may be associated with the up-regulation of CFTR, the inhibition of ERK pathway and decreased pro-inflammatory cytokine. Applicability of Research to Practice This research may be used in practice in the future to regulate the metabolism after severe burns.

Cystic fibrosis transmembrane conductance regulator modulates enteric cholinergic activities and is abnormally expressed in the enteric ganglia of patients with slow transit constipation.

Cystic fibrosis transmembrane conductance regulator (CFTR) was recently found in the enteric nervous system, where its role is unclear. We aimed to identify which enteric neuronal structures express CFTR, whether CFTR modulates enteric neurotransmission and if altered CFTR expression is associated with slow transit constipation (STC). Immunofluorescence double labeling was performed to localize CFTR with various neuronal and glial cell markers in the human colon. The immunoreactivity (IR) of CFTR and choline acetyltransferase (ChAT) on myenteric plexus of control and STC colon was quantitatively analyzed. In control colonic muscle strips, electrical field stimulation (EFS) evoked contractile responses and the release of acetylcholine (ACh) was measured in the presence of the CFTR channel inhibitor, CFTR(inh)-172. CFTR-IR was densely localized to myenteric ganglia, where it was co-localized with neuronal markers HuC/D and β-tubulin, and glial marker S-100 but little with glial fibrillary acidic protein. Vesicular ACh transport was almost exclusively co-localized with CFTR, but neurons expressing nitric oxide synthase were CFTR negative. Significant reductions of CFTR-IR (P < 0.01) and ChAT-IR (P < 0.05) were observed on myenteric ganglia of STC compared to control. Pre-treatment of colonic muscle strips with CFTR(inh)-172 (10µM) significantly inhibited EFS-evoked contractile responses (P < 0.01) and ACh release (P < 0.05). Co-localization of CFTR-IR with cholinergic markers, inhibition of EFS-induced colonic muscle contractility and ACh release by CFTR(inh)-172 suggest that CFTR modulates enteric cholinergic neurotransmission. The downregulation of CFTR and ChAT in myenteric ganglia of STC correlated with the impaired contractile responses to EFS.

Colonic bile acids regulate epithelial wound healing

BackgroundEpithelial restitution is an essential process for maintenance of intestinal barrier function. While previously published data have shown bile acids to be involved in regulation of epithelial barrier function in in vivo models of colonic inflammation, their roles in regulating restitution are not yet known. Here, we investigated the effects of bile acids on epithelial restitution and the molecular pathways involved in colonic epithelial healing.MethodsWounds were created by pinch biopsy in C5BL/6 mice during colonic endoscopy. Animals received daily topical application of deoxycholic acid (DCA; 150 μM) or the “therapeutic” bile acid, ursodeoxycholic acid (UDCA; 150 μM). Images of the wounds were taken at day 2 and day 6 post wounding and the area of the wounds was measured by Image J ®software. T84 colonic epithelial cells, grown as monolayers on transparent permeable supports, were wounded at T=0. Cells were treated with DCA (50 – 150 μM), UDCA (50 – 150 μM, a farnesoid X receptor (FXR) agonist, GW4064 (10 μM), or the cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers, CFTR(inh)‐172 (10 μM) or GlyH‐101 (25 μM). Restitution was measured as wound area after 48 h and expressed as % T=0 wound area. HEK‐293 cells were transfected with vectors expressing FXR and the luciferase gene under control of the CFTR promoter. Protein expression was assessed by western blotting, mRNA levels were assessed by q‐PCR and cell migration by Boyden chamber assay.ResultsWound healing was significantly delayed in animals treated with DCA compared to control (Day 6 Wound Area −0.41 ± 0.15 mm2, p= 0.0287, N=5). DCA also inhibited wound closure in cultured epithelial monolayers and attenuated cell migration in Boyden chamber assays, without significantly altering cell proliferation. Nuclear accumulation of FXR was detected upon DCA stimulation, while activation of FXR by GW4064 inhibited epithelial wound closure to 35.1 ± 1.1%, compared to 51.6 ± 5 % in vehicle‐treated controls (n = 5; p &lt; 0.05) and decreased T84 cell migration in Boyden chambers to 57 ± 9.6 % (n = 3; p &lt;0.05) of that in controls. To address the mechanism by which DCA exerts its action on wound healing, levels of CFTR expression was measured. Both, DCA and GW4064, attenuated CFTR mRNA levels, along with total and surface CFTR protein expression, whereas promoter/reporter assays revealed that transcriptional downregulation of CFTR by DCA is mediated in an FXR specific manner. Furthermore, inhibition of CFTR activity with either CFTR(inh)‐172 or GlyH‐101 mimicked the effects of FXR on wound healing. In contrast, UDCA (50 – 150 μM) enhanced wound healing in both in vitro and in vivo models and prevented the effects of DCA in cell migration and restitution assays.ConclusionsIn conclusion, these studies suggest that bile acids are important regulators of epithelial wound healing and are therefore good targets for the development of new drugs to modulate intestinal barrier function in disease treatment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tanshinone IIA\xa0sulfonate protects against cigarette smoke-induced COPD and down-regulation of CFTR in mice

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by abnormal inflammation, persistent and progressive lung function decline. The anti-inflammatory actions of tanshinone IIA, which is the most important active component from Chinese herbal medicine Danshen, have been well studied. However, it remains unknown whether sodium tanshinone IIA sulfonate (STS) protects against the development of COPD. Here we found that STS inhalation (5 mg/kg, 30 min per session, twice a day) significantly attenuated lung function decline, airspace enlargement, mucus production, bronchial collagen deposition, inflammatory responses and oxidative stress caused by cigarette smoke (CS) and lipopolysaccharide (LPS) exposures in mice. Moreover, treatment with STS (10 μg/ml) reduced CS extract (CSE)-induced IL-6 and IL-8 secretion in human bronchial epithelial (16HBE) cells. The anti-inflammatory actions of STS were associated with inhibition of ERK1/2 and NF-κB activations. Interestingly, STS inhibited CS-induced reduction of cystic fibrosis transmembrane conductance regulator (CFTR) in mouse lungs and in 16HBE cells. Treatment with a specific CFTR inhibitor CFTR-Inh172 augmented CSE-induced ERK1/2 and NF-κB-dependent inflammatory responses, but abolished the inhibitory action of STS on IL-6 and IL-8 secretion in 16HBE cells. These results demonstrate that CS-induced COPD and down-regulation of CFTR are prevented by STS.

Defective CFTR leads to aberrant \u03b2-catenin activation and kidney fibrosis

Cystic fibrosis transmembrane conductance regulator (CFTR), known as a cAMP-activated Cl− channel, is widely expressed at the apical membrane of epithelial cells in a wide variety of tissues. Of note, despite the abundant expression of CFTR in mammalian kidney, the role of CFTR in kidney disease development is unclear. Here, we report that CFTR expression is downregulated in the UUO (unilateral ureteral obstruction)-induced kidney fibrosis mouse model and human fibrotic kidneys. Dysfunction or downregulation of CFTR in renal epithelial cells leads to alteration of genes involved in Epithelial-Mesenchymal Transition (EMT) and kidney fibrosis. In addition, dysregulation of CFTR activates canonical Wnt/β-catenin signaling pathways, whereas the β-catenin inhibitor reverses the effects of CFTR downregulation on EMT marker. More interestingly, CFTR interacts with Dishevelled 2 (Dvl2), a key component of Wnt signaling, thereby suppressing the activation of β-catenin. Compared to wild type, deltaF508 mice with UUO treatment exhibit significantly higher β-catenin activity with aggregated kidney fibrogenesis, which is reduced by forced overexpression of CFTR. Taken together, our study reveals a novel mechanism by which CFTR regulates Wnt/β-catenin signaling pertinent to progression of kidney fibrosis and indicates a potential treatment target.

CFTR protects against vascular inflammation and atherogenesis in apolipoprotein E-deficient mice.

Atherosclerosis is a chronic inflammatory disease of the vascular wall. Dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to result in inflammatory responses in cystic fibrosis (CF) patients. However, little is known about the role of CFTR in vascular inflammation and atherogenesis. Our results showed that CFTR was dominantly expressed in macrophages of atherosclerotic plaque and reduced in aorta and aortic sinus from atherosclerotic apolipoprotein E-deficient (apoE−/−) mice. In vivo administration of adenovirus encoding CFTR (Ad-CFTR) with apoE−/− mice fed on high-fat diet (HFD) improved plaque stability by decreasing lipid accumulation and necrotic area and increasing smooth muscle cell content and collagen. The Ad-CFTR-treated mice also displayed reduced proinflammatory cytokines levels in aorta and peritoneal macrophages, whereas the anti-inflammatory M2 macrophage markers were increased. Confocal microscopy revealed that the infiltration of T lymphocytes, neutrophils, and macrophages in aortic sinus was markedly attenuated in Ad-CFTR-treated apoE−/− mice. Moreover, in vitro experiments showed that overexpression of CFTR inhibited ox-LDL-induced the migration of peritoneal macrophages. Finally, it was observed that CFTR up-regulation suppressed NFκB and MAPKs activity induced by ox-LDL. Inhibition of JNK or ERK abrogated CFTR down-regulation induced NFκB activation, whereas NFκB inhibitor had no effect on JNK or ERK activation. Taken together, these results demonstrate that CFTR prevents inflammation and atherogenesis via inhibition of NFκB and MAPKs activation. Our data suggest that CFTR may present a potential therapeutic target for the treatment of vascular inflammation and development of atherosclerotic disease.

Down-regulated CFTR During Aging Contributes to Benign Prostatic Hyperplasia.

Benign prostatic hyperplasia (BPH) is a hyper-proliferative disease of the aging prostate; however, the exact mechanism underlying the development of BPH remains incompletely understood. The present study investigated the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR), which has been previously shown to negatively regulate nuclear factor-κB (NF-κB)/cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway, in the pathogenesis of BPH. Our results showed decreasing CFTR and increasing COX2 expression in rat prostate tissues with aging. Furthermore, suppression of CFTR led to increased expression of COX2 and over-production of PGE2 in a normal human prostate epithelial cell line (PNT1A) with elevated NF-κB activity. PGE2 stimulated the proliferation of primary rat prostate stromal cells but not epithelial cells, with increased PCNA expression. In addition, the condition medium from PNT1A cells after inhibition or knockdown of CFTR promoted cell proliferation of prostate stromal cells which could be reversed by COX2 or NF-κB inhibitor. More importantly, the involvement of CFTR in BPH was further demonstrated by the down-regulation of CFTR and up-regulation of COX2/NF-κB in human BPH samples. The present results suggest that CFTR may be involved in regulating PGE2 production through its negative regulation on NF-κB/COX2 pathway in prostate epithelial cells, which consequently stimulates cell growth of prostate stromal cells. The overstimulation of prostate stromal cell proliferation by down-regulation of CFTR-enhanced PGE2 production and release during aging may contribute to the development of BPH.

High level of CFTR expression is associated with tumor aggression and knockdown of CFTR suppresses proliferation of ovarian cancer in vitro and in vivo.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP-binding cassette (ABC) transporter family, members of which are involved in various types of cancer. The relationship between CFTR and ovarian cancer remains to be elucidated. The aim of the present study was to investigate the expression of CFTR in human ovarian cancer tissues and its clinical significance in the progression of ovarian cancer. The role of CFTR in the malignant invasion, migration and proliferation of ovarian cancer in vitro and in vivo was also investigated. Immunohistochemical staining analysis was performed to detect the expression of CFTR in 83 cases of human epithelial ovarian cancer specimens. Moreover, SKOV3 and A2780 stable cell lines containing shRNA gene specific for CFTR were established. Cell proliferation and motility were observed and compared with CFTR-RNAi cells. Tumorigenicity of CFTR-RNAi cells was investigated by tumor xenograft experiments conducted subcutaneously in nude mice. The expresssion of CFTR in ovarian cancer was significantly higher than that in benign ovarian tumor and normal ovaries (P<0.05). In ovarian cancer, CFTR expression was significantly associated with advanced FIGO stage, poor histopathological grade and serum Ca-125 (P<0.05). Furthermore, we observed that CFTR staining was stronger in the serous type as compared to the other types (P<0.05). Compared with the negative control, decreased cell invasion, migration, proliferation, adhesion and colony formation were observed in CFTR-RNAi cells in vitro. In vivo, tumorigenic abilities of CFTR-RNAi cells were significantly repressed compared with that of the control groups. CFTR overexpression may play an important role in the development and progression of ovarian cancer. Additionally, the downregulation of CFTR suppresses aggressive malignant biological behaviors of ovarian cancer cells in vitro and in vivo.

Abstract 170: CFTR as a Novel Target and Mechanism for High-Fructose Diet and Salt-Induced Hypertension

Objective: High fructose and salt diet (HFSD) consumption is very common in modern lifestyle. Fructose not only stimulates intestinal salt and water absorption but also decreases renal sodium (Na+) and chloride (Cl-) excretion. High fructose intake induces rapid and sustained increase in blood pressure (BP). But a Cl--free diet could block fructose-induced hypertension. The underlying mechanism for this Cl_dependence of high fructose and salt induced hypertension is not clear. This study was designed to test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel may play a critical role in the HFSD-induced hypertension. Approach and Results: The DSI HD-X11 transmitters for telemetry recordings of both blood pressure and ECG were implanted into CFTR-knockout (CFTR-/-) mice (8-week old, male) and their age- and gender-matched wild-type (CFTR+/+) mice. The systolic (SBP), diastolic (DBP) and mean BP (MBP) and ECG of the mice before and after feeding with HFSD (%65 fructose, 2% salt) were monitored daily for 8 weeks. Continuous 8-week HFSD caused a significant increase in aortic stiffness, mesenteric and renal artery resistance, SBP, DBP, and MBP in CFTR+/+ mice. Real-time PCR and Western blot analysis revealed a significant decrease (~90%) in CFTR mRNA and protein expression in aorta and mesenteric arteries but not in the heart and brain. This HFSD-induced specific down-regulation of CFTR was highly correlated with the HFSD-induced reduction in WINK1 and WINK4 expression in aorta and mesenteric artery. In the mice fed with normal diet the aortic stiffness, mesenteric and renal artery resistance and the SDP, DBP, and MBP at either daytime or nighttime was significantly higher in CFTR-/- mice (n=8) than in CFTR+/+ mice (n=15), implicating an important functional role for CFTR in the regulation of vascular resistance and BP. Conclusions: 1) CFTR plays a pivotal functional role in the regulation of arterial resistance and BP; 2) HFSD may cause hypertension through a specific WINKs-CFTR signaling pathway. These results provide compelling evidence that diet may cause changes in expression of specific genes in the vasculature and CFTR may represent a novel molecular target and mechanism for the HFSD-induced hypertension.

Decreased expression of cystic fibrosis transmembrane conductance regulator impairs sperm quality in aged men

Sperm quality declines with aging; however, the underlying molecular mechanism remains elusive. The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to play an essential role in fertilizing capacity of sperm and male fertility. This study aimed to investigate the involvement of age-dependent CFTR downregulation in lowering sperm quality in old age. Two hundred and one healthy fertile men of three age groups (20-40 years, n=64; 40-60 years, n=61; and >60 years, n=76) were recruited. Expression of CFTR was determined by RT-PCR, western blot, and immunofluorescence staining. Collected sperm were treated with CFTR inhibitor or potentiator. Sperm quality was assessed by motility and bicarbonate-induced capacitation. The results showed that the expression of CFTR on the equatorial segment and neck region of sperm was significantly decreased in an age-dependent manner. Reduction of CFTR expression in sperm from old men was correlated with lowered forward motility and decreased HCO3(-) sensitivity required for sperm capacitation. Activation of CFTR by genistein partially rescued the decreased forward motility in sperm from old men. Decreased CFTR expression in sperm was also found to be associated with lowered sperm quality in aging mice. These results suggest that age-dependent downregulation of CFTR in sperm leads to lowered sperm quality in old age sperm. CFTR may be a pontential target for rescuing sperm motility as well as a fertility indicator in old age men.

Downregulation of CFTR promotes epithelial-to-mesenchymal transition and is associated with poor prognosis of breast cancer

The epithelial-to-mesenchymal transition (EMT), a process involving the breakdown of cell–cell junctions and loss of epithelial polarity, is closely related to cancer development and metastatic progression. While the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl− and HCO3− conducting anion channel expressed in a wide variety of epithelial cells, has been implicated in the regulation of epithelial polarity, the exact role of CFTR in the pathogenesis of cancer and its possible involvement in EMT process have not been elucidated. Here we report that interfering with CFTR function either by its specific inhibitor or lentiviral miRNA-mediated knockdown mimics TGF-β1-induced EMT and enhances cell migration and invasion in MCF-7. Ectopic overexpression of CFTR in a highly metastatic MDA-231 breast cancer cell line downregulates EMT markers and suppresses cell invasion and migration in vitro, as well as metastasis in vivo. The EMT-suppressing effect of CFTR is found to be associated with its ability to inhibit NFκB targeting urokinase-type plasminogen activator (uPA), known to be involved in the regulation of EMT. More importantly, CFTR expression is found significantly downregulated in primary human breast cancer samples, and is closely associated with poor prognosis in different cohorts of breast cancer patients. Taken together, the present study has demonstrated a previously undefined role of CFTR as an EMT suppressor and its potential as a prognostic indicator in breast cancer.

Letter from the editor

Letter from the editor

Influenza A matrix protein M2 downregulates CFTR: inhibition of chloride transport by a proton channel of the viral envelope

in a recent issue of the Journal , Londino et al. ([15][1]) report that the influenza matrix protein M2 decreases the activity of the human cystic fibrosis transmembrane conductance regulator (CFTR) by increasing secretory organelle pH, which results in downregulation of CFTR in a ubiquitination-

Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air-liquid but not liquid-liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr(∆F508/∆F508) immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor-mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.

Cryptorchidism-induced CFTR down-regulation results in disruption of testicular tight junctions through up-regulation of NF- B/COX-2/PGE2

Does elevated temperature-induced cystic fibrosis transmembrane conductance regulator (CFTR) down-regulation in Sertoli cells in cryptorchid testis disrupt testicular tight junctions (TJs) through the nuclear factor kappa B (NF-κB)/cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway? Our results suggest that CFTR may be involved in regulating testicular TJs and the blood-testis barrier (BTB) through its negative regulation of the NF-κB/COX-2/PGE(2) pathway in Sertoli cells, a defect of which may result in the spermatogenesis defect in cryptorchidism. Cryptorchidism, or undescended testes, is known to result in defective spermatogenesis. Although an elevated testicular temperature is regarded as an important factor affecting spermatogenesis in cryptorchidism, the exact mechanism remains elusive. It is known that the expression of functional CFTR is temperature sensitive. Our previous study has demonstrated that CFTR negatively regulates NF-κB/COX-2/PGE(2) in bronchial epithelial cells. Disruption of TJs by COX-2/PGE(2) has been found in tumour cells. Expression of CFTR, NF-κB, COX-2 and TJ proteins was examined in the testes of a surgical-induced cryptorchidism mouse model and a testicular hyperthermia mouse model, as well as in control or CFTR-inhibited/knocked down primary rat Sertoli cells. PGE(2) production was measured by ELISA. Sertoli cell barrier function was determined by transepethelial resistance (TER) measurements in rat Sertoli cell primary cultures. BTB integrity in the cryptorchidism model was monitored by examining tracker dye injected into seminiferous tubules. Down-regulation of CFTR accompanied by activation of NF-κB, up-regulation of COX-2 and down-regulation of TJ proteins, including ZO-1 and occludin, was observed in a cryptorchidism mouse model. BTB leakage revealed impaired BTB integrity in cryptorchid testes, confirming the destruction of TJs. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and CFTR knockout mouse model. Culturing primary Sertoli cells at 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression and PGE(2) production compared with the culture at the physiological 32°C. Inhibition or knockdown of CFTR led to increased COX-2 but decreased ZO-1 and occludin expression in Sertoli cells, which could be mimicked by PGE(2), but reversed by NF-κB or COX-2 inhibitor, suggesting that the regulation of TJs by CFTR is mediated by a NF-κB/COX-2/PGE(2) pathway. Inhibition of CFTR or administration of PGE(2) significantly decreased Sertoli cell TER. This study has tested only the CFTR/NF-κB/COX-2/PGE(2) pathway in mouse testes in vivo and in rat Sertoli cells in vitro, and thus, it has some limitations. Further investigations in other species, especially humans, are needed. Our study may shed more light on one of the aspects of the complicated underlying mechanisms of defective spermatogenesis induced by cryptorchidism.