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.

CD147 deficiency is associated with impairedsperm motility/acrosome reaction and offersa therapeutic target for asthenozoospermia.

Patients with asthenozoospermia often present multiple defects in sperm functions apart from a decrease in sperm motility. However, the etiological factors underlying these multifaceted defects remain mostly unexplored, which may lead to unnecessary treatment and unsatisfactory assisted reproductive technologies (ART) outcome. Here, we show that the protein levels of CD147 were lowered in sperm obtained from asthenozoospermic infertile patients exhibiting defects in both sperm motility and the acrosome reaction. Whereas CD147 maintained sperm motility before capacitation, female tract-derived soluble CD147 interacted with sperm-bound CD147 to induce an acrosome reaction in capacitated sperm. Soluble CD147 treatment restored the acrosome reaction and improved the fertility of sperm from patients with asthenozoospermia. Mechanistically, CD147 promotes sperm motility and acrosome reaction (AR) by eliciting Ca2+ influx through soluble CD147 binding to sperm-bound CD147. Notably, the level of soluble CD147 in seminal plasma was positively correlated with the fertilization rate and pregnancy outcome in infertile couples undergoing in vitro fertilization. Our study has identified a marker for the diagnosis and a therapeutic target for the defective AR capability in asthenozoospermia and a candidate for the prediction of in vitro fertilization outcomes for male infertile patients that facilitates the development of precision medicine in ART.

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.

CCR6 is required for ligand-induced CatSper activation in human sperm.

CatSper channel has been considered the principal sperm Ca2+ channel responsible for the cytosolic Ca2+ elevation required for various sperm functions necessary for fertilization [1-4]. However, the mechanism underlying the activation of CatSper channel by various physiological ligands remain incompletely understood. We have recently demonstrated the expression of C-C chemokine receptor 6 (CCR6) in sperm and Ca2+ influx upon binding of human ß-defensin 1 (DEFB1) to CCR6, which is important for sperm motility [5]. In the present study, we have demonstrated that CCR6 receptor and CatSper channel are both required for the Ca2+ entry/current induced by physiological ligands DEFB1, chemokine (C-C motif) ligand 20 (CCL20) and progesterone in human sperm. CCR6 is co-localized and interacts with CatSper in human sperm. Ca2+ influx mediated by CCR6 and CatSper is required for essential sperm functions, including motility, hyperactivation and acrosome reaction, which are impaired in infertile sperm showing reduced levels of CCR6 and CatSper. The present finding suggests a critical role of CCR6 receptor in mediating ligand-induced, CatSper-dependent Ca2+ influx required for various sperm functions and thus male fertility.

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.

Hepatitis C virus NS5A protein cooperates with phosphatidylinositol 4-kinase IIIα to induce mitochondrial fragmentation.

Hepatitis C virus (HCV) has long been observed to take advantage of the host mitochondria to support viral replication and assembly. The HCV core protein has been implicated to fragment host mitochondria. In this report, we have discovered that the non-structural protein 5A (NS5A) plays an instructive role in attaching ER with mitochondria, causing mitochondrial fragmentation. Dynamin-related protein 1(Drp1), a host protein essential to mitochondrial membrane fission, does not play a role in NS5A-induced mitochondrial fragmentation. Instead, phosphatidylinositol 4-kinase IIIα (PI4KA), which has been demonstrated to bind to NS5A and is required to support HCV life cycle, is required for NS5A to induce mitochondrial fragmentation. Both NS5A and core are required by HCV to fragment the mitochondria, as inhibiting either of their respective downstream proteins, PI4KA or Drp1, resulted in lengthening of mitochondria tubules in HCVcc-infected cells. By fragmenting the mitochondria, NS5A renders the cells more resistant to mitochondria mediated apoptosis. This finding indicates previously-ignored contribution of NS5A in HCV-induced mitochondria dysfunction.

FSH regulates fat accumulation and redistribution in aging through the Gαi/Ca(2+)/CREB pathway.

Increased fat mass and fat redistribution are commonly observed in aging populations worldwide. Although decreased circulating levels of sex hormones, androgens and oestrogens have been observed, the exact mechanism of fat accumulation and redistribution during aging remains obscure. In this study, the receptor of follicle-stimulating hormone (FSH), a gonadotropin that increases sharply and persistently with aging in both males and females, is functionally expressed in human and mouse fat tissues and adipocytes. Follicle-stimulating hormone was found to promote lipid biosynthesis and lipid droplet formation; FSH could also alter the secretion of leptin and adiponectin, but not hyperplasia, in vitro and in vivo. The effects of FSH are mediated by FSH receptors coupled to the Gαi protein; as a result, Ca(2+) influx is stimulated, cAMP-response-element-binding protein is phosphorylated, and an array of genes involved in lipid biosynthesis is activated. The present findings depict the potential of FSH receptor-mediated lipodystrophy of adipose tissues in aging. Our results also reveal the mechanism of fat accumulation and redistribution during aging of males and females.

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.

Deficient human ß-defensin 1 underlies male infertility associated with poor sperm motility and genital tract infection.

Genital tract infection and reduced sperm motility are considered two pivotal etiological factors for male infertility associated with leukocytospermia and asthenozoospermia, respectively. We demonstrate that the amount of human ß-defensin 1 (DEFB1) in sperm from infertile men exhibiting either leukocytospermia or asthenozoospermia, both of which are associated with reduced motility and reduced bactericidal activity in sperm, is much lower compared to that in normal fertile sperm. Interference with DEFB1 function also decreases both motility and bactericidal activity in normal sperm, whereas treatment with recombinant DEFB1 markedly restores DEFB1 expression, bactericidal activity, sperm quality, and egg-penetrating ability in sperm from both asthenozoospermia and leukocytospermia patients. DEFB1 interacts with chemokine receptor type 6 (CCR6) in sperm and triggers Ca(2+) mobilization, which is important for sperm motility. Interference with CCR6 function also reduces motility and bactericidal activity of normal sperm. The present finding explains a common defect in male infertility associated with both asthenozoospermia and leukocytospermia, indicating a dual role of DEFB1 in defending male fertility. These results also suggest that the expression of DEFB1 and CCR6 may have diagnostic potential and that treatment of defective sperm with recombinant DEFB1 protein may be a feasible therapeutic approach for male infertility associated with poor sperm motility and genital tract infection.

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.

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.

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.

TRAPPC9 mediates the interaction between p150 and COPII vesicles at the target membrane.

BACKGROUND: The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150(Glued), a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown. PRINCIPLE FINDINGS: We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150(Glued) away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150(Glued) via the same carboxyl terminal domain of p150(Glued) that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150(Glued) and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150(Glued) from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane. CONCLUSIONS: These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.