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.

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.

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.

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.

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-ß1-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.

Flavonoid Myricetin Modulates GABA(A) Receptor Activity through Activation of Ca(2+) Channels and CaMK-II Pathway.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA(A) receptor. This effect of myricetin was not blocked by the GABA(A) receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca(2+)/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca(2+) imaging studies found that myricetin could increase Ca(2+) current and intracellular Ca(2+) concentration, respectively, via T- and L-type Ca(2+) channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA(A) receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA(A) receptor.

STK31 maintains the undifferentiated state of colon cancer cells.

The expression of serine/threonine kinase (STK) family is frequently altered in human cancers. However, the functions of these kinases in cancer development remain elusive. Here, we report that STK31 is robustly and heterogeneously expressed in colon cancer tissues and plays a critical role in determining the differentiation state of colon cancer cells. Knockdown or overexpression of STK31 induced or inhibited differentiation of colon cancer cells, respectively. Deletion of the STK domain abolished the inhibiting effect of STK31. Associated with differentiation, knockdown of STK31 resulted in significant suppression of tumorigenicity both in vitro and in vivo. Genome microarray analysis showed that knockdown of STK31 altered the expression profile of genes that are known to be involved in germ cell and cancer differentiation. Taken together, these results suggest that STK31 is able to control the differentiation state of colon cancer cells, which critically depends on its STK domain. The present findings may shed light on the new therapeutic approach against cancer by targeting STK31 and cancer differentiation.

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.

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.

Lymphocyte CFTR promotes epithelial bicarbonate secretion for bacterial killing.

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in lymphocytes has been reported for nearly two decades; however, its physiological role remains elusive. Here, we report that co-culture of lymphocytes with lung epithelial cell line, Calu-3, promotes epithelial HCO(3)- production/secretion with up-regulated expression of carbonic anhydrase 2 and 4 (CA-2, CA-4) and enhanced bacterial killing capability. The lymphocyte-enhanced epithelial HCO(3)- secretion and bacterial killing activity was abolished when Calu3 cells were co-cultured with lymphocytes from CFTR knockout mice, or significantly reduced by interfering with E-cadherin, a putative binding partner of CFTR. Bacterial lipopolysaccharide (LPS)-induced E-cadherin and CA-4 expression in the challenged lung was also found to be impaired in CFTR knockout mice compared to that of the wild-type. These results suggest that the interaction between lymphocytes and epithelial cells may induce a previously unsuspected innate host defense mechanism against bacterial infection by stimulating epithelial HCO(3)- production/secretion, which requires CFTR expression in lymphocytes.

Bak Foong Pills induce an analgesic effect by inhibiting nociception via the somatostatin pathway in mice.

Dysmenorrhoea, defined as cramping pain in the lower abdomen occurring before or during menstruation, affects, to varying degrees, up to 90% of women of child-bearing age. We investigated whether BFP (Bak Foong Pills), a traditional Chinese medicine treatment for dysmenorrhoea, possesses analgesic properties. Results showed that BFP was able to significantly reduce pain responses following subchronic treatment for 3 days, but not following acute (1 h) treatment in response to acetic acid-induced writhing in C57/B6 mice. The analgesic effect was not due to inhibition of COX (cyclo-oxygenase) activity, evidenced by the lack of inhibition of prostacyclin and PGE2 (prostaglandin E2) production. Molecular analysis revealed that BFP treatment modulated the expression of a number of genes in the spinal cord of mice subjected to acetic acid writhing. RT-PCR (reverse transcription-PCR) analysis of spinal cord samples showed that both sst4 (somatostatin receptor 4) and sst2 receptor mRNA, but not µOR (µ-opiate receptor) and NK1 (neurokinin-1) receptor mRNA, were down-regulated following BFP treatment, thus implicating somatostatin involvement in BFP-induced analgesia. Administration of c-som (cyclo-somatostatin), a somatostatin antagonist, prior to acetic acid-induced writhing inhibited the analgesic effect. Thus subchronic treatment with BFP has anti-nociceptive qualities mediated via the somatostatin pathway.

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.

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.

A protective mechanism against antibiotic-induced ototoxicity: role of prestin.

Hearing loss or ototoxicity is one of the major side effects associated with the use of the antibiotics, particularly aminoglycosides (AGs), which are the most commonly used antibiotics worldwide. However, the molecular and cellular events involved in the antibiotic-induced ototoxicity remains unclear. In the present study, we test the possibility that prestin, the motor protein specifically expressed in the basolateral membrane of outer hair cells (OHCs) in the cochlea with electromotility responsible for sound amplification, may be involved in the process of AG-induced apoptosis in OHCs. Our results from both mice model and cultured cell line indicate a previously unexpected role of prestin, in mediating antibiotic-induced apoptosis, the effect of which is associated with its anion-transporting capacity. The observed downregulation of prestin mRNA prior to detectable apoptosis in OHCs and hearing loss in the antibiotic-treated mice is interesting, which may serve as a protective mechanism against hearing loss induced by AGs in the early stage.

Abnormally enhanced cystic fibrosis transmembrane conductance regulator-mediated apoptosis in endometrial cells contributes to impaired embryo implantation in controlled ovarian hyperstimulation.

OBJECTIVE: To investigate the effects and underlying mechanism of controlled ovarian hyperstimulation (COH)-induced supraphysiologic concentration of E2 on the endometrium and outcome of embryo implantation. DESIGN: Prospective experimental study. SETTING: University-based laboratory. ANIMAL(S): Imprinting control region female mice, 8-10 weeks old with regular estrous cycles. INTERVENTION(S): Intraperitoneal injection of 10 IU of pregnant mare serum gonadotropin (PMSG) at noon followed by an additional injection of 10 IU hCG 48 hours later. MAIN OUTCOME MEASURE(S): Uteri were collected from either superovulated or control mice (natural cycle) the morning after hCG administration on day 4 to evaluate and count blastocysts. A mouse blastocyst-endometrium coculture model was used to evaluate blastocyst adhesion to control or COH-treated endometrium. The cystic fibrosis transmembrane conductance regulator (CFTR) expression was determined by immunofluorescence, Western blot, and apoptosis determined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay in both natural cycle and COH cycle endometrium. Primary culture of mouse endometrial epithelial cells was established to further determine the effects of various concentrations of E2 on apoptosis. RESULT(S): We demonstrated that COH had adverse effects on blastocyst adhesion. In addition, COH endometrium exhibited an aberrant up-regulation of CFTR expression and a higher apoptotic rate compared with normal endometrium during the implantation period. Administration of exogenous supraphysiologic concentration of E2 to primary mouse endometrial epithelial cells mimicked the COH-induced up-regulation of CFTR and apoptosis observed in vivo. Furthermore, inhibition of CFTR activity abrogated E2-induced apoptosis. CONCLUSION(S): The COH-induced supraphysiologic concentration of E2 aberrantly up-regulates CFTR, which leads to increased apoptosis in endometrial epithelial cells, resulting in impaired embryo implantation.

Involvement of calpain-I and microRNA34 in kanamycin-induced apoptosis of inner ear cells.

Inner ear cells, including hair cells, spiral ganglion cells, stria vascularis cells and supporting cells on the basilar membrane, play a major role in transducing hearing signals and regulating inner ear homoeostasis. However, their functions are often damaged by antibiotic-induced ototoxicity. Apoptosis is probably involved in inner ear cell injury following aminoglycoside treatment. Calpain, a calcium-dependent protease, is essential for mediating and promoting cell death. We have therefore investigated the involvement of calpain in the molecular mechanism underlying ototoxicity induced by the antibiotic kanamycin in mice. Kanamycin (750 mg/kg) mainly induced cell death of cochlear cells, including stria vascularis cells, supporting cells and spiral ganglion cells, but not hair cells within the organ of Corti. Cell death due to apoptosis occurred in a time-dependent manner with concomitant up-regulation of calpain expression. Furthermore, the expression levels of two microRNAs, mir34a and mir34c, were altered in a dose-dependent manner in cochlear cells. These novel findings demonstrated the involvement of both calpain and microRNAs in antibiotic-induced ototoxicity.

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.

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.