Androgen-induced upregulation of CFTR in pancreatic ß-cell contributes to hyperinsulinemia in PCOS model.

PURPOSE: Polycystic ovarian syndrome (PCOS) is an endocrine-metabolic condition affecting 5-10% of reproductive-aged women and characterized by hyperandrogenism, insulin resistance (IR), and hyperinsulinemia. CFTR is known to be regulated by steroid hormones, and our previous study has demonstrated an essential role of CFTR in ß-cell function. This study aims to investigate the contribution of androgen and CFTR to hypersecretion of insulin in PCOS and the underlying mechanism. METHODS: We established a rat PCOS model by subcutaneously implanting silicon tubing containing Dihydrotestosterone (DHT). Glucose tolerance test with insulin levels was performed at 9 weeks after implantation. A rat ß-cell line RINm5F, a mouse ß-cell line ß-TC-6, and mouse islets were treated with DHT, and with or without the androgen antagonist flutamide for CFTR and insulin secretion-related functional assays or mRNA/protein expression measurement. The effect of CFTR inhibitors on DHT-promoted membrane depolarization, glucose-stimulated intracellular Ca2+ oscillation and insulin secretion were examined by membrane potential imaging, calcium imaging and ELISA, respectively. RESULTS: The DHT-induced PCOS model showed increased body weight, impaired glucose tolerance, and higher blood glucose and insulin levels after glucose stimulation. CFTR was upregulated in islets of PCOS model and DHT-treated cells, which was reversed by flutamide. The androgen receptor (AR) could bind to the CFTR promoter region, which was enhanced by DHT. Furthermore, DHT-induced membrane depolarization, enhanced glucose-stimulated Ca2+ oscillations and insulin secretion, which could be abolished by CFTR inhibitors. CONCLUSIONS: Excessive androgen enhances glucose-stimulating insulin secretion through upregulation of CFTR, which may contribute to hyperinsulinemia in PCOS.

CFTR Modulates Hypothalamic Neuron Excitability to Maintain Female Cycle.

Cystic fibrosis transmembrane conductance regulator (CFTR), known as an epithelial Cl- channel, is increasingly noted to be expressed in the nervous system, although whether and how it plays a role in neuronal excitability is unclear. Given the association of CFTR with fertility, we tested here possible involvement of CFTR in regulating hypothalamic neuron excitability. Patch-clamp and Ca2+ imaging showed that pharmacological inhibition of CFTR evoked electrical pulses and Ca2+ spikes in primary rat hypothalamic neurons, which was dependent on extracellular Cl-. Hypothalamic neurons in brain-slice preparations from adult female mice with CFTR mutation (DF508) exhibited significantly reduced electrical pulses as compared to the wild-type controls. Removal of extracellular Cl- eliminated hypothalamic electrical pulses in the wild-type brain slices, which was reversible by subsequent addition of Cl-. In adult female mice, Ca2+ indicator (GCaMP6s)-based fiber-photometry showed that hypothalamic Ca2+ activities in vivo were enhanced at the proestrus/estrus phase as compared to the diestrus phase of the female cycle. Such estrus-associated hypothalamic activities were largely diminished in DF508 female mice, together with delayed puberty and disturbed female cycles. Therefore, these findings suggest a critical role of CFTR in modulating hypothalamic neuron excitability, which may account for the disturbed female cycles and reduced female fertility associated with CFTR mutations.

Targeting GSTP1 as Therapeutic Strategy against Lung Adenocarcinoma Stemness and Resistance to Tyrosine Kinase Inhibitors.

Glutathione S-transferase pi (GSTP1), a phase II detoxification enzyme, is known to be overexpressed and mediates chemotherapeutic resistance in lung cancer. However, whether GSTP1 supports cancer stem cells (CSCs) and the underlying mechanisms in lung adenocarcinoma (LUAD) remain largely unknown. This study unveiled that GSTP1 is upregulated in lung CSCs and supports tumor self-renewal, metastasis, and resistance to targeted tyrosine kinase inhibitors of LUAD both in vitro and in vivo. Mechanistically, CaMK2A (calcium/calmodulin-dependent protein kinase 2 isoform A)/NRF2 (nuclear factor erythroid 2-related factor 2)/GSTP1 is uncovered as a regulatory axis under hypoxia. CaMK2A increased GSTP1 expression through phosphorylating the Sersine558 residue of NRF2 and promoting its nuclear translocation, a novel mechanism for NRF2 activation apart from conventional oxidization-dependent activation. Upregulation of GSTP1 in turn suppressed reactive oxygen species levels and supported CSC phenotypes. Clinically, GSTP1 analyzed by immunohistochemistry is upregulated in a proportion of LUAD and serves as a prognostic marker for survival. Using patient-derived organoids from an ALK-translocated LUAD, the therapeutic potential of a specific GSTP1 inhibitor ezatiostat in combination treatment with the ALK inhibitor crizotinib is demonstrated. This study demonstrates GSTP1 to be a promising therapeutic target for long-term control of LUAD through targeting CSCs.

Biphasic regulation of CFTR expression by ENaC in epithelial cells: The involvement of Ca2+-modulated cAMP production.

The regulatory interaction between two typical epithelial ion channels, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC), for epithelial homeostasis has been noted, although the underlying mechanisms remain unclear. Here, we report that in a human endometrial epithelial cell line (ISK), shRNA-based stable knockdown of ENaC produced a biphasic effect: a low (∼23%) degree of ENaC knockdown resulted in significant increases in CFTR mRNA and protein levels, CFTR-mediated Cl- transport activity as well as intracellular cAMP concentration, while a higher degree (∼50%) of ENaC knockdown did not further increase but restored CFTR expression and cAMP levels. The basal intracellular Ca2+ level of ISK cells was lowered by ENaC knockdown or inhibition in a degree-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator that lowers free Ca2+ concentration, elevated cAMP level and CFTR mRNA expression at a low (5 µM) but not a high (50 µM) dose, mimicking the biphasic effect of ENaC knockdown. Moreover, KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC), abolished the CFTR upregulation induced by low-degree ENaC knockdown or Ca2+ chelation, suggesting the involvement of sAC-driven cAMP production in the positive regulation. A luciferase reporter to indicate CFTR transcription revealed that all tested degrees of ENaC knockdown/inhibition stimulated CFTR transcription in ISK cells, suggesting that the negative regulation on CFTR expression by the high-degree ENaC deficiency might occur at post-transcription stages. Additionally, similar biphasic effect of ENaC knockdown on CFTR expression was observed in a human bronchial epithelial cell line. Taken together, these results have revealed a previously unidentified biphasic regulatory role of ENaC in tuning CFTR expression involving Ca2+-modulated cAMP production, which may provide an efficient mechanism for dynamics and plasticity of the epithelial tissues in various physiological or pathological contexts.

Human pluripotent stem cell-derived ectomesenchymal stromal cells promote more robust functional recovery than umbilical cord-derived mesenchymal stromal cells after hypoxic-ischaemic brain damage.

Aims: Hypoxic-ischaemic encephalopathy (HIE) is one of the most serious complications in neonates and infants. Mesenchymal stromal cell (MSC)-based therapy is emerging as a promising treatment avenue for HIE. However, despite its enormous potential, the clinical application of MSCs is limited by cell heterogeneity, low isolation efficiency and unpredictable effectiveness. In this study, we examined the therapeutic effects and underlying mechanisms of human pluripotent stem cell-derived ectomesenchymal stromal cells (hPSC-EMSCs) in a rat model of HIE. Methods: hPSC-EMSCs were induced from either human embryonic stem cells or induced pluripotent stem cells. Stem cells or the conditioned medium (CM) derived from stem cells were delivered intracranially or intranasally to neonatal rats with HIE. Human umbilical cord-derived MSCs (hUC-MSCs) were used as the therapeutic comparison control and phosphate-buffered saline (PBS) was used as a negative control. Lesion size, apoptosis, neurogenesis, astrogliosis and microgliosis were evaluated. The rotarod test and Morris water maze were used to determine brain functional recovery. The PC-12 cell line, rat primary cortical neurons and neural progenitor cells were used to evaluate neurite outgrowth and the neuroprotective and neurogenesis effects of hPSC-EMSCs/hUC-MSCs. RNA-seq and enzyme-linked immunosorbent assays were used to determine the secretory factors that were differentially expressed between hPSC-EMSCs and hUC-MSCs. The activation and suppression of extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) were characterised using western blotting and immunofluorescent staining. Results: hPSC-EMSCs showed a higher neuroprotective potential than hUC-MSCs, as demonstrated by a more significant reduction in lesion size and apoptosis in the rat brain following hypoxia-ischaemia (HI). Compared with PBS treatment, hPSC-EMSCs promoted endogenous neurogenesis and alleviated astrogliosis and microgliosis. hPSC-EMSCs were more effective than hUC-MSCs. hPSC-EMSCs achieved a greater recovery of brain function than hUC-MSCs and PBS in rats with HIE. CM derived from hPSC-EMSCs had neuroprotective and neurorestorative effects in vitro through anti-apoptotic and neurite outgrowth- and neurogenesis-promoting effects. Direct comparisons between hPSC-EMSCs and hUC-MSCs revealed the significant enrichment of a group of secretory factors in hPSC-EMSCs, including nerve growth factor (NGF), platelet-derived growth factor-AA and transforming growth factor-ß2, which are involved in neurogenesis, synaptic transmission and neurotransmitter transport, respectively. Mechanistically, the CM derived from hPSC-EMSCs was found to potentiate NGF-induced neurite outgrowth and the neuronal differentiation of NPCs via the ERK/CREB pathway. Suppression of ERK or CREB abolished CM-potentiated neuritogenesis and neuronal differentiation. Finally, intranasal delivery of the CM derived from hPSC-EMSCs significantly reduced brain lesion size, promoted endogenous neurogenesis, mitigated inflammatory responses and improved functional recovery in rats with HIE. Conclusion: hPSC-EMSCs promote functional recovery after HI through multifaceted neuromodulatory activities via paracrine/trophic mechanisms. We propose the use of hPSC-EMSCs for the treatment of HIE, as they offer an excellent unlimited cellular source of MSCs.

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.

Angiotensin(1-7) activates MAS-1 and upregulates CFTR to promote insulin secretion in pancreatic ß-cells: the association with type 2 diabetes.

Objective: The beneficial effect of angiotensin(1-7) (Ang(1-7)), via the activation of its receptor, MAS-1, has been noted in diabetes treatment; however, how Ang(1-7) or MAS-1 affects insulin secretion remains elusive and whether the endogenous level of Ang(1-7) or MAS-1 is altered in diabetic individuals remains unexplored. We recently identified an important role of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in the regulation of insulin secretion. Here, we tested the possible involvement of CFTR in mediating Ang(1-7)'s effect on insulin secretion and measured the level of Ang(1-7), MAS-1 as well as CFTR in the blood of individuals with or without type 2 diabetes. Methods: Ang(1-7)/MAS-1/CFTR pathway was determined by specific inhibitors, gene manipulation, Western blotting as well as insulin ELISA in a pancreatic ß-cell line, RINm5F. Human blood samples were collected from 333 individuals with (n = 197) and without (n = 136) type 2 diabetes. Ang(1-7), MAS-1 and CFTR levels in the human blood were determined by ELISA. Results: In RINm5F cells, Ang(1-7) induced intracellular cAMP increase, cAMP-response element binding protein (CREB) activation, enhanced CFTR expression and potentiated glucose-stimulated insulin secretion, which were abolished by a selective CFTR inhibitor, RNAi-knockdown of CFTR, or inhibition of MAS-1. In human subjects, the blood levels of MAS-1 and CFTR, but not Ang(1-7), were significantly higher in individuals with type 2 diabetes as compared to those in non-diabetic healthy subjects. In addition, blood levels of MAS-1 and CFTR were in significant positive correlation in type-2 diabetic but not non-diabetic subjects. Conclusion: These results suggested that MAS-1 and CFTR as key players in mediating Ang(1-7)-promoted insulin secretion in pancreatic ß-cells; MAS-1 and CFTR are positively correlated and both upregulated in type 2 diabetes.

Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.

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.

Progenitor Cells Derived from Drain Waste Product of Open-Heart Surgery in Children.

Human cardiac progenitor cells isolated from the same host may have advantages over other sources of stem cells. The aim of this study is to establish a new source of human progenitor cells collected from a waste product, pericardiac effusion fluid, after open-heart surgery in children with congenital heart diseases. The fluid was collected every 24 h for 2 days after surgery in 37 children. Mononuclear cells were isolated and expanded in vitro. These pericardial effusion-derived progenitor cells (PEPCs) exhibiting cardiogenic lineage markers, were highly proliferative and enhanced angiogenesis in vitro. Three weeks after stem cell transplantation into the ischemic heart in mice, cardiac ejection fraction was improved significantly without detectable progenitor cells. Gene expression profiles of the repaired hearts revealed activation of several known repair mechanisms including paracrine effects, cell migration, and angiogenesis. These progenitor cells may have the potential for heart regeneration.

Elevation of antimüllerian hormone in women with polycystic ovary syndrome undergoing assisted reproduction: effect of insulin.

OBJECTIVE: To measure blood and follicular antimüllerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) undergoing assisted reproductive technologies (ART) and to examine the direct action of insulin on AMH expression in human granulosa cells. DESIGN: Prospective clinical and experimental study. SETTING: University Hospital-based laboratory. PATIENT(S): Women with (n = 86) and without (n = 172) PCOS in ART. INTERVENTION(S): Blood, follicular fluid, and luteinized granulosa cells were collected from PCOS and non-PCOS women in ART. MAIN OUTCOME MEASURE(S): Hormone levels in blood and fluid, and gene expression in granulosa cells. RESULT(S): Serum levels of AMH were elevated and inversely correlated with embryo cleavage rate in PCOS women in ART. Significant higher levels of AMH were also found in small and large follicles collected from PCOS women compared with non-PCOS women. Luteinized granulosa cells from PCOS women showed higher expression of AMH and its receptor AMHR2. Direct effect of insulin in increasing the expression of AMH in the isolated luteinized granulosa cells was observed, with the PCOS granulosa cells responding to a high dose of insulin. Cotreatment with AMH attenuated insulin-induced aromatase expression in the luteinized granulosa cells. CONCLUSION(S): These results suggest that insulin may contribute to AMH elevation in PCOS and that AMH counteracts insulin-promoted aromatase expression in granulosa cells.

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.

Retinoic acid promotes stem cell differentiation and embryonic development by transcriptionally activating CFTR.

Retinoic acid (RA) plays a pivotal role in many cellular processes; however, the signaling mechanisms mediating the effect of RA are not fully understood. Here, we show that RA transcriptionally upregulates cystic fibrosis transmembrane conductance regulator (Cftr) by promoting the direct binding of its receptor RARα to Cftr promoter in mouse spermatogonia and embryonic stem (ES) cells. The RA/CFTR pathway is involved in the differentiation of spermatogonia and organogenesis during the embryo development of Xenopus laevis. Loss of CFTR by siRNA-mediated knockdown blunts the RA-induced spermatogonial differentiation. Overexpression of CFTR mimics the effect of RA on the induction of spermatogonial differentiation or restores the developmental defects induced by the knockdown of RARα in spermatogonial cells and Xenopus laevis. Analysis of the human database shows that the expression of CFTR positively correlates with RARα in brain tissues, stem cells as well as cancers, supporting the role of RA/CFTR pathway in various developmental processes in humans. Together, our study discovers an essential role of CFTR in mediating the RA-dependent signaling for stem cell differentiation and embryonic development.

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.

MRP4 regulates ENaC-dependent CREB/COX-2/PGE2 signaling during embryo implantation.

Multi-drug resistance protein 4 (MRP4), a potential chemotherapeutic target as well as a transporter for endogenous signaling molecules (e.g. prostaglandins), is known to be expressed in the endometrium, although its possible role(s) in the physiology of the endometrium remains unknown. Here, we show that MRP4 is upregulated at implantation window and localized to the basolateral membrane of the endometrial epithelium, the interface between the epithelium and stroma in mice. In human endometrial epithelial cells, MRP4 expression is upregulated by ENaC activation and the inhibition of MRP4 blocks ENaC-dependent PGE2 release as well as phosphorylation of CREB. Intrauterine injection of MRP4 inhibitor in mice prior to implantation significantly downregulated implantation markers COX-2, Claudin4 and Lif, and reduced implantation rate. These results in together have revealed a previously undefined role of MRP4 in mediating ENaC-dependent CREB/COX-2/PGE2 signaling essential to embryo implantation with implication in cancer progression as well.

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.

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response.

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

XCI-escaping gene KDM5C contributes to ovarian development via downregulating miR-320a.

Mechanisms underlying female gonadal dysgenesis remain unclarified and relatively unstudied. Whether X-chromosome inactivation (XCI)-escaping genes and microRNAs (miRNAs) contribute to this condition is currently unknown. We compared 45,X Turner Syndrome women with 46,XX normal women, and investigated differentially expressed miRNAs in Turner Syndrome through plasma miRNA sequencing. We found that miR-320a was consistently upregulated not only in 45,X plasma and peripheral blood mononuclear cells (PBMCs), but also in 45,X fetal gonadal tissues. The levels of miR-320a in PBMCs from 45,X, 46,XX, 46,XY, and 47,XXY human subjects were inversely related to the expression levels of XCI-escaping gene KDM5C in PBMCs. In vitro models indicated that KDM5C suppressed miR-320a transcription by directly binding to the promoter of miR-320a to prevent histone methylation. In addition, we demonstrated that KITLG, an essential gene for ovarian development and primordial germ cell survival, was a direct target of miR-320a and that it was downregulated in 45,X fetal gonadal tissues. In conclusion, we demonstrated that downregulation of miR-320a by the XCI-escaping gene KDM5C contributed to ovarian development by targeting KITLG.

Epidermal CFTR Suppresses MAPK/NF-κB to Promote Cutaneous Wound Healing.

BACKGROUND: CFTR is implicated in cutaneous wound healing although the underlying mechanisms are not fully understood. In other cell types, CFTR is reported to regulate MAPK/ NF-κB signaling. We undertook the present study to explore a possible role of CFTR in regulating MAPK/NF-κB during cutaneous wound healing. Methods& Results: The splint-excisional and incisional wound healing models were used in CFTR mutant (DF508) mice. The cell-scratch model was used in a human keratinocyte line, HaCaT, in conjunction with CFTR knockdown or overexpression. The epidermal inflammation, keratinocyte proliferation and differentiation, as well as MAPK/NF-κB signaling were examined. Inhibitors of MAPK/NF-κB were also used. RESULTS: Both DF508 mice and HaCaT cells with CFTR knockdown exhibited delayed cutaneous wound healing with exuberant inflammation, increased proliferation and aberrant differentiation. Knockdown of CFTR in HaCaT cells resulted in phosphorylation of ERK, p38 and IκBα. The disturbance of inflammation, proliferation and differentiation in HaCaT cells were reversed by CFTR overexpression or inhibition of MAPK or NF-κB. CONCLUSION: CFTR plays a role in suppressing MAPK/NF-κB to relieve inflammation, reduce proliferation and promote differentiation of keratinocytes, and thus promotes cutaneous wound healing.

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.