Jing Hui Guo

Activation of the epithelial Na + channel triggers prostaglandin E 2 release and production required for embryo implantation

Embryo implantation remains a poorly understood process. We demonstrate here that activation of the epithelial Na+ channel (ENaC) in mouse endometrial epithelial cells by an embryo-released serine protease, trypsin, triggers Ca2+ influx that leads to prostaglandin E2 (PGE2) release, phosphorylation of the transcription factor CREB and upregulation of cyclooxygenase 2, the enzyme required for prostaglandin production and implantation. We detected maximum ENaC activation, as indicated by ENaC cleavage, at the time of implantation in mice. Blocking or knocking down uterine ENaC in mice resulted in implantation failure. Furthermore, we found that uterine ENaC expression before in vitro fertilization (IVF) treatment is markedly lower in women with implantation failure as compared to those with successful pregnancy. These results indicate a previously undefined role of ENaC in regulating the PGE2 production and release required for embryo implantation, defects that may be a cause of miscarriage and low success rates in IVF.

Glucose-induced electrical activities and insulin secretion in pancreatic islet β-cells are modulated by CFTR

The cause of insulin insufficiency remains unknown in many diabetic cases. Up to 50% adult patients with cystic fibrosis (CF), a disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), develop CF-related diabetes (CFRD) with most patients exhibiting insulin insufficiency. Here we show that CFTR is a regulator of glucose-dependent electrical acitivities and insulin secretion in β-cells. We demonstrate that glucose elicited whole-cell currents, membrane depolarization, electrical bursts or action potentials, Ca2+ oscillations and insulin secretion are abolished or reduced by inhibitors or knockdown of CFTR in primary mouse β-cells or RINm5F β-cell line, or significantly attenuated in CFTR mutant (DF508) mice compared with wild-type mice. VX-809, a newly discovered corrector of DF508 mutation, successfully rescues the defects in DF508 β-cells. Our results reveal a role of CFTR in glucose-induced electrical activities and insulin secretion in β-cells, shed light on the pathogenesis of CFRD and possibly other idiopathic diabetes, and present a potential treatment strategy.

CFTR suppresses tumor progression through miR-193b targeting urokinase plasminogen activator (uPA) in prostate cancer

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in the epithelial cells of a wide range of organs/tissues from which most cancers are derived. Although accumulating reports have indicated the association of cancer incidence with genetic variations in CFTR gene, the exact role of CFTR in cancer development and the possible underlying mechanism have not been elucidated. Here, we report that CFTR expression is significantly decreased in both prostate cancer cell lines and human prostate cancer tissue samples. Overexpression of CFTR in prostate cancer cell lines suppresses tumor progression (cell growth, adhesion and migration), whereas knockdown of CFTR leads to enhanced malignancies both in vitro and in vivo. In addition, we demonstrate that CFTR knockdown-enhanced cell proliferation, cell invasion and migration are significantly reversed by antibodies against either urokinase plasminogen activator (uPA) or uPA receptor (uPAR), which are known to be involved in various malignant traits of cancer development. More interestingly, overexpression of CFTR suppresses uPA by upregulating the recently described tumor suppressor microRNA-193b (miR-193b), and overexpression of pre-miR-193b significantly reverses CFTR knockdown-enhanced malignant phenotype and abrogates elevated uPA activity in prostate cancer cell line. Finally, we show that CFTR gene transfer results in significant tumor repression in prostate cancer xenografts in vivo. Taken together, the present study has demonstrated a previously undefined tumor-suppressing role of CFTR and its involvement in regulation of miR-193b in prostate cancer development.

Regulation of smooth muscle contractility by the epithelium in rat vas deferens: role of ATP-induced release of PGE2

Recent studies suggest that the epithelium might modulate the contractility of smooth muscle. However, the mechanisms underlying this regulation are unknown. The present study investigated the regulation of smooth muscle contraction by the epithelium in rat vas deferens and the possible factor(s) involved. Exogenously applied ATP inhibited electrical field stimulation (EFS)‐evoked smooth muscle contraction in an epithelium‐dependent manner. As the effects of ATP on smooth muscle contractility were abrogated by inhibitors of prostaglandin synthesis, but not by those of nitric oxide synthesis, prostaglandins might mediate the effects of ATP. Consistent with this idea, PGE2 inhibited EFS‐evoked smooth muscle contraction independent of the epithelium, while ATP and UTP induced the release of PGE2 from cultured rat vas deferens epithelial cells, but not smooth muscle cells. The ATP‐induced PGE2 release from vas deferens epithelial cells was abolished by U73122, an inhibitor of phospholipase C (PLC) and BAPTA AM, a Ca2+ chelator. ATP also transiently increased [Ca2+]i in vas deferens epithelial cells. This effect of ATP on [Ca2+]i was independent of extracellular Ca2+, but abolished by the P2 receptor antagonist RB2 and U73122. In membrane potential measurements using a voltage‐sensitive dye, PGE2, but not ATP, hyperpolarized vas deferens smooth muscle cells and this effect of PGE2 was blocked by MDL12330A, an adenylate cyclase inhibitor, and the chromanol 293B, a blocker of cAMP‐dependent K+ channels. Taken together, our results suggest that ATP inhibition of vas deferens smooth muscle contraction is epithelium dependent. The data also suggest that ATP activates P2Y receptor‐coupled Ca2+ mobilization leading to the release of PGE2 from epithelial cells, which in turn activates cAMP‐dependent K+ channels in smooth muscle cells leading to the hyperpolarization of membrane voltage and the inhibition of vas deferens contraction. Thus, the present findings suggest a novel regulatory mechanism by which the epithelium regulates the contractility of smooth muscle.

CFTR negatively regulates cyclooxygenase-2-PGE2 positive feedback loop in inflammation

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over‐inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased cyclooxygenase‐2 (COX‐2) expression and over‐production of prostaglandin E2 (PGE2) in human CF bronchial epithelia cell line (CFBE41o−) with elevated NF‐κB activity compared to a wild‐type airway epithelial cell line (16HBE14o−). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX‐2 and NF‐κB activity, supporting the notion that lack of CFTR results in hyper‐inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE2 involving PKA and transcription factor, CREB. More importantly, overexpression of wild‐type CFTR significantly suppressed COX‐2 expression in CFBE41o− cells, and wild‐type CFTR protein expression was significantly increased when 16HBE14o− cells were challenged with LPS as well as PGE2, indicating possible involvement of CFTR in negative regulation of COX‐2/PGE2. In conclusion, CFTR is a negative regulator of PGE2‐mediated inflammatory response, defect of which may result in excessive activation of NF‐κB, leading to over production of PGE2 as seen in inflammatory CF tissues. J. Cell. Physiol. 227: 2759–2766, 2012.

Involvement of CFTR in oviductal HCO3− secretion and its effect on soluble adenylate cyclase-dependent early embryo development

BACKGROUND The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in electrolyte and fluid transport in epithelial cells, and women with cystic fibrosis (CF), caused by CFTR gene mutations, have a higher incidence of infertility. METHODS In the present study, we investigated the expression of CFTR in porcine oviduct and its functional role in oviductal HCO(3)(-) secretion and embryo development with RT-PCR, western blot, patch-clamp, short-circuit current (I(sc)), pH measurement and embryo culture. RESULTS RT-PCR and western blot analysis showed the expression of CFTR mRNA and protein in the oviduct with its localization demonstrated by immunohistochemistry. The whole-cell patch-clamp recording revealed a forskolin (FSK)-activated current with electrophysiological and pharmacological characteristics of CFTR. The I(sc) measurement showed that FSK-stimulated an increase in the I(sc), which could be significantly reduced by CFTR inhibitor or removal of both CO(2) and HCO(3)(-). pH measurement showed a FSK stimulated alkalization at the apical surface, which could be inhibited by CFTR inhibitor, indicating CFTR-mediated HCO(3)(-) secretion. Mouse embryo development from 2-cell to morula or blastocyst stage was significantly inhibited in the absence of HCO(3)(-) or when co-cultured with HCO(3)(-) secretion-deficient CFTR mutant cells as compared with the wild-type. RT-PCR, western blot and immunostaining showed the expression of soluble adenylate cyclase (sAC), the known HCO(3)(-) sensor, in embryos. Treatment with its inhibitors, 2-hydroxyestradiol and KH7, prevented the HCO(3)(-) dependent embryo development. CONCLUSION The present results suggest that CFTR-mediated oviductal HCO(3)(-) secretion may be vital for sAC-dependent early embryo development, a defect of which may contribute to the reduced fertility seen in women with CF.

Impaired CFTR-Dependent Amplification of FSH-Stimulated Estrogen Production in Cystic Fibrosis and PCOS

CONTEXT Estrogens play important roles in a wide range of physiological and pathological processes, and their biosynthesis is profoundly influenced by FSH that regulates the rate-limiting enzyme aromatase-converting estrogens from androgens. Abnormal estrogen levels are often seen in diseases such as ovarian disorders in polycystic ovarian syndrome (PCOS), an endocrine disorder affecting 5-10% of women of reproductive age, and cystic fibrosis (CF), a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). OBJECTIVES We undertook the present study to investigate the mechanism underlying these ovarian disorders, which is not well understood. RESULTS FSH-stimulated cAMP-responsive element binding protein phosphorylation, aromatase expression, and estradiol production are found to be enhanced by HCO3- and a HCO3- sensor, the soluble adenylyl cyclase, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/ΔF508 mutant mice. CFTR expression is found positively correlated with aromatase expression in human granulosa cells, supporting its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in PCOS rodent models and human patients. CONCLUSIONS CFTR regulates ovarian estrogen biosynthesis by amplifying the FSH-stimulated signal via the nuclear soluble adenylyl cyclase. The present findings suggest that defective CFTR-dependent regulation of estrogen production may underlie the ovarian disorders seen in CF and PCOS.

Regulation of miR-101/miR-199a-3p by the epithelial sodium channel during embryo implantation: involvement of CREB phosphorylation

In our previous study, we have demonstrated that the epithelial sodium channel (ENaC) mediates the embryo-derived signals leading to the activation of CREB and upregulation of cyclooxygenase type 2 (COX2) required for embryo implantation. This study aims to investigate whether microRNAs (miRNAs) are involved in the ENaC-induced upregulation of COX2 during embryo implantation. The results show that the levels of miR-101 and miR-199a-3p, two COX2 targeting miRNAs, are reduced by ENaC activation, and increased by ENaC inhibition or knock-down of ENaC subunit (ENaCα) in human endometrial surface epithelial (HES) cells or in mouse uteri during implantation. Phosphorylation of CREB is induced by the activation of ENaC, and blocked by ENaC inhibition or knockdown in HES cells. Knockdown of ENaCα or CREB in HES cells or in mouse uterus in vivo results in increases in miR-101 and miR-199a-3p, accompanied with decreases in COX2 protein levels and reduction in implantation rate. The downregulation of COX2 caused by knockdown of ENaC or CREB can be recovered by the inhibitors of miR-101 or miR-199a-3p in HES cells. These results reveal a novel molecular mechanism modulating COX2 expression during embryo implantation via ENaC-dependent CREB activation and COX2-targeting miRNAs.

Defective CFTR-regulated granulosa cell proliferation in polycystic ovarian syndrome

Polycystic ovarian syndrome (PCOS) is one of the most frequent causes of female infertility, featured by abnormal hormone profile, chronic oligo/anovulation, and presence of multiple cystic follicles in the ovary. However, the mechanism underlying the abnormal folliculogenesis remains obscure. We have previously demonstrated that CFTR, a cAMP-dependent Cl(-) and HCO3 (-) conducting anion channel, is expressed in the granulosa cells and its expression is downregulated in PCOS rat models and human patients. In this study, we aimed to investigate the possible involvement of downregulation of CFTR in the impaired follicle development in PCOS using two rat PCOS models and primary culture of granulosa cells. Our results indicated that the downregulation of CFTR in the cystic follicles was accompanied by reduced expression of proliferating cell nuclear antigen (PCNA), in rat PCOS models. In addition, knockdown or inhibition of CFTR in granulosa cell culture resulted in reduced cell viability and downregulation of PCNA. We further demonstrated that CFTR regulated both basal and FSH-stimulated granulosa cell proliferation through the HCO3 (-)/sAC/PKA pathway leading to ERK phosphorylation and its downstream target cyclin D2 (Ccnd2) upregulation. Reduced ERK phosphorylation and CCND2 were found in ovaries of rat PCOS model compared with the control. This study suggests that CFTR is required for normal follicle development and that its downregulation in PCOS may inhibit granulosa cell proliferation, resulting in abnormal follicle development in PCOS.

Glucose-Sensitive CFTR Suppresses Glucagon Secretion by Potentiating KATP Channels in Pancreatic Islet α Cells

&NA; The secretion of glucagon by islet &agr; cells is normally suppressed by high blood glucose, but this suppressibility is impaired in patients with diabetes or cystic fibrosis (CF), a disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate‐activated Cl‐ channel. However, precisely how glucose regulates glucagon release remains controversial. Here we report that elevated glucagon secretion, together with increased glucose‐induced membrane depolarization and Ca2+ response, is found in CFTR mutant (DF508) mice/islets compared with the wild‐type. Overexpression of CFTR in AlphaTC1‐9 cells results in membrane hyperpolarization and reduced glucagon release, which can be reversed by CFTR inhibition. CFTR is found to potentiate the adenosine triphosphate‐sensitive K+ (KATP) channel because membrane depolarization and whole‐cell currents sensitive to KATP blockers are significantly greater in wild‐type/CFTR‐overexpressed &agr; cells compared with that in DF508/non‐overexpressed cells. KATP knockdown also reverses the suppressive effect of CFTR overexpression on glucagon secretion. The results reveal that by potentiating KATP channels, CFTR acts as a glucose‐sensing negative regulator of glucagon secretion in &agr; cells, a defect of which may contribute to glucose intolerance in CF and other types of diabetes.