Magnesium implantation or supplementation ameliorates bone disorder in CFTR-mutant mice through an ATF4-dependent Wnt/ß-catenin signaling.

Magnesium metal and its alloys are being developed as effective orthopedic implants; however, the mechanisms underlying the actions of magnesium on bones remain unclear. Cystic fibrosis, the most common genetic disease in Caucasians caused by the mutation of CFTR, has shown bone disorder as a key clinical manifestation, which currently lacks effective therapeutic options. Here we report that implantation of magnesium-containing implant stimulates bone formation and improves bone fracture healing in CFTR-mutant mice. Wnt/ß-catenin signaling in the bone is enhanced by the magnesium implant, and inhibition of Wnt/ß-catenin by iCRT14 blocks the magnesium implant to improve fracture healing in CFTR-mutant mice. We further demonstrate that magnesium ion enters osteocytes, increases intracellular cAMP level and activates ATF4, a key transcription factor known to regulate Wnt/ß-catenin signaling. In vivo knockdown of ATF4 abolishes the magnesium implant-activated ß-catenin in bones and reverses the improved-fracture healing in CFTR-mutant mice. In addition, oral supplementation of magnesium activates ATF4 and ß-catenin as well as enhances bone volume and density in CFTR-mutant mice. Together, these results show that magnesium implantation or supplementation may serve as a potential anabolic therapy for cystic fibrosis-related bone disease. Activation of ATF4-dependent Wnt/ß-catenin signaling in osteocytes is identified as a previously undefined mechanism underlying the beneficial effect of magnesium on bone formation.

MRP4 sustains Wnt/ß-catenin signaling for pregnancy, endometriosis and endometrial cancer.

Rationale: Abnormal Wnt/ß-catenin signaling in the endometrium can lead to both embryo implantation failure and severe pathogenic changes of the endometrium such as endometrial cancer and endometriosis. However, how Wnt/ß-catenin signaling is regulated in the endometrium remains elusive. We explored possible regulation of Wnt/ß-catenin signaling by multi-drug resistance protein 4 (MRP4), a potential target in cancer chemotherapy, and investigated the mechanism. Methods: Knockdown of MRP4 was performed in human endometrial cells in vitro or in a mouse embryo-implantation model in vivo. Immunoprecipitation, immunoblotting and immunofluorescence were used to assess protein interaction and stability. Wnt/ß-catenin signaling was assessed by TOPflash reporter assay and quantitative PCR array. Normal and endometriotic human endometrial tissues were examined. Data from human microarray or RNAseq databases of more than 100 participants with endometriosis, endometrial cancer or IVF were analyzed. In vitro and in vivo tumorigenesis was performed. Results: MRP4-knockdown, but not its transporter-function-inhibition, accelerates ß-catenin degradation in human endometrial cells. MRP4 and ß-catenin are co-localized and co-immunoprecipitated in mouse and human endometrium. MRP4-knockdown in mouse uterus reduces ß-catenin levels, downregulates a series of Wnt/ß-catenin target genes and impairs embryo implantation, which are all reversed by blocking ß-catenin degradation. Analysis of human endometrial biopsy samples and available databases reveals significant and positive correlations of MRP4 with ß-catenin and Wnt/ß-catenin target genes in the receptive endometrium in IVF, ectopic endometriotic lesions and endometrial cancers. Knockdown of MRP4 also inhibits in vitro and in vivo endometrial tumorigenesis. Conclusion: A previously undefined role of MRP4 in stabilizing ß-catenin to sustain Wnt/ß-catenin signaling in endometrial cells is revealed for both embryo implantation and endometrial disorders, suggesting MRP4 as a theranostic target for endometrial diseases associated with Wnt/ß-catenin signaling abnormality.

Defective CFTR promotes intestinal proliferation via inhibition of the hedgehog pathway during cystic fibrosis.

Hyperproliferation occurs in a variety of tissues and organs during cystic fibrosis (CF). However, the associated molecular mechanisms remain elusive. We investigated the molecular link between cystic fibrosis transmembrane conductance regulator (CFTR) defects and hyperproliferation, and showed that the length of the entire gastrointestinal tract was longer and the intestinal crypts were deeper in CF mice compared to those in wild-type animals. PCNA expression increased in CF mouse intestines and CFTR-knockdown cells. Villin1, an intestinal differentiation marker, was downregulated in CF mice. Ihh and Gli1 were significantly downregulated, whereas TCF4 was activated in CF mouse intestines and CFTR-knockdown Caco2 cells. Importantly, ß-catenin activators rescued Gli1 suppression, suggesting that hedgehog signaling might be mediated by the Wnt/ß-catenin pathway in the absence of functional CFTR. Moreover, PCNA positivity in the crypts of CF mice was alleviated by LiCl, which activates Wnt/ß-catenin signaling. Further, a strong positive correlation was observed between the expression of CFTR and Ihh in intestines. Our study revealed a previously unidentified role of CFTR in regulating hedgehog signaling through ß-catenin, providing novel insights into the physiological function of CFTR and CF-related diseases.

Activation of the epithelial sodium channel (ENaC) leads to cytokine profile shift to pro-inflammatory in labor.

The shift of cytokine profile from anti- to pro-inflammatory is the most recognizable sign of labor, although the underlying mechanism remains elusive. Here, we report that the epithelial sodium channel (ENaC) is upregulated and activated in the uterus at labor in mice. Mechanical activation of ENaC results in phosphorylation of CREB and upregulation of pro-inflammatory cytokines as well as COX-2/PGE2 in uterine epithelial cells. ENaC expression is also upregulated in mice with RU486-induced preterm labor as well as in women with preterm labor. Interference with ENaC attenuates mechanically stimulated uterine contractions and significantly delays the RU486-induced preterm labor in mice. Analysis of a human transcriptome database for maternal-fetus tissue/blood collected at onset of human term and preterm births reveals significant and positive correlation of ENaC with labor-associated pro-inflammatory factors in labored birth groups (both term and preterm), but not in non-labored birth groups. Taken together, the present finding reveals a pro-inflammatory role of ENaC in labor at term and preterm, suggesting it as a potential target for the prevention and treatment of preterm labor.

Abnormal CFTR Affects Glucagon Production by Islet α Cells in Cystic Fibrosis and Polycystic Ovarian Syndrome.

Glucagon, produced by islet α cells, functions to increase blood glucose. Abnormal glucose levels are often seen in cystic fibrosis (CF), a systematic disease caused by mutations of the CF transmembrane conductance regulator (CFTR), and in polycystic ovarian syndrome (PCOS), an endocrine disorder featured with hyperandrogenism affecting 5-10% women of reproductive age. Here, we explored the role of CFTR in glucagon production in α cells and its possible contribution to glucagon disturbance in CF and PCOS. We found elevated fasting glucagon levels in CFTR mutant (DF508) mice compared to the wildtypes. Glucagon and prohormone convertase 2 (PC2) were also upregulated in CFTR inhibitor-treated or DF508 islets, as compared to the controls or wildtypes, respectively. Dihydrotestosterone (DHT)-induced PCOS rats exhibited significantly lower fasting glucagon levels with higher CFTR expression in α cells compared to that of controls. Treatment of mouse islets or αTC1-9 cells with DHT enhanced CFTR expression and reduced the levels of glucagon and PC2. The inhibitory effect of DHT on glucagon production was blocked by CFTR inhibitors in mouse islets, and mimicked by overexpressing CFTR in αTC1-9 cells with reduced phosphorylation of the cAMP/Ca2+ response element binding protein (p-CREB), a key transcription factor for glucagon and PC2. These results revealed a previously undefined role of CFTR in suppressing glucagon production in α-cells, defects in which may contribute to glucose metabolic disorder seen in CF and PCOS.

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

The secretion of glucagon by islet α 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 α 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 α cells, a defect of which may contribute to glucose intolerance in CF and other types of diabetes.

Sperm gamma-aminobutyric acid type A receptor delta subunit (GABRD) and its interaction with purinergic P2X2 receptors in progesterone-induced acrosome reaction and male fertility.

The mechanism underlying the non-genomic action of progesterone in sperm functions and related Ca2+ mobilisation remains elusive. Herein we report the expression of gamma-aminobutyric acid type A receptor delta subunit (GABRD) in human and rodent sperm and its involvement in mediating the progesterone-induced acrosome reaction. GABRD was localised in the sperm head/neck region. A δ(392-422)-specific inhibitory peptide against GABRD blocked the progesterone-induced acrosome reaction and the associated increase in intracellular Ca2+. Similarly, an inhibitory effect against both progesterone-induced Ca2+ influx and the acrosome reaction was observed with a P2X2 receptor antagonist. The lack of synergism between the GABRD and P2X2 inhibitors suggests that these two receptors are playing a role in the same pathway. Furthermore, a co-immunoprecipitation experiment demonstrated that GABRD could undergo protein-protein interactions with the Ca2+-conducting P2X2 receptor. This interaction between the receptors could be reduced following progesterone (10µM) inducement. Significantly reduced GABRD expression was observed in spermatozoa from infertile patients with reduced acrosome reaction capacity, suggesting that normal expression of GABRD is critical for the sperm acrosome reaction and thus male fertility. The results of the present study indicate that GABRD represents a novel progesterone receptor or modulator in spermatozoa that is responsible for the progesterone-induced Ca2+ influx required for the acrosome reaction through its interaction with the P2X2 receptor.

CFTR-ß-catenin interaction regulates mouse embryonic stem cell differentiation and embryonic development.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated anion channel capable of conducting both Cl- and HCO3-, mutations of which cause cystic fibrosis (CF), a common autosomal recessive disease. Although CF patients are known to have varied degree of developmental problems, the biological role of CFTR in embryonic development remains elusive. Here, we show that CFTR is functionally expressed in mouse ESCs. CFTR-/- mESCs exhibit dramatic defect in mesendoderm differentiation. In addition, CFTR physically interacts with ß-catenin, defect of which leads to premature degradation of ß-catenin and suppressed activation of ß-catenin signaling. Furthermore, knockdown of CFTR retards the early development of Xenopus laevis with impaired mesoderm/endoderm differentiation and ß-catenin signaling. Our study reveals a previously undefined role of CFTR in controlling ESC differentiation and early embryonic development via its interaction with ß-catenin, and provides novel insights into the understanding of embryonic development.

Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats.

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.

Dynamically Regulated CFTR Expression and Its Functional Role in Cutaneous Wound Healing.

The physiological role of cystic fibrosis transmembrane conductance regulator (CFTR) in keratinocytes and skin wound healing is completely unknown. The present study shows that CFTR is expressed in the multiple layers of keratinocytes in mouse epidermis and exhibits a dynamic expression pattern in a dorsal skin wound healing model, with diminishing levels observed from day 3 to day 5 and re-appearing from day 7 to day 10 after wounding. Knockdown of CFTR in cultured human keratinocytes promotes cell migration but inhibits differentiation, while overexpression of CFTR suppresses migration but enhances differentiation, indicating an important role of CFTR in regulating keratinocyte behavior. In addition, we have demonstrated a direct association of CFTR with epithelial junction formation as knockdown of CFTR downregulates the expression of adhesion molecules, such as E-cadherin, ZO-1 and ß-catenin, and disrupts the formation of cell junction, while overexpression of CFTR enhances cell junction formation. More importantly, we have shown that ΔF508cftr-/- mice with defective CFTR exhibit delayed wound healing as compared to wild type mice, indicating that normal function of CFTR is critical for wound repair. Taken together, the present study has revealed a previously undefined role of CFTR in regulating skin wound healing processes, which may have implications in injury repair of other epithelial tissues.

Glucose-induced electrical activities and insulin secretion in pancreatic islet ß-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 ß-cells. We demonstrate that glucose elicited whole-cell currents, membrane depolarization, electrical bursts or action potentials, Ca(2+) oscillations and insulin secretion are abolished or reduced by inhibitors or knockdown of CFTR in primary mouse ß-cells or RINm5F ß-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 ß-cells. Our results reveal a role of CFTR in glucose-induced electrical activities and insulin secretion in ß-cells, shed light on the pathogenesis of CFRD and possibly other idiopathic diabetes, and present a potential treatment strategy.

Activation of the epithelial Na+ channel triggers prostaglandin E2 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 Ca²âº 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.

CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development.

Although HCO(3)(-) is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO(3)(-) acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO(3)(-) on preimplantation embryo development can be suppressed by interfering the function of a HCO(3)(-)-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO(3)(-) or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO(3)(-) removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO(3)(-) to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.

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.

CFTR negatively regulates cyclooxygenase-2-PGE(2) 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 E(2) (PGE(2)) 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 PGE(2) 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 PGE(2), indicating possible involvement of CFTR in negative regulation of COX-2/PGE(2). In conclusion, CFTR is a negative regulator of PGE(2)-mediated inflammatory response, defect of which may result in excessive activation of NF-κB, leading to over production of PGE(2) as seen in inflammatory CF tissues.

Dedifferentiation-reprogrammed mesenchymal stem cells with improved therapeutic potential.

Stem cell transplantation has been shown to improve functional outcome in degenerative and ischemic disorders. However, low in vivo survival and differentiation potential of the transplanted cells limits their overall effectiveness and thus clinical usage. Here we show that, after in vitro induction of neuronal differentiation and dedifferentiation, on withdrawal of extrinsic factors, mesenchymal stem cells (MSCs) derived from bone marrow, which have already committed to neuronal lineage, revert to a primitive cell population (dedifferentiated MSCs) retaining stem cell characteristics but exhibiting a reprogrammed phenotype distinct from their original counterparts. Of therapeutic interest, the dedifferentiated MSCs exhibited enhanced cell survival and higher efficacy in neuronal differentiation compared to unmanipulated MSCs both in vitro and in vivo, with significantly improved cognition function in a neonatal hypoxic-ischemic brain damage rat model. Increased expression of bcl-2 family proteins and microRNA-34a appears to be the important mechanism giving rise to this previously undefined stem cell population that may provide a novel treatment strategy with improved therapeutic efficacy.

Defective CFTR-dependent CREB activation results in impaired spermatogenesis and azoospermia.

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disease among Caucasians caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) with over 95% male patients infertile. However, whether CFTR mutations could affect spermatogenesis and result in azoospermia remains an open question. Here we report compromised spermatogenesis, with significantly reduced testicular weight and sperm count, and decreased cAMP-responsive element binding protein (CREB) expression in the testes of CFTR knockout mice. The involvement of CFTR in HCO(3) (-) transport and the expression of the HCO(3) (-) sensor, soluble adenylyl cyclase (sAC), are demonstrated for the first time in the primary culture of rat Sertoli cells. Inhibition of CFTR or depletion of HCO(3) (-) could reduce FSH-stimulated, sAC-dependent cAMP production and phosphorylation of CREB, the key transcription factor in spermatogenesis. Decreased CFTR and CREB expression are also observed in human testes with azoospermia. The present study reveals a previously undefined role of CFTR and sAC in regulating the cAMP-CREB signaling pathway in Sertoli cells, defect of which may result in impaired spermatogenesis and azoospermia. Altered CFTR-sAC-cAMP-CREB functional loop may also underline the pathogenesis of various CF-related diseases.

Switching from bone marrow-derived neurons to epithelial cells through dedifferentiation and translineage redifferentiation.

While the ability of stem cells to switch lineages has been suggested, the route(s) through which this may happen is unclear. To date, the best characterized adult stem cell population considered to possess transdifferentiation capacity is BM-MSCs (bone marrow mesenchymal stem cells). We investigated whether BM-MSCs that had terminally differentiated into the neural or epithelial lineage could be induced to transdifferentiate into the other phenotype in vitro. Our results reveal that neuronal phenotypic cells derived from adult rat bone marrow cells can be switched to epithelial phenotypic cells, or vice versa, by culture manipulation allowing the differentiated cells to go through, first, dedifferentiation and then redifferentiation to another phenotype. Direct transdifferentiation from differentiated neuronal or epithelial phenotype to the other differentiated phenotype cannot be observed even when appropriate culture conditions are provided. Thus, dedifferentiation appears to be a prerequisite for changing fate and differentiating into a different lineage from a differentiated cell population.

Rescue of defective pancreatic secretion in cystic-fibrosis cells by suppression of a novel isoform of phospholipase C.

BACKGROUND: Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived. METHODS: Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohistochemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion. FINDINGS: We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion. INTERPRETATION: NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.

Involvement of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in a wide variety of epithelial cells, mutations of which are responsible for the hallmark defective chloride secretion observed in cystic fibrosis (CF). Although CFTR has been implicated in bicarbonate secretion, its ability to directly mediate bicarbonate secretion of any physiological significance has not been shown. We demonstrate here that endometrial epithelial cells possess a CFTR-mediated bicarbonate transport mechanism. Co-culture of sperm with endometrial cells treated with antisense oligonucleotide against CFTR, or with bicarbonate secretion-defective CF epithelial cells, resulted in lower sperm capacitation and egg-fertilizing ability. These results are consistent with a critical role of CFTR in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm, providing a link between defective CFTR and lower female fertility in CF.